BIOL1106

   

 
 
 
 
 
 
 
 
 

Introduction  to  Organismic  and    

                                                 

                                           Evolutionary  Biology  
                                                     Biology  1106  
 
                                         Laboratory  Manual  
                                                             
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Liliana  Busconi,  Ph.D    
Joel  Kowit,  Ph.D  
Anupama  Seshan  ,  Ph.D  
 
 
                                                                                                                                                                                     Spring  2016  

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Lab  activities  
 
Lab  1:  Introduction  to  Scientific  Method:  Fast  Plants  
 
Lab  2:  Natural  Selection/  Fast  Plant  Experiment  continue    
 
Lab  3:  Comparative  Genomics  /  Fast  Plant  Experiment  continue    
 
Lab  4:  Domains  Bacteria  /Archaea    
 
Lab  5:  Domain  Eukarya:  Protists      
 
Lab  6:  Domain  Eukarya:  Plants:  GMO  
 
Lab  7:  Domain  Eukarya:  Plants:  GMO  continue  /Fungi  Kingdom  
 
Lab  8:  Domain  Eukarya:  Animal  Kingdom  I:  Sponges,  Cnidarians,  Bilaterians  
 
Lab  9:  Domain  Eukarya:  Animal  Kingdom  III:  Insect  Life  Cycle    
 
Lab  10:  Domain  Eukarya  Animal  Kingdom  II  Comparative  Analysis  of  Myosin  
 
Students  will  visit  the  Harvard  Museum  of  Natural  History  on  their  own.  
 
 
Lab  Sections  
01     Tuesday  9:25am  -­‐  12:05pm                                  Roy  
02     Tuesday  1:40pm  -­‐  4:20pm     Belanger   belangera@emmanuel.edu  
03     Tuesday  4:30pm  -­‐  7:20pm     Heller     hellerd@emmanuel.edu  
04     Thursday  9:25am  -­‐  12:05pm     Ennis     ennist@emmanuel.edu  
05     Thursday  1:40pm  -­‐  4:20pm     Slavitskiy     slavitskiyv@emmanuel.edu  
06     Thursday  4:30Ppm  -­‐  7:10pm     Slavitskiy   slavitskiyv@emmanuel.edu    
07     Wednesday  9:00am  -­‐  11:40am     Busconi     busconil@emmanuel.edu  
08   Wednesday  12:00pm  -­‐  2:40pm     Slavitskiy     slavitskiyv@emmanuel.edu  
10                    Friday  12:00pm  -­‐  2:40pm     Ennis     ennist@emmanuel.edu  
   

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Labs  on:   Topic:   Due  

  Students  will  visit  the  Harvard  Museum  Natural   If  you  do  not  include  A  PICTURE  OF  
History  on  their  own.  W3  Due  week  2/16-­‐2/19   YOURSELF  in  front  a  specific  part  of  the  
You  will  find  a  copy  of  W3  on  ECLearn   exhibition  indicated  in  the  worksheet  and  
  if  the  answers  ARE  NOT  HANDWRITTEN,  
the  worksheet  will  not  be  graded  and  will  
count  as  zero.    
 
1/26-­‐1/29   Lab  1  Introduction  to  Scientific  Method:  Fast  Plants      
 
2/2-­‐2/5   Lab  2    Fast  Plants  data  collection     BRING  YOUR  COMPUTER  TO  LAB  CLASS  
                     Natural  Selection  –  A  simulation    
 
2/9-­‐2/12   Lab  3    Fast  Plants  data  collection     o W1:  Natural  Selection    
                     Comparative  Genomics   BRING  YOUR  COMPUTER  TO  LAB  CLASS  
 
2/16-­‐2/19   Lab  4    Fast  Plants  data  collection   o W2:  Comparative  Genomics  
                     Domains  Bacteria/Archaea     o W3:  Harvard  HMNH  questionnaire  
 
2/23-­‐2/26   Lab  5    Domain  Eukarya:  Protists   o W4:  Bacteria/Archaea    
o Lab  report  #1  Effect  of  Fertilizer  on  
Fast  Plants  Growth.  
3/1-­‐3/4   Lab  6    Domain  Eukarya:  Plant  (GMO)   o W5:  Protists  
                       
 
3/8-­‐3/6   No  labs  -­‐  Spring  Break      
 
3/15-­‐3/18   Lab  7  Domain  Eukarya:  Complete  GMO  Lab   o Lab  report  #2  Phagocytosis  and  
                   Domain  Eukarya:  Fungi   Vacuole  Formation  in  Tetrahymena  
  o W6a:  GMO  
3/22-­‐3/25   No  labs  –  Easter    
   
3/29-­‐4/1   Lab  8  Domain  Eukarya:  Animal  Kingdom  I:     o W6b:GMO  
Sponges,  Cnidarians,  and  Bilaterians   o W7  Fungi    
Lab  10  Domain  Eukarya:  Animal  Kingdom  III:  Insect  
Life  Cycle    
 
4/5-­‐4/8   Lab  10a  Domain  Eukarya:  Animal  Kingdom  II   o W8:    Sponges,  Cnidarians,  and  
Comparative  Analysis  of  Myosin:  Western  blot   Bilaterians  
Sample  Preparation   BRING  YOUR  COMPUTER  TO  LAB  CLASS    
Lab  8  Continue:  Initial  Planaria  observations  
 
4/12-­‐4/15   Lab  10b  Comparative  Analysis  of  Myosin:  SDS-­‐   o Lab10a:  Quiz  on  SDS-­‐PAGE  and  
PAGE/Transfer     nitrocellulose  transfer    
Lab  8  continue:    Final  Planaria  observations    
   

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4/19-­‐4/22   Lab  10c  Comparative  Analysis  of  Myosin:  Developing   o Lab  report  #3:  Planaria  experiment  
a  Western  blot  and  analysis  of  results   o W10a  Myosin  Comparative  Genomics  
o Lab10b:  Quiz  on  antibody  detection  
method  
 
4/26-­‐4/29   No  labs     o W9:  Insect  Life  Cycle    
o W10b:Myosin  Western  Blot  
Experiment  
 
Laboratory  Meeting  Schedule:    All  labs  will  be  held  in  the  Wilkens  Science  Center  Room  208.  If  any  
changes  are  made  to  the  schedule  below,  they  will  be  announced  in  class  and  posted  on  ECLearn.  
   

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Lab  1:    Design  of  an  Experiment  and  Data  Analysis.  

Effect  of  Fertilizers  on  Fast  Plants  Growth.  
 
For  the  next  several  weeks,  your  lab  group  will  perform  an  experiment  that  will  examine  the  
growth  of  Wisconsin  Fast  Plants  TM  treated  with  different  amounts  of  fertilizer.    
 
Background  
Wisconsin  Fast  Plants   TM  are  a  variety  of  wild  mustard  (Brassica  rapa)  chosen  because  they  
complete  their  life  cycle  quickly  and  are  ideally  suited  for  laboratory  experiments.  Brassica  is  
a  member  of  the  crucifer  family,  which  includes  broccoli,  cabbage,  cauliflower,  turnips  and  
other  foods  you  can  find  at  the  market.    
 
They   are   called   Fast   Plants   because   they   go   through   a   complete   life   cycle   in   just   over   40  
days.  At  48-­‐72  hours  a  little  root,  hypocotyl  (primitive  stem)  and  cotyledons  are  visible.  True  
leaves  start  showing  after  a  week  or  so.  By  the  end  of  the  second  week,  the  flowers  are  fully  
opened  and  ready  to  pollinate.  For  optimal  growth  in  the  laboratory  they  require  continuous  
water,  fertilizer,  and  lighting.  Their  optimal  temperature  is  18-­‐26  °C.  For  more  information  
about  fast  plants  visit  the  following  website:  http://www.fastplants.org/  
 

 
 
 
 
 
 
 
 
 
 

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Lab  activities  
Today,  you  will  set  up  an  experiment  that  investigates  the  effect  of  varying  amounts  of  
fertilizer  on  plant  growth.  Before  setting  up  today’s  experiment  there  will  be  a  class  
discussion  about  how  to  best  design  the  experiment.    
 
• Develop  a  hypothesis  and  write  it  down  in  your  notebook.  
• Specify   the   variables   of   the   experiment   (dependent   and   independent   variables,  
constant  parameters)  and  write  it  down  in  your  notebook.  
o Independent  variable  
o Dependent  variable  
o Constant  parameters  
• Define  the  control  and  experimental  groups.  
• Define  the  number  of  repetitions.  
• Make  predictions.  
This   experimental   design   provides   a   range   of   fertilizer   from   none   to   three   times  
the  normal  level.  What  do  you  predict  will  the  result  be?  
• Discuss  how  data  will  be  collected  and  analyzed.  
 
Procedure:  
The   basic   unit   for   growth   of   Fast   Plants   is   the   quad,   a   cube   of   Styrofoam   with   four   openings  
(cells)   one   for   each   plant.   You   will   add   soil,   fertilizer   and   seeds   to   each   of   the   cells   and   then  
place  the  quads  on  a  reservoir  of  water  designed  to  irrigate  the  plants  on  a  continuous  basis.  
The   reservoirs   will   be   placed   under   continuous   illumination   by   fluorescent   lighting.   On   a  
weekly   basis   you   will   be   responsible   for   measuring   the   plants   and   keeping   track   of   their  
growth.   The   standard   procedure   calls   for   2-­‐3   fertilizer   pellets   per   plant.     For   this   experiment  
you  will  set  up  a  quad  with  three  different  conditions.  
 
Setting  up  your  experiment  
Each  pair  of  students  will  prepare  one  quad  as  follows:    
• Obtain  a  quad,  four  wicks,  seeds,  soil  and  fertilizer  pellets.  
• Drop  one  wick  into  each  cell  so  that  the  tip  extends  halfway  out  of  the  hole  in  the  
bottom  (about  2  cm).    
• Moisten  soil  slightly.  Fill  each  cell  halfway  with  soil.  
• Add  fertilizer  pellets  to  each  quad  cell  (0,  3,  6,  9  pellets  as  described  above)  
• Fill  each  cell  to  the  top  with  moistened  soil.    
• Make  shallow  depressions  on  top  of  each  cell.  Do  not  press  hard  and  compact  soil.  
• Drop  2-­‐3  seeds  in  each  depression.    
• Sprinkle  enough  potting  mix  to  cover  seeds  in  each  cell.  
• Water   very   gently   with   a   wash   bottle   until   water   drips   from   each   wick.   Be   careful  
NOT  TO  WASH  seeds  OUT  of  cells.  
• Label   each   quad   with   today’s   date,   lab   section,   your   initials,   and   the   experimental  
conditions.  
• Go   to   the   illuminated   growth   chamber   and   place   your   quad   on   the   reservoir.   Be  
sure  that  the  mat  dips  into  the  reservoir  of  water  and  is  wet.    Also  be  sure  that  the  
wicks  protruding  from  the  bottom  of  the  quads  are  in  contact  with  the  wet  mat.  The  
success  of  the  quad  system  rests  to  a  large  extent  on  the  continuous  flow  of  water  
from  the  reservoir,  through  the  mat,  to  the  wick  and  into  the  soil  in  the  cell.  

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• Each  cell  needs  to  be  watered  from  the  top  with  a  wash  bottle  for  the  first  3  days  (It  
will  be  TAs  will  responsibility).  
• Reservoirs  should  remained  full  of  water.    
 
Data  Collection  
Week  one    
One  week  from  the  date  of  planting  
• Remove  extra  plants  but  CUTTING  them  with  scissors.  
• Leave  only  ONE  plant/cell.  
• Measure  plants  height  in  cm.  
• Write  down  this  value  and  any  observation  in  your  notebook  (yellow  pages  will  be  
added  to  your  Lab  Report).  
• UPLOAD  your  data  on  ECLearn:  
o Go  to  Home  page    
o In  class  schedule  go  to  Lab  1  Introduction  to  Scientific  Method:  Fast  Plants  
o On  the  right  column  
o CLICK  on  DATA  Section  and  upload  the  height  in  cm  and  any  observation  that  
you  had  made.  
 
Week  two  
• Measure   the   height   of   the   plants   in   each   cell   and   record   the   results   in   your   lab  
notebook.  
• Take  notes  describing  the  appearance  of  the  plants.  
• UPLOAD  your  data  on  ECLearn.  
 
Week  three  
• Measure  the  height  of  the  plants.  
• Take  notes  describing  the  appearance  of  the  plants.  
• UPLOAD  your  data  on  ECLearn.  
 
• Harvest  plants  and  wash  the  roots  carefully  to  remove  dirt.  
• Collect  the  plants  from  the  entire  class  and  divide  them  in  four  groups:  no  fertilizer,  3  
pellets,  6  pellets,  9  pellets.  
• Weigh  each  group  of  plants.  Do  not  forget  to  count  the  number  of  plants  in  each  group.  
Then,  determine  the  mass  per  plant  for  each  group.  
 
Experimental  data  
Record  experimental  values  in  your  notebook.    
Complete  Table  1  with  the  experimental  data  from  YOUR  GROUP.  
 
Table  1:  Effect  of  Fertilizer  on  Fast  Plants  height  (cm).  ADD  a  legend  describing  briefly  how  
the  experiment  was  set  up.  
Number  of  pellets   WEEK  1   WEEK  2   WEEK  3  
0        
3        
6        
9        

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Complete  Tables  2  and  3  using  CLASS  experimental  DATA.    
• The  data  of  the  other  groups  will  be  posted  on  EClearn.    
• Students   that   do   not   post   their   data   on   time   on   ECLearn   will   have   points   reduced   in  
their  final  Lab  Report  grade.  
• Calculate  the  average  value  of  all  experimental  points  and  the  corresponding  standard  
deviation  (STDEV)  values  using  Excel.    
 
Table   2:   Effect   of   Fertilizer   on   Fast   Plants   Height   (cm).   Add   a   legend   indicating   that   this  
table   contains   data   from   the   entire   class.   DO   NOT   include   average   an   STDEV   values   with  
more  than  two  decimals.  
                                                                                                                   WEEK1                    WEEK2                                                      WEEK3  
Number  of  pellets   Average   STDEV   Average   STDEV   Average   STDEV  
Height     Height   Height  
0              
3              
6              
9              
 
 
Table   3:   Effect   of   Fertilizer   on   Fast   Plants   Weight   after   3   Weeks   (g).   ADD   a   legend  
describing  briefly  how  samples  were  collected.  Total  Mass  and  Average  Mass  Weight/Plant  
should  have  only  two  decimals.  
 
Number  of  pellets    Total  Mass  (grams)   Number  of  Plants      Average  Mass  Weight/Plant  
0        
3        
6        
9        
 
 
Lab  Report:  Effect  of  Fertilizers  on  Fast  Plants  Growth.    
 
• Include  a  cover  sheet  and  notebook  yellow  pages  with  the  Lab  report  
• Write  the  Lab  report  as  a  scientific  paper.  
Parts  of  a  Lab  Report  
• Introduction  
• Materials  and  Methods  
• Results  
• Discussion  and  Conclusions  
• References  
The  Lab  Report  should  include  all  these  sections  with  their  corresponding  subtitles.  
 
 
 
 

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Guidelines  
Introduction  (Total:  25pts)  
(Total  1.5-­‐2  pages  double-­‐spaced)  
1. Context    
Introduce  the  reader  to  the  subject  matter  
Some  suggestions:  What  are  fertilizers?  Different  types  of  fertilizers.  What  are  the  
advantages/disadvantages  of  using  fertilizers?  Include  introductory  information  about  
Wisconsin  Fast  Plants.  What  are  the  advantages  of  using  Fast  Plants  as  your  experimental  
system?    
2.      Outline  your  experiments  in  your  last  paragraph:  be  brief!!    
1)  Describe  what  you  were  interested  in  determining      
2)  Describe  how  the  experiment  was  done  and  what  the  results  were  (briefly!)  
3)  Describe  your  hypothesis/predictions    
 
Materials  and  Methods  (Total:  10pts)  
Do  not  copy  the  handout  instructions.  Write  a  paragraph  (in  past  tense)  describing  how  the  
experiment  was  done,  including  in  your  description  all  the  materials  used  (do  not  list  them  
separately).  Describe  what  are  the  variables  and  the  control  of  the  experiment.  
 
Results  (Total:  40pts)        
This   section   should   be   written   in   paragraph   format   and   include   3   Tables   and   2   Figures   (Each  
figure/table  should  be  numbered,  have  a  title,  and  a  legend).    
 
o Start   with   a   brief   description   of   the   purpose   of   the   experiments   (rationale,  why   the  
experiments  were  done)  and  how  they  were  done.    
o Briefly  describe  the  experiment  without  repeating  experimental  details  already  
described  in  Materials  and  Methods  section.  
o Finish  with  ONE  sentence  with  the  conclusion  of  the  experiment.  Then,  in  the  
Discussion  section  the  conclusion  will  be  expanded  in  a  more  general  context.  
o Use  data  in  Tables  2  and  3  to  do  the  analysis  of  the  experiment.    
Explain  why  is  better  to  use  the  data  of  the  whole  class  instead  of  your  group  data.  
 
Make  two  figures  using  data  from  Tables  2  and  3.    
• Do  not  include  the  title  inside  of  the  figure.  Title  should  be  informative    
• Figure  legend  is  not  the  same  as  the  “legend”  Excel  asks  for,  which  is  really  a  title.  
The  legend  of  the  figure  is  a  brief  description  of  how  the  experiment  was  done.  It  is  
NOT  the  conclusion  of  the  experiment.  
• Figure/Table  number,  title  and  legend  should  go  above  or  below  the  figure  or  table.  
 
Example  

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Figure  1:  Effect  of  Different  Amounts  of  Fertilizer  on  Plant  Height.  Plants  were  grown  in  the  
absence  or  presence  of  different  amounts  of  fertilizer.    Plants  were  kept  at  room  
temperature  and  under  constant  illumination  for  three  consecutive  weeks.    
 
Figure  1:  Make  a  bar  graph  using  the  data  from  Table  2.  Include  a  title  and  legend.      
Y  axis:  Fast  Plants  Height  (Average  values  from  Table  2)  
X  axis:  Time  (in  weeks)  
• Label  both  axes  and  add  the  units  
• In  the  same  graph  show  the  results  of  the  three  weeks  that  the  experiment  lasted.  
The   data   of   each   week   (average   values)   should   include   the   experimental   data  
obtained  with  the  different  amounts  of  fertilizer  used    
• Add  error  bars  to  each  column  using  the  calculated  STDEV.  Use  the  option  Custom  
to  incorporate  the  calculated  STDEV.  
   
Figure  2:  Make  a  bar  graph  using  the  data  from  Table  3.  Include  a  title  and  legend.      
Y  axis:  Fast  Plants  Weight    (Average  values  from  Table  3)  
X  axis:  Number  of  pellets  
 
Conclusions  and  Discussion  (Total:  20pts)      
o DO  NOT  repeat  the  information  presented  in  the  Results  section.  Analyze  your  data.    
o Start  with  one  short  paragraph  explaining  what  was  the  main  goal  of  the  project  (study  
the  effect  of  fertilizers  in  plant  growth),  what  you  did  (measure  changes  in  height  and  
weight),  and  what  the  results  indicate  
o Do  not  explain  the  experiments  again  (this  was  done  in  results  section)    
What  was  the  effect  of  the  fertilizer  on  plants  growth?  (Describe  any  differences  in  the  
appearance  of  the  plants  during  weeks  1,  2  and  3).  Does  the  appearance  of  the  plants  
correlate   with   the   amount   of   fertilizer?     Can   the   same   conclusions   be   obtained   using  
height   or   weight   data?   Why   or   why   not?   Compare   your   results   with   your   predictions.  
Was   your   hypothesis   supported   or   rejected?   If   applicable   discuss   any   possible   sources   of  
error  in  your  experiment.  Be  specific  about  the  source  of  error.  DO  NOT  SAY  because  of  
“human  error”.  
How  do  fertilizes  affect  the  environment?  Are  there  any  other  alternatives  to  the  use  of  
fertilizers?      
o End   describing   what   would   your   next   experiment   be   to   better   understand   the  
experimental  system?  What  other  question  would  you  like  to  address  using  Fast  Plants  
as  your  experimental  system?    

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References  (Total:  5pts/  1  point  reduction  if  not  incorporated  in  the  text)      
• Include  at  least  three  references  (make  sure  to  use  at  least  one  book  and  one  primary  
article).  
• List   books,   journals   and   websites   from   which   you   have   gathered   information   for   this  
project.    
• List  the  references  at  the  end  of  the  paper  but  also  make  sure  to  incorporate  them  in  
the  text.    
 
List  all  references  used  in  writing  the  lab  report  using  MLA/APA  or  Chicago  format.  
 
o A   primary   research   article   reports   on   an   empirical   research   study   conducted   by   the  
authors.  It  is  almost  always  published  in  a  peer-­‐reviewed  journal.  
The  reference  of  a  primary  article  should  include:  
• Authors  listed  by  last  name  followed  by  initial,  separated  by  commas  
• Latin  names  of  organisms  in  italics  
• Title  of  the  article  
• Title  of  the  journal  (periodical)  
• Year  of  publication  
• Volume  of  journal  (and  issue  in  parenthesis  if  available)  
• Page  numbers  
See  example  below:  
Evans  RN,  Blaha  G,  Bailey  S,  Steitz  TA.  The  structure  of  LepA,  the  ribosomal  back  translocase.  
Proceedings  of  the  National  Academy  of  Sciences  of  the  United  States  of  America.  
2008;105(12):4673-­‐4678.  
 
If  you  get  the  scientific  paper  reference  from  the  web  DO  NOT  included  doi.    
Evans  RN,  Blaha  G,  Bailey  S,  Steitz  TA.  The  structure  of  LepA,  the  ribosomal  back  translocase.  
Proceedings  of  the  National  Academy  of  Sciences  of  the  United  States  of  America.  
2008;105(12):4673-­‐4678.  doi:10.1073/pnas.0801308105.  

 
o Websites  
Do  not  use  Wikipedia  as  a  source  of  information    
You  can  go  to  the  following  websites  to  look  for  books  or  scientific  papers:    
PubMed  http://www.ncbi.nlm.nih.gov/pubmed/  
Google  scholar  
 
If  you  use  a  website  include  the  URL  and  the  author  name  in  case  that  the  web  page  has  an  
author.  Use  credible  websites,  for  example  those  having  the  following  extensions:  edu,  gov,  
etc.  
http://www.fastplants.org/life_cycle/  
 
http://www.nal.usda.gov/fnic/foodcomp/search/  
 
DO  NOT  INCLUDE  the  day  that  you  download  the  reference.  
 

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Lab  Report  #1:  Effect  of  Fertilizers  on  Fast  Plants  Growth  
 
 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  
 
 
YELLOW  PAGES  INCLUDED  __________  
 
   

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Lab  2:  Natural  Selection  

The  Chips  are  Down:  Natural  Selection  Simulation  
 
Before  coming  to  lab  
• Write  in  your  notebook  Goal/purpose  of  the  lab  activity.    
• Flow  Chart/  Protocol(s)    
 
BRING  YOUR  COMPUTER  TO  THE  LAB.  As  part  of  the  lab  activities  you  will  make  the  
graphs  in  class.  
 
Background:  
The  process  of  natural  selection  occurs  because  organisms  vary  in  their  heritable  
characteristics.  The  genome  is  affected  by  mutations  creating  genetic  diversity  but  in  
organisms  that  reproduce  sexually,  this  is  an  important  source  of  genetic  diversity.  Because  
of  the  genetic  diversity,  some  individuals  have  traits  that  will  allow  them  to  survive  and  
reproduce  better  than  others.  As  a  result,  the  genetic  structure  of  a  population  changes  
through  time,  which  is  a  factor  in  evolution.  Although  evolution  may  be  defined  in  terms  of  
genetic  change,  natural  selection  occurs  by  the  interaction  of  the  environment  and  whole  
organisms.  Over  time,  natural  selection  can  increase  the  match  between  organisms  and  
their  environment.    
 
In  this  exercise,  we  want  to  reinforce  the  concept  of  natural  selection  with  a  demonstration  
of  how  natural  selection  works.  It  is  far  too  time-­‐consuming  to  observe  natural  selection  at  
work  in  natural  populations,  so  we  will  use  an  artificial  population  consisting  of  paper  chips  
living  in  two  different  environments.  This  population  will  be  followed  over  three  generations  
after  three  cycles  of  predation.  
 
Before  starting  with  today  activities,  collect  data  for  the  Fast  Plants  experiment  (Week  1).  
 
Week  one    
• Remove  excess  plants  so  it  remains  only  one  healthy  plant  per  cell    
• Measure   the   height   of   the   plants   in   each   cell   and   record   the   results   in   your   lab  
notebook.  
• Take  notes  describing  the  appearance  of  the  plants.  
• Upload  your  data  on  ECLearn.  

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Natural  Selection.  Procedure  

There  are  two  backgrounds  on  the  tabletop  representing  two  different  environments.  
 
1.  Start  using  one  of  the  two  background  papers  as  the  habit  for  the  chips.  
2.  Assume  that  you  have  one  population  of  100  individuals,  and  that  the  population  is  
composed  of  25  chips  of  each  color.  
3.  One  person  spread  the  chips  out  randomly  over  the  entire  fabric,  making  sure  the  chips    
do  not  stick  together.  
4.  Different  members  of  the  group  (Predators)  take  turns  picking  off  the  prey  (chips)  one  by  
one  until  only  25  individuals  remain.    
5.  Carefully  shake  off  the  fabric  to  remove  survivors  (25  chips).  
6.  Group  the  survivors  according  to  color.  Count  and  record  these  numbers  in  your  
notebook.  
7.  Assume  that  each  survivor  produces  three  offspring.  Using  the  reserved  chips,  place  
three  chips  of  the  same  color  with  the  survivors  (i.e.,  take  the  number  of  survivors  
multiplied  by  4).  
8.  Before  starting  the  next  round  of  predation,  make  sure  that  the  population  has  100  
individuals    
9.  Mix  these  chips  together  and  re-­‐distribute  them  as  in  step  3.  
10.  Repeat  the  entire  process  two  more  times,  making  a  total  of  three  generations  of  prey  
being  preyed  upon.  
 
10.  Repeat  the  activity  using  the  second  background.  
   

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Worksheet  1:  The  Chips  are  Down:  a  Natural  Selection  Simulation  

 
 
 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  
 
 
 

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1a.  Complete  the  tables  below  using  your  experimental  data  (10pts).    
 
         1b.  Add  a  title  (Title  5pts)  and  write  a  legend  (5  pts)  for  Table  1.  The  legend  is  a  brief  description  
of  how  the  experiment  was  done.    
 
 
 
 
 
 
Environment  1  

   
 RED    GREEN    YELLOW    BROWN  
Number  at  start-­‐-­‐>  
               

#  after  1st  predation-­‐-­‐>  

               

#  after  1st  reproduction-­‐-­‐>  

               

#  after  2nd  predation-­‐-­‐>  

               

#  after  2nd  reproduction-­‐-­‐>  

               

#  after  3rd  predation-­‐-­‐>  

               

 
Environment  2  
   
 RED    GREEN    YELLOW    BROWN  
Number  at  start-­‐-­‐>  
               

#  after  1st  predation-­‐-­‐>  

               

#  after  1st  reproduction-­‐-­‐>  

               

#  after  2nd  predation-­‐-­‐>  

               

#  after  2nd  reproduction-­‐-­‐>  

               

#  after  3rd  predation-­‐-­‐>  

               

 
 
 

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2-­‐  (Total  45  pts)  Make  two  bar  graphs  that  illustrate  the  number  of  individuals  (color)  remaining  
after  each  round  of  predation  and  reproduction.    
Include  both  graphs  on  the  same  page  one  on  top  of  the  other  (part  a  and  b),  so  you  can  
compare  the  results  in  both  environments.  
Use  the  same  scale  in  both  graphs.  
Write  a  title  (5pts)  and  a  legend  (5pts)  that  include  both  graphs.  Label  both  axes.  (5pts)  
 
3-­‐  Conclusions  (20pts)  
Write  a  paragraph  with  your  conclusions.  Use  the  following  questions  as  a  guide  to  draw  
conclusions  from  the  natural  selection  simulation  (Do  not  answer  the  questions  individually).      
 
Describe  the  two  environments  and  how  differences  in  the  environment  affected  the  final  
composition  of  the  population.  
Compare  the  original  and  survivor  populations.  Is  there  any  color  from  the  original  population  
that  is  missing?  
How  do  the  colors  of  the  survivors  are  related  to  their  habitat?  Compare  the  results  obtained  
using  the  two  different  backgrounds.    
Extrapolate  the  results  of  this  activity  by  giving  an  example  in  nature.    
Is  natural  selection  working  mainly  in  the  phenotype  or  the  genotype?  Are  the  individuals  or  the  
population  evolving?  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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4-­‐  Camouflage  is  an  example  of  evolutionary  adaptation.  The  pictures  below  represent  two  
different  species  of  insects  called  mantids  (15pts)  
 

                                                 
                   Praying  mantid              African  flower  mantid  
 
 
Explain  how  those  insects  demonstrate  the  three  key  observations  about  life:    
• The  match  between  organisms  and  their  environments  
• Unity  
• Diversity  
 
You  might  need  to  do  some  research  about  features  shared  by  different  mantids  species.  
Include  your  references.    
   

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Lab  3:  Comparative  Genomics  

 
The  goal  of  this  lab  is  to  use  molecular  data  analysis  to  study  the  level  of  genetic  subdivision  
in  the  species  Trimerotropis  saxatilis  (lichen  grasshopper)  
 
BRING  YOUR  COMPUTER  TO  THE  LAB.  
On  ECLearn  you  will  find  a  word  file  Comparative  Genomics  Exercises  that  you  will  use  in  class.  
Please  download  this  file  to  your  computer  before  coming  to  class.  
 
 Background    
               The  lichen  grasshopper  Trimerotropis  
saxatilis  lives  in  dry  rocky  areas,  known  as  
glades,  ranging  as  far  west  as  Oklahoma  and  
east  into  Georgia.  This  medium  sized  
grasshopper  occurs  exclusively  on  acidic  
glades  where  its  cryptic  coloration  makes  it  
nearly  invisible  against  the  lichen  covered  
granite,  rhyolite  or  sandstone  dominating  
 
these  habitats.    
 
  In  the  Ozarks  of  Missouri  the  glades  are  habitat  islands  separated  by  patches  of  forest.  
Historically  the  glades  were  maintained  by  the  frequent  occurrence  (around  every  four  years)  of  
forest  fires.  Recently,  human  suppression  of  forest  fires  has  resulted  in  the  loss,  reduction,  and  
fragmentation  of  glade  habitats.  
 
  The  fragmentation  of  the  lichen  grasshoppers’  habitat  could  have  a  substantial  impact  on  
the  way  genetic  variation  is  distributed  throughout  the  population.  As  the  habitat  becomes  
patchier,  gene  flow  may  be  reduced,  causing  genetic  subdivision  of  the  population.  Although  
this  grasshopper  can  fly,  it  is  doubtful  that  the  grasshopper  disperses  across  large  tracts  of  forest  
to  get  from  one  rocky  area  to  another.  Recent  work  to  preserve  glade  habitats  has  prompted  
controlled  burns  that  enlarge  and  preserve  the  glades  and  may  provide  temporary  corridors  for  
lichen  grasshoppers  to  disperse.  
 
In  this  lab,  you  will  study  the  level  of  genetic  subdivision  (directly  related  to  the  level  of  
gene  flow)  in  this  species  by  comparing  a  300  base-­‐pair  segment  of  its  mitochondrial  DNA  
(mtDNA).  Although  most  bases  in  this  region  are  invariable,  there  are  a  few  bases  that  show  
variation  in  preliminary  studies.  You  will  use  an  on-­‐line  computational  program  to  identify  the  
three  alleles  in  this  population.  The  effects  of  gene  flow  vs.  genetic  drift  will  be  examined  using  a  
statistic,  called  FST.  You  will  compare  this  statistic  between  glades  occurring  in  burned  and  
unburned  areas  to  examine  whether  controlled  burns  have  an  impact  on  population  subdivision.  
Finally,  you  will  use  a  BLAST  search  (Basic  Local  Alignment  Search  Tool)  to  identify  organisms  
that  have  mtDNA  and  genomic  DNA  sequences  that  are  similar  to  the  grasshopper  T.  saxatilis.  
 
 
 
 
 
 

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BLAST  (Basic  Local  Alignment  Search  Tool)    

 
BLAST  was  developed  in  1989  by  the  National  Center  for  Biotechnology  Information  (NCBI)  at  
the  National  Institutes  of  Health  (NIH).    It  is  a  free  online  service  available  at  
http://blast.ncbi.nlm.nih.gov.  BLAST  exchange  data  with  the  European  Molecular  Biology  
Laboratory  Nucleotide  Sequence  Database  and  the  DNA  Data  Bank  of  Japan  to  ensure  global  
coverage  and  consensus  across  the  major  databases.  
 
BLAST  is  an  algorithm  to  compare  the  nucleotides  of  DNA  sequences  or  the  amino-­‐acid  
sequences  of  different  proteins  to  DNA  or  protein  databases  respectively.      
The  sequence  used  in  the  search  is  called  the    “query  sequence”  and  is  compared  to  a  database  
of  sequences  (“subject  sequences”).  The  program  then  return  those  sequences  that  have  a  
significant  level  of  similarity  to  the  query  sequence.    
 
BLAST  can  be  used  to  identify  an  unknown  sequence,  build  a  homology  tree  for  a  protein,  get  
clues  about  protein  structure  by  finding  similar  proteins  with  known  structures,  and  map  a  
sequence  in  a  genome  among  other  uses.  
 
NCBI  has  created  the  "BLAST  Program  Selection  Guide”,  a  site  that  contains  information  that  
allows  answer  the  question  “Which  BLAST  program  should  I  use?”  
Seelink:http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=
ProgSelectionGuide  
 
There  are  two  commonly  used  BLAST  algorithms:  
Nucleotide  blast  (blastn)  
• Search  a  nucleotide  database  using  a  nucleotide  query  (the  input).  The  program  will  
return  the  most  similar  DNA  sequences  from  the  DNA  database  that  the  user  specifies.  
Algorithms:  blastn,  megablast,  discontiguous  megablast    
• MEGABLAST  is  specifically  designed  to  efficiently  find  long  alignments  between  very  
similar  sequences  and  thus  is  the  best  tool  to  use  to  find  the  identical  match  to  your  
query  sequence.  
 
• BLAST2  Sequences  
• Is  an  algorithm  used  to  align  two  sequences  
• helpful  for  observing  differences  between  two  sequences  
 
Protein  blast  (blastp)  
• Search  a  protein  database  using  amino-­‐acid  or  protein  sequences  as  query.  The  
program  will  return  the  most  similar  protein  sequences  from  the  protein  database  that  
the  user  specifies  
 
 
 
 
 
 
 
 
 
 
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BIOL1106    

When  trying  to  compare  two  or  more  sequences  you  will  have  access  to  the  following  screen:  
 

 
 
The  BLAST  algorithm  calculates  similarity  scores  for  local  alignments  (i.e.,  the  most  similar  
regions  between  2  sequences)  between  the  query  sequence  and  subject  sequences  and  returns  
a  table  of  the  best  matches  (“hits”)  from  the  database.    
 
The  hit  table  includes  several  useful  pieces  of  information,  including  the  similarity  score,  query  
coverage,  E-­‐value,  and  max  identity.  These  measures  treat  each  aligned  segment  
independently.  

Analyzing  the  results  

• E  Value:  describes  the  chance  of  randomly  achieving  the  same  alignment  in  a  database  
of  a  particular  size.  An  E  Value  is  used  to  describe  the  significance  (instead  of  a  P  value)  
of  each  sequence  alignment  hit  to  the  query.    
• The  lower  the  E  value  is,  the  more  significant  the  alignment  is.  
 

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• The  expect  value  is  the  default  sorting  metric  and  normally  gives  the  same  sorting  order  

•
as  Max  Score.  Typically,  E  <  .05  is  required  to  be  considered  significant.  
Max  score:  Score  of  the  single  best  aligned  sequence  from  the  same  database  sequence.
The  higher  the  Max  Score,  the  better  the  alignment  between  the  hit  and  the  query  
 
 
• Query  coverage:  The  amount  of  the  query  sequence,  expressed  as  a  percent,  that  
overlaps  the  subject  sequence  
• Maximum  identity:  Percent  similarity  between  the  query  and  subject  sequences  over  
the  length  of  the  coverage  area.  
 
• Score:  Normalized  score  of  alignment.  Can  be  compared  across  searches.  The  higher  the  
alignment  score,  the  better  the  alignment  
• Total  score:  Sum  of  scores  of  all  aligned  sequences  
 
Retrieved  information  

 
 
Before  starting  today  activities,  collect  data  for  Fast  Plant  Experiment:  
Week  two  
• Measure   the   height   of   the   plants   in   each   cell   and   record   the   results   in   your   lab  
notebook.  
• Take  notes  describing  the  appearance  of  the  plants.  
• UPLOAD  your  data  on  ECLearn.  
 
 
   
 
 
 
 
 
 
 
 
 
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BIOL1106    

Lab  3:  Comparative  Genomics  Exercises  

PART  I:  SEQUENCE  ALIGNMENTS  
 
Allele  1    
 
GTTCGAGTTC  TAACTAGAGC  ATCAGATGTT  TTACATTCAT  GAGCCGTACC  
CGCTCTAGGA  ATTAAAATAG  ATGCAACTTC  TGGACGTTTA  AATCAAGGAA  
CATTTTTAAT  TAAACGTCCA  GGATTATTTT  TTGGACAATG  TTCAGAAATT  
TGTGGAGCAA  TTCATAGATT  TATACCAATT  GTAATTGAAA  GAACATCAGT  
AAAATTATTT  ATTAAATGAT  TATCAAGTAT  AATATAAGGA  GTTAGTTAAA  
ATATAACATT  AGAATGTCAA  TCTAAAATAA  CTAAATATTA  GTACACCTTG  
 
Allele  2    
 
GTTCGAGTTC  TAACTAGAGC  ATCAGATGTT  TTACATTCAT  GAGCCGTACC  
CGCTCTAGGA  ATTAAAATGG  ATGCAACTTC  TGGACGTTTA  AATCAAGGAA  
CATTTTTAAT  TAAACGTCCA  GGATTATTTT  TTGGACAATG  TTCAGAAATT  
TGTGGAGCAA  TTCATAGATT  TATACCAATT  GTAATTGAAA  GAACATCAGT  
AAAATTATTT  ATTAAATGAT  TATCAAGTAT  AATATAAGGA  GTTAGTTAAA  
ATATAACATT  AGAATGTCAA  TCTAAAATAA  CTAAATATTA  GTACACCTTG  
 
Allele  3    
 
GTTCGAGTTC  TAACTAGAGC  ATCAGATGTT  TTACATTCAT  GAGCCGTACC  
CGCTCTAGGA  ATTAAAATGG  ATGCAACTTC  TGGACGTTTA  AATCAAGGAA  
CATTTTTAAT  TAATCGTCCA  GGATTATTTT  TTGGACAATG  TTCAGAAATT  
TGTGGAGCAA  TTCATAGATT  TATACCAATT  GTAATTGAAA  GAACATCAGT  
AAAATTATTT  ATTAAATGAT  TATCAAGTAT  AATATAAGGA  GTTAGTTAAA  
ATATAACATT  AGAATGTCAA  TCTAAAATAA  CTAAATATTA  GTACACCTTG  
 
Use  the  NCBI  Nucleotide  Blast  tool  in  order  to  align  the  sequences  and  identify  the  3  alleles  in  
these  grasshopper  populations.  Here  are  instructions  on  how  to  do  this:  
 
1)  Paste  this  web  address  into  your  browser:  
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PR
OG_DEF=megaBlast&SHOW_DEFAULTS=on&BLAST_SPEC=blast2seq&LINK_LOC=align2seq  
 
2)  You  will  see  two  text  boxes:  ‘Enter  Query’  and  ‘Enter  Subject.’  You  will  compare  allele  
sequences  2  and  3  to  allele  sequence  1  by  using  sequence  1  as  your  Query.    
NOTE:  Make  sure  the  ‘Query  subrange’  next  to  this  box  does  not  have  any  numbers  in  it.  If  
there  are  numbers,  delete  them.    
3)  Paste  the  ‘Allele  1’  sequence  into  the  ‘Enter  Query’  box.  Next  paste  the  ‘Allele  2’  sequence  
into  the  ‘Enter  subject’  box.  Do  not  change  any  other  settings.  Click  the  blue  ‘BLAST’  button  at  
the  bottom.  This  will  open  a  sequence  alignment  in  your  browser.    
 
4)  All  of  the  nucleotides  that  are  identical  will  have  a  line  connecting  the  query  and  subject  
sequences.  Only  the  nucleotides  that  are  different  will  not  have  a  line  connecting  the  two  
sequences.    

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BIOL1106    

5)  Repeat  steps  1  –  4  using  the  Allele  1  sequence  as  your  Query  and  the  Allele  3  sequence  as  the  
Subject.    
Figure  1.  Copy  both  alignments  and  paste  it  into  a  Word  file  as  a  figure  to  submit  as  part  of  
your  worksheet  for  this  lab.  Make  sure  to  label  it  with  an  appropriate  title.  (15pts+  5pts  title)  
 
Question  1:  What  are  the  3  alleles?  (You  can  report  nucleotide  changes  between  sequences  as  
follows:  if  nucleotide  300  in  your  query  sequence  is  A,  while  nucleotide  300  in  your  subject  
sequence  is  T,  you  would  call  the  query  allele  A300A  and  the  subject  allele  A300T).  Be  sure  to  
include  your  alignments  as  figures.  (6pts)    
 
 
PART  II:  GENE  FLOW  VS.  GENETIC  DRIFT  
The  Fst  statistic  was  designed  by  Sewall  Wright  to  measure  the  amount  of  genetic  variation  
found  among  subpopulations  relative  to  the  total  population  (hence,  the  subscript  “st”).  Wright  
used  a  model  of  the  balance  between  gene  flow  (genetic  interchange  among  the  glade  
populations  in  this  case)  versus  genetic  drift  (due  to  the  small  populations  sizes  on  each  glade,  
between  20-­‐100  individuals  in  this  case).  If  gene  flow  were  very  common  among  the  glade  
populations,  each  glade  population  should  have  the  same  alleles  at  the  same  frequency.  
However,  if  gene  flow  were  not  very  common,  genetic  drift  should  predominate  and  allele  
frequencies  would  be  different  between  glades.    
 
The  Fst  statistic  can  be  calculated  using  the  following  equation:  
 
Fst  =  (HT  –  HS)/HT  
 
In  the  above  equation,  HT  refers  to  the  probability  that  any  two  alleles  in  the  total  population  
being  considered  (e.g.  all  glades  in  the  unburned  area)  are  different  from  each  other  if  chosen  at  
random.  The  term  HS  is  a  measure  of  the  probability  that  two  alleles  drawn  at  random  within  
one  glade  are  different  from  each  other.    
 
Here  are  two  extreme  examples  to  illustrate  the  meaning  of  the  FST  statistic:  
 
i)  If  HT  =  HS,  then  this  means  that  the  allele  frequencies  are  identical  in  all  glades  and  that  gene  
flow  dominates  over  genetic  drift.  In  this  case,  the  value  of  FST  =  0.  
 
ii)  If,  however,  each  glade  has  only  1  of  3  alleles,  and  therefore  the  allele  frequencies  are  very  
different  between  glades  (e.g.  Glade  1  has  only  allele  1,  Glade  12  has  only  allele  2,  and  Glade  17  
has  only  allele  3),  then  HS  would  be  equal  to  0,  and  FST  =  1.  In  this  case,  genetic  drift  dominates  
and  gene  flow  is  absent.    
 
In  order  to  compare  the  effects  of  gene  flow  and  genetic  drift,  one  must  compare  how  close  FST  
is  to  0  or  to  1.    
Below  are  the  values  of  HT  and  HS  for  the  glades  in  the  burned  areas  and  those  in  the  unburned  
areas.    
 
  HS   HT  
Burned  Glades  (3,  6,  9)   0.518   0.554  
Unburned  Glades  (1,  12,  17)   0.453   0.499  
 

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Question  2.  Calculate  the  FST  statistic  for  the  burned  and  unburned  areas.  Contrast  the  values  
you  obtained.  Which  FST  is  smaller  (closer  to  0)?  (10pts)  
 
Question  3.  What  does  this  imply  about  the  impact  of  forest  fires  on  the  dispersal  of  
grasshoppers  in  this  area?  Do  you  think  that  the  controlled  burns  are  necessary  to  prevent  
genetic  drift  in  these  grasshopper  glades?  (10pts)  
 
PART  III:  SEQUENCE  COMPARISIONS  
Perform  a  BLAST  search  to  see  whether  you  can  identify  other  organisms  with  similar  mtDNA  
sequences.    
 
Question  4:  What  is  the  difference  between  nucleotide  blast  and  protein  blast?  (4pts)    
 
1) You  will  do  a  nucleotide  blast.  To  do  this,  go  to  a  BLAST  site  that  allows  you  to  query  whole  
genome  sequencing  projects:  
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&QUERY=NC_0
07420.2&DATABASE=nr&MEGABLAST=on&BLAST_PROGRAMS=megaBlast&LINK_LOC=nuccor
e&PAGE_TYPE=BlastSearch&QUERY_FROM=9960642&QUERY_TO=9962376  
 
2) You  will  see  a  box  that  says  ‘Enter  Query  Sequence.’  Copy  and  paste  the  sequence  of  allele  1  
into  this  box.    
 
3)  You  do  not  need  to  change  any  other  parameters.  (The  Database  should  be  set  to  
‘Nucleotide  collection  nr/nt’  that  will  allow  you  to  search  all  the  sequences  in  the  entire  NCBI  
database).  Click  on  the  blue  ‘BLAST’  button.    
 
4)      A  Blast  report  will  open,  which  gives  you  the  accession  numbers  for  sequences  from  other    
organisms  that  are  similar  to  the  Query  sequence.  Click  on  ‘Accession  Number’  next  to  an  
identified  sequence  to  get  more  information  on  the  organisms  you  have  identified.    Under  
‘Organism’  you  will  see  the  full  lineage  of  the  identified  organism.    
 
Question  5.  What  type  of  organism(s)  did  you  identify?  Name  the  top  5  species  that  you  
obtained  in  your  search.  Is  this  to  be  expected?  Why  or  why  not?  (10pts)    
 
The  gene  sequence  below  codes  for  an  odorant  receptor  protein  in  the  red  flour  beetle  
Tribolium  castaneum.  The  whole  genome  of  this  pest  of  stored  agriculture  products  was  
sequenced  in  2008  and  it  is  now  proving  to  be  an  important  model  organism  for  studying  both  
insect  development  and  the  identification  of  targets  for  insect  control.    
 
1  atgaaaatct  ccactttagt  cgcaatcctt  gtgctagctg  gaagtgcggt  ttgtgcggac                61  gaagataatt  tgaacaccga  
aaatgtccag  agtatagaag  aggattgtca  gaaagaaaca              121  ggggtttctg  atgaatcact  tcaagaacta  
agcgaaacgg  gggattctga  cgatcccctt              181  gtgaaaaaga  atgcactttg  catactaaaa  gcttatggag  ttattgatga  
ccagggagaa              241  atttctgaag  ataaattaga  agaaaagtta  gagcctgacc  gtggcaaaga  agaagcagaa              301  
aaggtagcca  aaagttgcgc  tgttaaaaag  gactcacctg  aagaaacagc  acatgaagcc              361  ctgttgtgta  
tgcaacagaa  atcacaaaag  tag    
 
Follow  the  steps  above  to  do  a  BLAST  using  the  red  flour  beetle  odorant  receptor  gene  
sequence.    

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[IMPORTANT:  this  time,  you  will  need  to  change  the  parameters  in  Step  3.  In  the  box  above  the  
BLAST  button  called  ‘Program  Selection,’  choose  ‘Somewhat  similar  sequences  (blastn).’  This  will  
allow  for  some  more  mismatches  between  the  sequences  that  you  will  receive  in  your  search,  
and  you  will  find  genes  that  have  some  similar  regions,  and  some  different  regions].  
 
Question  6a.What  types  of  organisms  did  you  identify  within  your  top  20  hits?  Were  they  all  
insects?  (7pts)  
 
Q6b.  Take  a  screen  shot  of  your  search.  (3pts)    
 
Question  7.  You  are  doing  the  search  with  a  sequence  that  has  405  nucleotides  (query).  
Analyze  the  information  corresponding  to  the  sequences  of  Tribolium  castaneum  similar  to  12  
kDa  hemolymph  protein  e  (hit#3)  and  the  human  chromosome  5  (hit#47)  and  determine  how  
similar  or  different  they  are  from  the  query  sequence.  
Q7a.  Complete  the  following  information  for  each  hit:  (5pts)  
 
What  is  the  score  of  the  match?    
What  is  the  query  coverage?      
How  many  nucleotides  are  identical  to  the  query?    
Identity    
Are  there  any  gaps?    
 
Q7b.  Go  to  the  sequence  corresponding  to  Homo  sapiens  chromosome  5  clone  RP11-­‐302K24,  
complete  sequence,  and  complete  the  following  information  (5pts)  
 
What  is  the  score  of  the  match?    
What  is  the  query  coverage?      
How  many  nucleotides  are  identical  to  the  query?    
Identity    
Are  there  any  gaps?    
 
Q7c.  Include  a  picture  of  the  Distribution  of  101  Blast  Hits  on  the  Query  Sequence  containing  the  first  20  hits.  
Highlight  hits  #  4  and  17.  (5pts)  
 
Question  8  Which  of  the  two  sequences  corresponding  to  hits  4  and  17  is  more  similar  to  the  
query?  Explain.  (10pts)  
 
Question  9.  Given  your  BLAST  results  and  your  answer  to  question  8,  do  you  think  it  would  be  
easy  to  develop  a  pesticide  that  would  specifically  block  the  red  flour  beetle  odorant  receptor  
from  detecting  odors  without  being  toxic  to  humans  or  pets?  Why  or  why  not?    (10pts)  
   

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Lab  5:  Domains  Bacteria  /Archaea    
 
 
 
 

     
                   
Introduction:  
 
Based   on   genetic   data,   biologists   have   adopted   a   three-­‐domain   system   to   classify   all   known  
species.   The   three   domains   are:   Bacteria,   Archaea   and   Eukarya.   This   classification   has   been  
validated  after  completing  the  sequencing  of  more  than  100  genomes  of  different  species.  The  
purpose  of  this  laboratory  is  to  acquaint  you  with  the  domains  Bacteria  and  Archaea.  Members  
of   these   two   domains,   which   are   among   the   simplest   organisms   known,   are   prokaryotic  
organisms.  Archaea  share  some  traits  with  bacteria  and  other  traits  with  eukaryotes,  but  they  
also   have   some   special   characteristics   not   present   in   any   of   the   species   in   the   other   two  
domains.    
Prokaryotic  cells  are  characterized  by  their  lack  of  a  true  nucleus  and  the  absence  of  membrane-­‐
bound   organelles.   They   have   a   single,   double   stranded   molecule   of   DNA   suspended   in   the  
cytoplasm.  Since  the  prokaryotes  have  no  organelles,  the  cell  membrane  often  serves  as  a  site  
for  metabolic  reactions  such  as  respiration,  photosynthesis,  and  DNA  replication.    
 
Read   through   the   instructions   for   the   exercise,   making   appropriate   observations,   drawings   (D-­‐1,  
D-­‐2,  etc.  Make  drawings  directly  in  the  worksheet)  and  answering  questions  where  called  for.  
Keep   any   observations   or   experimental   data   in   your   lab   notebook   because   they   will   form   the  
basis  for  your  worksheet.  Whenever  you  make  drawings,  be  sure  to  label  them  and  indicate  the  
magnification.  
 
 
Before   coming   to   the   lab,   write   in   your   notebook   the   purpose   of   the   lab.   Make   a   list   of   the  
samples  that  you  will  observe.  Write  the  protocol  for  bacteria  transformation.    
 
 
 

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BIOL1106    

PART  I    
Bacteria   and   Archaea   are   the   two   prokaryotic   domains   of   microscopic   organisms   that   are  
essential  to  the  cycling  of  carbon  and  other  elements  important  to  life.  These  organisms  lack  a  
complex  cell  structure.  However,  bacteria  are  the  result  of  almost  4  million  years  of  evolution,  
which  led  to  a  great  diversity  and  ability  to  survive  in  different  environments.  
The  compound  light  microscope  available  to  you  in  the  lab  can  only  reveal  the  basic  shape  of  the  
bacterial   cells.   There   are   3   cell   types:   the   bacilli   (rod-­‐shaped),   the   cocci   (sphere-­‐shaped),   and  
spirilli   (helix-­‐shaped).   Traditionally,   bacterial   species   were   identified   according   to   their   shapes  
and   metabolic   capacities,   among   other   characteristics.   With   the   development   of   technologies  
that   allow   the   sequencing   of   different   genomes   in   short   periods   of   time,   bacterial   species   are  
now  characterized  by  their  DNA  sequences.    
 
Cyanobacteria    
Cyanobacteria  are  the  only  prokaryotes  that  carry  out  photosynthesis.  They  contain  pigments  
like  chlorophyll  that  give  the  cells  color.  The  pigments  absorb  sunlight  and  allow  these  cells  to  
synthesize  their  own  organic  compounds  through  the  process  of  photosynthesis.  Consequently  
these  organisms  are  known  as  autotrophs.  Cyanobacteria  are  components  of  freshwater  and  
marine  phytoplankton.  In  addition  to  a  cell  wall,  some  cyanobacteria  are  surrounded  by  a  
prominent  mucilaginous  sheath.  Although  these  organisms  are  all  unicellular,  large  numbers  of  
cells  are  often  contained  within  a  single  sheath  giving  the  appearance  of  multicellular  filaments,  
plates  or  spheres.  
 
Gloeocapsa:  This  species  is  commonly  found  growing  on  moist  rocks  and  flowerpots  in  
greenhouses.  It  consists  of  single  cells  within  a  single  sheath.  When  one  cell  divides,  the  two  
progeny  may  continue  to  reside  within  the  same  sheath  or  they  may  separate.  
Oscillatoria:   People   swimming   in   tropical   oceans   where   there   have   been   blooms   of   these  
organisms   have   suffered   skin   rashes.   The   Red   Sea   takes   its   name   from   the   fact   that   this  
organism  grows  there  and  produces  a  red  pigment  periodically.  These  cells  take  the  form  of  fine  
filaments  that  seem  to  wave  or  oscillate  back  and  forth  in  the  water.    
 
     
Observe  the  two  cyanobacteria  living  cultures  (Gloeocapsa  and  Oscillatoria)  with  the  aid  of  the  
microscope.  
• Prepare  a  wet  mount  
• Use   a   Pasteur   pipette   to   transfer   a   drop   of   the   culture   from   the   bottom   of   the   tube   to   a  
depression  slide.    
• Cover  the  drop  with  a  cover  slip.    
• Examine  the  cyanobacteria  with  the  microscope  increasing  the  magnification  as  
necessary.  
• Compare  what  you  see  with  the  photographs  in  your  Photo  Atlas.  
 
Make  drawings  (Drawing  1:  Gloeocapsa  and  Drawing  2  Oscillatoria)  and  answer  the  questions  
below.  
Drawings  should  contain  features  clearly  identified  and  labeled.  Indicate  the  magnification  
used  for  the  drawings  
 
Gloeocapsa    
• Include  an  example  of  their  grouping  in  your  drawing.  

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• Can  you  see  the  sheath?  If  not,  try  reducing  the  amount  of  light  passing  through  the  
slide.  Note  the  arrangement  of  the  cells.  Are  they  single  or  in  groups?    
 
Oscillatoria    
• Do   you   see   any   oscillation?   Describe   the   motion.   To   be   able   to   see   it,   do   not   touch   the  
slide.  By  adjusting  the  amount  of  light  see  if  you  can  determine  where  one  cell  ends  and  the  
next  begins.  
 
When  you  finish  with  the  DEPRESSION  slides,  wipe  them  with  Kimwipes  and  return  them  to  the  
front  desk.  DO  NOT  THROW  THEM  IN  GLASSWARE  TRASH.  
 
Clean  the  100X  and  40X  objectives  with  lens  paper  to  remove  the  oil.  
 
 
PART  II.  Genetic  transformation    
Introduction  
Genetic  transformation  literally  means  change  caused  by  genes,  and  involves  the  insertion  of  a  
gene  into  an  organism  in  order  to  change  the  organism’s  trait.  Genetic  transformation  is  used  in  
many  areas  of  biotechnology.  In  agriculture,  genes  coding  for  traits  such  as  frost,  pest,  or  
spoilage  resistance  can  be  genetically  transformed  into  plants.  In  bioremediation,  bacteria  can  
be  genetically  transformed  with  genes  enabling  them  to  digest  oil  spills.  In  medicine,  diseases  
caused  by  defective  genes  are  beginning  to  be  treated  by  gene  therapy;  that  is,  by  genetically  
transforming  a  sick  person’s  cells  with  healthy  copies  of  the  defective  gene  that  causes  the  
disease.  

In  this  activity,  you  will  learn  about  the  process  of  moving  genes  from  one  organism  to  
another  with  the  aid  of  a  plasmid.    

1.  Which  organism  is  better  suited  for  total  genetic  transformation—  one  composed  of  many  
cells,  or  one  composed  of  a  single  cell?    

2.  Scientists  often  want  to  know  if  the  genetically  transformed  organism  can  pass  its  new  traits  
on  to  its  offspring  and  future  generations.  To  get  this  information,  which  would  be  a  better  
candidate  for  your  investigation,  an  organism  in  which  each  new  generation  develops  and  
reproduces  quickly,  or  one  that  does  this  more  slowly?    

3.  Safety  is  another  important  consideration  in  choosing  an  experimental  organism.  What  traits  
or  characteristics  should  the  organism  have  (or  not  have)  to  be  sure  will  not  harm  you  or  the  
environment?    

4.  Based  on  the  above  considerations,  which  would  be  the  best  choice  for  a  genetic  
transformation:  a  bacterium,  earthworm,  fish,  or  mouse?  Describe  your  reasoning.  

 
 
 
 
 
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Background

Bacteria,  in  addition  to  one  large  

chromosome,  naturally  contain  one  or  more  
small  circular  pieces  of  DNA  called  plasmids.  
Plasmid  DNA  usually  contains  genes  for  one  
or  more  traits  that  may  be  beneficial  to  
bacterial  survival.  In  nature,  bacteria  can  
transfer  plasmids  back  and  forth  allowing  
them  to  share  these  beneficial  genes.  This  
natural  mechanism  allows  bacteria  to  adapt  
to  new  environments.

You  will  use  a  procedure  to  transform  bacteria  with  a  plasmid  that  contains  a  gene  that  codes  
for  Green  Fluorescent  Protein  (GFP).  The  real-­‐life  source  of  this  gene  is  the  bioluminescent  
jellyfish  Aequorea  victoria.  Green  Fluorescent  Protein  causes  the  jellyfish  to  fluoresce  and  glow  
in  the  dark.  The  pGLO  plasmid  carries  the  GFP  gene  that  codes  for  the  green  fluorescent  protein  
and  a  gene  (bla)  that  codes  for  a  protein  that  gives  the  bacteria  resistance  to  an  antibiotic.  
Following  the  transformation  procedure,  the  bacteria  express  their  newly  acquired  jellyfish  gene  
and  produce  the  fluorescent  protein,  which  causes  them  to  glow  a  brilliant  green  color  under  
ultraviolet  light.  

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7.    While  the  tubes  are  sitting  on  ice,  label  two  agar  plates  on  the  bottom  (not  the  lid)    
• Label  LB/amp  nutrient  agar  plate  with  your  initials,  lab  section  +pGLO      
• Label  LB  nutrient  agar  plate  with  your  initials,  lab  section  -­‐pGLO    
 

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                                                 LB/amp                                                                                                                                          LB                        
 
 

 
 

 
 
12.  Stack  the  plates  upside  down  in  the  37°C  incubator  until  the  next  day.  
 
 
 
 
 
 
 
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Make  predictions  
1.  In  which  of  the  two  plates  would  you  expect  to  find  non-­‐transformed  E.  coli  colonies?  Explain  
your  predictions.  
2.  If  there  are  any  genetically  transformed  bacterial  cells,  on  which  of  the  two  plates  would  they  
be  located?  Explain  your  predictions.  
3.    What  is  meant  by  a  control  plate?  What  purpose  does  a  control  serve?  

13.  Data  analysis  

• Observe  the  results  obtained  from  the  transformation  lab  under  normal  room  lighting.    
• Then  turn  out  the  lights  and  hold  the  ultraviolet  light  over  the  plates.  
• Record  your  results  and  complete  Worksheet  5.  

Information  reproduced  from  pGLO  Manual.  BioRad  

   

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Worksheet  4:  Domains  Bacteria/Archaea  

 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  

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PART  I  
Include  references  for  each  of  the  four  questions.  (4pts)  
1.  (10pts)  Write  a  paragraph  comparing  similarities  and  differences  between  Bacteria  and  
Archaea  domains.    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.   (5pts)   Give   one   example   of   one   bacterial   species   that   is   beneficial   (explain   why)   and   one   that  
is  harmful  (explain  why)  to  the  environment  or  humans.    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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 3.   (5pts)   Many   of   the   organisms   that   belong   to   Archaea   domain   live   in   environments   with  
extreme   conditions   (high   salt,   high   temperatures).   Give   an   example   of   archaebacteria   living   in  
extreme   conditions,   indicating   what   are   the   adaptations   that   allow   them   to   live   in   this  
particular  environment.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Drawings  should  contain  features  clearly  identified  and  labeled.    
Drawing  1(7.5pts):  Make  a  detailed  drawing  of  Gloeocapsa.  Include  an  example  of  their  
grouping.  
o Include   magnification   by   indicating   the   power   of   the   ocular   and   the   power   of   the  
objective.  
Magnification  _______X________  =  
 
Domain  _______________________  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Drawing  2  (7.5pts):  Make  a  labeled  drawing  of  Oscillatoria    

o Include   magnification   by   indicating   the   power   of   the   ocular   and   the   power   of   the  
objective  
 
                               Magnification  _______X________  =  
 
Domain  _______________________  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4.  (5pts)    
o Write  a  short  paragraph  describing  the  main  characteristics  of  cyanobacteria.    
o Explain  similarities  and  differences  between  Gleocapsa  and  Oscillatoria.    
 
 
 
 
 
 
 
 
 
 
 
 
PART  II  Bacterial  Transformation  
1.      What  is  a  plasmid?  (4pts)  
 
 
 
 
2. What  is  horizontal  gene  transfer?  Why  is  it  important?  (5pts)  
 
 
 
 

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3. What  is  a  transformation?  (5pts)  

 
 
 
 
 
 
 
 
4.        Give  an  example  horizontal  gene  transfer  besides  transformation.  (2pts)  
 
 
 
 
 
 
5.  (20pts)  Carefully  observe  and  draw  what  you  see  on  each  of  the  two  plates.  Write  down  the  
following  observations  for  each  plate  on  the  right  side  of  the  graph.  
A. How  much  bacterial  growth  do  you  see  on  each  plate,  relatively  speaking?    
B. What  color  are  the  bacteria  when  exposed  to  UV  light?  
C. How  many  bacterial  colonies  are  on  each  plate  (count  the  spots  you  see)  
LB  plate      -­‐pGLO  

 
A    
 
B    
 
C    
       

             

                                   LB/Amp    +  pGLO    

    A    

                        B    

  C    

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6-­‐  What  is  the  control  of  the  experiment?  Explain.  (3pts)  

 
 
 
 
 
 
7-­‐  Describe  TWO  PIECES  of  evidence  that  indicate  whether  your  attempt  at  performing  a  
genetic  transformation  was  successful  or  not.  (7pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
8-­‐  In  this  experiment  you  have  expressed  the  protein  GFP  from  jellyfish  Aequorea  victoria  
(eukaryotic  organism)  in  E.coli  (prokaryotic  organism).  What  does  this  observation  tell  you  
about  the  genetic  code  in  different  organisms?  (5pts)  
 
 
 
 
 
 
 
 
 
 
 
 
9-­‐  (5pts)  Today,  the  term  "biotechnology"  refers  to  the  use  of  living  organisms  or  their  products  
to  influence  human  health  and  the  human  environment.  However,  over  centuries  humans  have  
used  yeast  cells  to  raise  bread  dough  and  produce  alcoholic  beverages  by  fermentation,  and  
bacterial  cells  to  make  cheese  and  yogurt.  They  also  bred  animals  to  make  offspring  stronger  
and  more  productive.  
Give  one  example  of  a  protein  of  non-­‐bacterial  origin  that  has  biotechnological,  medical,  or  
pharmacological  importance  after  being  expressed  in  bacteria.  Explain  what  are  the  
advantages  of  expressing  this  protein  in  bacteria.  
 
 
 
 
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Cite  your  references  (4pts).    
 
 
 
Participation  in  Paper  Discussion  (25pts)  

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Lab  6  Domain  Eukarya:  Protists

The  lineages  that  are  dominated  by  multicellular  organisms  are  shown  in  red.  

Introduction:   become  acquainted  with  the  remarkable  

We  have  previously  studied  two  prokaryotic   diversity  of  protists.    
domains,  Bacteria  and  Archaea  that  consist    
entirely  of  single-­‐celled  organisms.  We  will   Animals
now  start  with  the  study  of  the  domain   Choanoflagellates Opisthokonts
Fungi
Eukarya.    
Dictyostelid slime molds
  Plasmodial slime molds
The  figure  on  the  right  shows  the   Archamoebae Amoebozoans
phylogenetic  tree  of  eukaryotes  as  is   Flabellinea
understood  today.  Molecular  analysis  of   Tubulinea
eukaryotes  revealed  that  they  comprise   Glaucocystophytes
Red algae Archaeplastids
seven  major  groups  called  superkingdoms.   Green algae (land plants)
The  eukarya  domain  includes  plants,  fungi,   Diatoms
and  animals,  which  are  predominantly   Brown algae Stramenopiles
composed  of  multicellular  organisms.  Other   Oomycetes
branches  of  the  Eukarya  domain  include   Dinoflagellates
Apicomplexans Alveolates
protists,  most  of  which  are  unicellular  
Ciliates
organisms.  In  the  past,  protists  were   Cercozoans
classified  as  part  of  the  kingdom  Protista.   Foraminifera Rhizarians
However,  the  use  of  genetic  analysis  in   Radiolaria
eukaryotic  systematics  has  shown  that   Cryptophytes
Haptophytes
some  protists  are  more  closely  related  to  
Centrohelid heliozoans
plants,  fungi  and  animals  than  to  other   Euglenids
protists,  and  this  has  led  to  the  elimination   Trypanosomes
of  the  kingdom  Protista.  Most  biologists  use   Diplomonads Excavates
the  term  protists  to  refer  to  eukaryotic   Parabasalids

organisms  that  are  not  plants,  fungi  or  

animals,  although  various  lineages  of    
 
protists  are  recognized  as  kingdoms  on  their  
Figure  27.9  from  Biology  How  Life  Works  
own.      
Before   coming   to   the   lab,   write   in   your   notebook   the   purpose   of   the   lab.   Make   a   list   of   the  
samples  that  you  will  observe.  Write  any  protocol  that  you  will  use.    

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PART  I  Observations  of  different  protists  under  the  microscope  

 
1-­‐  Amoebazoans    
This  group  includes  dictyostelid  and  plasmodial  slime  molds.    
Slime  molds  were  once  classified  as  fungi  because  of  the  formation  of  fruiting  bodies  
(structures  that  aid  in  spore  dispersal).  However,  more  recently,  the  use  of  molecular  
systematics  has  reclassified  them  as  protists.  Slime  molds  have  diverged  in  two  branches,  each  
of  them  having  a  unique  life  cycle:  cellular  and  plasmodial  slime  molds.  The  cellular  slime  mold  
Dictyostelium  discoideum  has  been  extensively  used  as  a  model  organism  to  study  the  evolution  
of  multicellularity.    
 
Physarum  polycephalum  exists  in  part  of  its  life  cycle  as  plasmodium,  a  giant,  multinucleated  
cell.  The  plasmodium  is  very  sensitive  to  the  environment.  It  is  able  to  collect  and  integrate  
environmental  information  and  respond  to  it  by  migrating  as  a  coordinated  whole  “body”.  
Plasmodia  behave  very  much  like  giant  amoeba  moving  towards  food  and  other  attractants,  and  
away  from  repellents.  Physarum  polycephalum  is  an  excellent  organism  to  study  chemotaxis.  
Chemotaxis  is  the  organism’s  ability  to  sense  chemical  gradients  and  move  in  response  to  them.  
 

 
 
 
 
Plasmodial  slime  mold  
o Use  the  dissecting  microscope  to  observe  a  living  plate  of  culture  of  Physarum  
polycephalum  (D-­‐1).      
Notice   the   fan   shaped   plasmodium   that   spreads   over   the   agar   and   obtains   nutrients   from   the  
oat  flakes.    If  you  look  very  carefully,  you  should  be  able  to  see  the  movement  of  cytoplasm  in  
the  plasmodium.    This  cytoplasmic  streaming  is  a  good  indication  that  the  organism  is  alive  and  
moves  to  find  its  food.  Watch  for  a  while,  and  determine  the  direction  of  the  streaming  changes.  
What  purpose  might  this  serve  in  a  large  organism  that  feeds  by  secreting  digestive  enzymes  
and  absorbing  the  products  of  digestion?    

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2.  Archeaplastids  
Green  algae  
o Observe  living  cultures  of  Volvox  and  make  a  drawing  (D-­‐2)  
 
3.  Stramenopiles  
This   group   is   very   diverse   and   includes   unicellular   organisms,   algae,   protozoa.   Some   are   free-­‐
living  cells  and  some  are  parasites.  
Diatoms   are   among   the   most   common   types   of   phytoplankton.   Most   diatoms   are   unicellular  
organisms.   A   unique   feature   of   diatoms   is   that   they   are   enclosed   by   a   cell   wall   made   of   silica.  
They  can  also  exist  as  colonies  in  the  shape  of  filaments  or  ribbons.    
o Observe  a  preserved  microscope  slide  of  a  diatom  strew  (these  are  shells  of  dead  diatoms)  
and  make  a  drawing  (D-­‐3)  
 
4.   Alveolates   includes   a   great   diversity   of   protists   that   have   been   grouped   together   based   on  
DNA  sequence  data.  
o Ciliates   All   species   within   the   group   have   cilia   for   movement   or   food   capture.     This   group  
includes  the  Tethrahymena  
 
   

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PART  II  Setting  experiments  

Amebozoans  
Experiment  1.  Observation  of  Chemotaxis  in  Physarum  polycephalum    
Materials:    
• Plasmodium  culture  
• Non-­‐nutrient  agar  plates  
• Test  substances  (plate  with  yeast,  plate  with  E  coli,  sugar,  oak  flakes,  salt)  
• Scalpel  
 
Choose  a  test  substance  and  make  a  hypothesis  of  how  the  plasmodium  will  behave  in  its  
presence.  Write  it  down  in  your  notebook.    
Work  in  groups  and  discuss  what  should  the  control  of  the  experiment  be.  
 
o Students  will  set  up  a  control  plate  (one  per  bench)  and  each  group  of  two  students  will  
set  up  an  experimental  plate.  
o Each  group  will  choose  a  different  test  substance  (plate  with  yeast,  plate  with  E  coli,  
sugar,  oak  flakes,  salt)  
 
Instructions  below:    
Prepare  ONLY  one  CONTROL  plate  per  bench  
1. Take  the  plate  containing  the  plasmodium  culture  and  cut  a  1-­‐cm2  block.  When  transferring  
Physarum  polycephalum,  select  a  thick  vein  or  solid  piece  of  “tissue”  located  near  the  edge  
of  the  plasmodium  
2. Transfer  the  piece  of  plasmodium  to  the  non-­‐nutrient  agar  (CONTROL  plate)  
3. Place  the  agar  block,  plasmodium  SIDE  DOWN,  in  the  CENTER  of  the  dish.  DO  NOT  transfer  
pieces  of  oatmeal  from  the  stock  culture  
4. Draw  a  line  down  the  middle  of  the  petri  dish  bottom  so  that  the  plasmodium  is  in  the  
middle  
5. Add  a  block  of  non-­‐nutrient  agar  in  position  1.    
What  type  of  control  is  this  one?  
 

 
 
EACH  GROUP  OF  TWO  STUDENTS  
6. Prepare  another  plate  as  described  before  but  this  time,  add  ONE  test  substance/organism  
in  position  1.  (EXPERIMENTAL  Plate)  
7. Wrap  the  dish  in  aluminum  foil  and  incubate  right  side  up  at  room  temperature  
8. Come  back  after  24h  to  observe  plasmodium  migration  and  record  its  position.    
9. In  your  notebook,  make  a  drawing  showing  the  results    

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Alveolates  
Tetrahymena  is  a  unicellular  free-­‐living  ciliate  that  can  be  found  in  fresh  water.  It  can  live  in  a  
wide  range  of  temperatures  and  adapt  to  diverse  environments.  Tetrahymena  is  an  important  
model  organism  and  its  use  has  contributed  to  the  discoveries  of  ribozymes,  telomere  function  
and  the  cytoskeletal  motor  protein  dynein.    
 
Part  A:  Observation  of  Tetrahymena’s  movement  
• Prepare  a  wet  mount  and  observe  Tetrahymena’s  movement  under  the  microscope.  
• Observe  the  behavior  of  the  cells  using  a  compound  microscope.  Tetrahymena  are  best  
observed  at  either  100  or  200X  
• Record  your  observations.  Pay  attention  to  how  the  cells  swim.  

PART  B:  Experiment  2-­‐  Observation  of  Phagocytosis  in  Tetrahymena  

 
Will  the  rate  of  phagocytosis  change  with  temperature?  Write  your  hypothesis  in  your  
notebook.      

o Before  starting  the  experiment  prepare  tubes  1b,  2b,  and  3b  by  adding  50ul  of  Lugol  solution  
to  each  tube.  
o Before  taking  any  aliquot  of  of  Tetrahymena  suspension,  shake  the  flask  to  make  sure  that  
Tetrahymena  are  not  settled  down  on  the  bottom  of  the  flask.  
o Follow  instructions  below  to  prepare  and  incubate  tubes  1a,  2a,  and  3a    
o After  adding  the  ink  solution  to  tubes  2  and  3,  MIX  GENTLY  and  start  the  timer  
IMMEDIATELY  
o Keep  the  tubes  OPEN  during  the  incubation  
 
Tube   Tetrahymena                    H2O          Ink  5%      Incubation    
     Conditions  
1a                    50ul                  52ul                  -­‐-­‐-­‐-­‐-­‐   NO  incubation    
MIX  and  transfer  immediately  50ul  
from  tube  1a  to  tube  1b  
2a                    50ul                2ul              50ul   Incubate  15min  RT.    
After  incubation,  MIX  and  transfer  
50ul  from  tube  2a  to  tube  2b  
3a                    50ul   2ul  Inhibitor              50ul   Incubate  15min  RT.    
Preincubate   After  incubation,  MIX  and  transfer  
10min  RT  before   50ul  from  tube  3a  to  tube  3b    
adding  ink  
 
 
o Before  starting  the  experiment  prepare  tubes  1b,  2b,  and  4b  by  adding  50ul  of  Lugol  solution  
to  each  tube.  
o Before  taking  any  aliquot  of  of  Tetrahymena  suspension,  shake  the  flask  to  make  sure  that  
Tetrahymena  are  not  settled  down  on  the  bottom  of  the  flask.  
o Follow  instructions  below  to  prepare  and  incubate  tubes  1a,  2a,  and  4a  
o After  adding  the  ink  solution  to  tubes  2  and  3,  MIX  GENTLY  and  start  the  timer  
IMMEDIATELY  
o Keep  the  tubes  OPEN  during  the  incubation  

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Tube   Tetrahymena                    H2O          Ink  5%      Incubation    
     Conditions  
1a                    50ul                52ul                  -­‐-­‐-­‐-­‐-­‐   NO  incubation    
MIX  and  transfer  immediately  50ul  
from  tube  1a  to  tube  1b  
2a                    50ul                2ul              50ul   15min  RT.  After  incubation,  MIX  and  
transfer  50ul  from  tube  2a  to  tube  
2b  
4a                    50ul                2ul              50ul   15min  4°C.  After  incubation,  MIX  
and  transfer  50ul  from  tube  4a  to  
tube  4b  
 
 
What  type  of  control  is  tube  1b?  
 
 
 
If  the  stock  solution  of  inhibitor  Cytochalasine  D  has  a  concentration  of  2mM,  how  much  is  the  
concentration  in  the  incubation  media?  
Tip:  use  the  equation  Ci  X  Vi=  Cf  X  Vf  
 
Sample  quantification  

• Place  a  drop  of  the  fixed  cells  on  a  microscope  slide  and  prepare  a  wet  mount  
• Use  the  compound  microscope  at  a  total  magnification  of  100-­‐400x  
• Count  the  number  of  vacuoles  containing  ink  in  5  different  cells    
• Determine  the  average  number  of  vacuoles  per  cell    
• Complete  the  table  below    
 
Table  1:  Add  a  title  and  legend  
                         Cell  Observed            Number  of                        Number  
  of  Vacuoles            Number  of    
Vacuoles  at  RT    at  RT  in  the  presence        Vacuoles  at  4°C  
 of  Cyth.  D  
1        
2        
3        
4        
5        
Average  Number  of  Vacuoles/Cell        
STDVE        
These  data  will  be  used  to  make  a  bar  graph.  Complete  the  column  corresponding  to  the  
experiment  that  you  had  not  done  using  data  from  another  group.    
 
 
 

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Lab  Report  2:  Phagocytosis  and  vacuole  formation  in  Tetrahymena    

Include  a  cover  sheet  and  yellow  pages  with  the  Lab  report  
Upload  a  version  on  ECLearn  
Parts  of  a  Lab  Report  
• Introduction  
• Materials  and  Methods  
• Results  
• Discussion  and  Conclusions  
• References  
 
Guidelines:    
Title  
Introduction    (Total  25pts)  
(Total  1.5  pages  double-­‐spaced)  
1. Context      
Introduce  the  reader  to  the  subject  matter  
Some  suggestions:  What  is  phagocytosis?  What  do  unicellular  eukaryotic  organisms  use  
phagocytosis  for?  What  is  the  role  of  phagocytosis  in  the  immune  system?  Is  the  cytoskeleton  
involved  in  vacuole  formation?  If  so,  what  role  does  it  play  and  what  type  of  proteins  are  
involved?  What  are  the  advantages  of  using  Tethrahymena  as  experimental  system?    
 
2.      Outline  your  experiments  in  your  last  paragraph:  be  brief!!    
1)  Describe  what  you  were  interested  in  determining      
2)  Describe  how  the  experiment  was  done  and  what  the  results  were  (briefly!)  
3)  Describe  your  hypothesis/predictions    
 
Materials  and  Methods    (Total  10pts)  
Do  not  copy  the  handout  instructions.  Write  a  paragraph  (in  past  tense)  describing  how  the  
experiment  was  done,  including  in  your  description  all  the  materials  used  (do  not  list  them  
separately).    
 
Results  (Total:  35pts)    
This  section  should  be  written  in  paragraph  format  and  include  the  table  with  the  experimental  
data  and  a  bar  graph    (Make  a  table  followed  by  a  bar  graph).    
 
o Start   with   a   brief   description   of   the   purpose   of   the   experiments   (rationale,   why   the  
experiments  were  done)  and  how  they  were  done.    
o Briefly  describe  the  experiment  without  repeating  experimental  details  already  
described  in  the  Materials  and  Methods  section.  
o Summarize  the  results  shown  in  Table  1  and  Figure  1.  
o Finish  with  ONE  sentence  with  the  conclusion  of  the  experiment.  Then,  in  the  Discussion  
section  the  conclusion  will  be  expanded  in  a  more  general  context.  
 
o Table  1  and  a  Figure1  should  have  a  title  (must  be  self-­‐explanatory)  and  a  legend  (brief  
description  of  how  experiment  was  done).  
o Figure  1  Use  a  bar  graph  to  show  the  three  conditions  analyzed:  Incubation  at  RT,  
Incubation  at  RT  in  the  presence  of  Cytochalasin  D,  Incubation  at  4°C.  
Insert  the  values  of  STDEV  as  error  bars  using  the  Custom  option.    

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Conclusions  and  Discussion  (Total  25pts)  
DO  NOT  repeat  the  information  presented  in  the  Results  section.  Analyze  your  data.    
What   are   the   normal   sources   of   food   for   Tetrahymena?   Explain   the   importance   of   vacuole  
formation   in   Tetrahymena   life?     Why   did   you   use   ink   instead   of   water?   Compare   your   results  
with   your   predictions.   Was   the   behavior   of   vacuole   formation   at   the   two   temperatures  
expected?   Why?   What   are   the   factors   affecting   vacuole   formation?   What   is   the   advantage   for  
the   eukaryotic   cell   to   have   a   cytoskeleton   instead   of   a   rigid   framework   as   the   one   present   in  
bacteria?   How   is   vacuole   formation   related   to   the   presence   of   a   dynamic   cytoskeleton   and  
dynamic   membranes   in   eukaryotic   cells?   If   applicable,   discuss   any   possible   sources   of   error   in  
your   experiment.   Be   specific,   do   not   attribute   result   variability   to   “human   error”.   What   other  
factors   might   have   influenced   your   results.   What   would   your   next   experiment   be   to   better  
understand  the  phenomenon  further?    
 
References  (Total  5pts)  
Include  at  least  three  references  (make  sure  to  use  at  least  one  book  and  one  primary  article)  
List  the  references  at  the  end  of  the  paper  but  also  include  them  in  the  text.    
For  more  detailed  information  referred  to  instructions  outlined  in  Lab  Report  #1.  
 
   

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Worksheet  5:  Domain  Eukarya:  Protists  
 
 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  

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1.  Once,  all  protists  were  classified  in  a  single  kingdom  Protista.  However,  today  the  kingdom  
Protista  has  been  abandoned  and  various  protist  lineages  are  considered  kingdoms  in  their  
own.  Explain  why.  (5  pts)  
 
 
 
 
 
 
 
 
2.  Do  some  research  and  give  at  least  three  examples  of  the  remarkable  diversity  exhibited  by  
protist  cells.  (6pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
Include  references  for  Q  1  and  2  (1pt)  
 
 
 
 
 
 
Drawings  should  contain  features  clearly  IDENTIFIED  and  LABELED.    
 
3.  (5  pts)  D-­‐1  Physarum  polycephalum    Magnification__Ocular  X  Objective  =_____        X      _____=  
 
 
 
 
 
 
 
 
 
 
 
 

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4.  (4  pts)  What  purpose  the  changes  in  the  direction  of  the  streaming  might  serve  in  a  large  
organism  that  feeds  by  secreting  digestive  enzymes  and  absorbing  the  products  of  digestion?    
 
 
 
 
 
 
 
 
 
 
 
5.   (5pts)   What   are   the   similarities   and   differences   between   plasmodial   slime   molds   and  
Plasmodium  falciparum?    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6.   (5pts)   Explain   why   the   development   of   vaccines   against   Plasmodium   falciparum   has   not  
been  successful.    
 
 
 
 
 
 
 
 
   
Include  references  for  Q  4,  5,  and  6  (1pt)  
 
 
 
 
 
 

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7.  (5  pts)  D2-­‐  Volvox                Magnification__Ocular  X  Objective  =_____        X      _____=  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
8.  (10  pts)  Is  the  green  alga  Volvox  unicellular  or  multicellular?  Explain.  
 
 
 
 
 
 
 
 
 
 
 
 
9.   (5pts)   Green   algae   fall   within   the   superkingdom   Archeaplastida.   What   is   the   defining  
characteristic  of  the  organisms  present  in  this  superkingdom.   What  is  the  implication  from  an  
evolutionary  point  of  view?      
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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10.  (5pts)  Are  plants  parts  of  the  superkingdom  Archeaplastida?  If  your  answer  is  yes,  explain  
why.  
 
 
 
 
 
 
 
 
       
 
 
11.  (5  pts)  Red  and  brown  algae  belong  to  two  different  superkingdoms:  Archaeplastida  and  
Stramenopila   respectively.   Describe   some   similarities   and   differences   between   these   two  
types  of  algae.    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Include  references  for  Q  8,  9,  10  and  11  (1pt)  
 
 
 
 
 
 
 
 
 
12a.  (2  pts)  In  California,  deposits  of  diatoms  300  feet  deep  are  mined  to  produce  
diatomaceous  earth,  a  fine  powdery  material.  It  is  used  as  a  filtering  material  in  swimming  
pools  and  as  a  fine  abrasive  in  silver  polishes  and  toothpastes.  Why  do  they  work  well  for  
these  purposes?    
 
 
 
 

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12b.    (3  pts)  D3-­‐  Diatoms        Magnification__Ocular  X  Objective  =_____        X      _____=  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
13.  (Total    35  pts)Experiment  1.  Observation  of  Chemotaxis  in  Physarum  polycephalum    
a.  (5pts)  Make  a  drawing  of  the  results  in  the  control  and  experimental  plate.  
     CONTROL  plate              EXPERIMENTAL  plate  
 

 
a. Describe  your  observations.  (5  pts)  
 
 
 
 
 
 
 
 
 
 
 

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c.  Write  the  answer  to  the  following  questions  in  paragraph  format.  (10  pts)  
What  is  chemotaxis?  What  do  prokaryotic  and  eukaryotic  organisms  use  chemotaxis  for?  How  do  
cells  sense  chemoattactants/  chemorepellents?  Be  specific  about  the  type  of  molecules  involved.  
Is  the  cytoskeleton  remodeled  during  chemotaxis?  
 
 
 
 
 
 
 
 
 
   
   
 
d.  Conclusions  and  Discussion  (10pts)  
Write  a  paragraph  addressing:  What  was  the  behavior  of  plasmodium  in  the  presence  of  the  
stimulus  used?  Compare  your  results  with  your  predictions.  Was  your  hypothesis  supported  or  
rejected?  Explain  the  importance  of  the  chemotactic  response  for  survival?  If  applicable,  discuss  
any  possible  sources  of  error  in  your  experiment.  What  other  factors  might  have  influenced  your  
results.  What  would  your  next  experiment  be  to  better  understand  the  phenomenon  further.  
 
References  (2  pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Lab  Report  #2:  Vacuole  formation  in  Tetrahymena  
 
 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  
 
 
YELLOW  PAGES  INCLUDED  _____  

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Lab  6  Genetically  Modified  organisms  (GMOs)  
 
Background  
 
With  the  world  population  exploding  and  farmable  land  disappearing,  agricultural  specialists  are  
concerned  about  the  world's  ability  to  produce  enough  food  to  feed  the  growing  population.  
Environmentalists  are  concerned  about  the  overuse  of  pesticides  and  herbicides  and  the  long-­‐
term  effects  of  these  chemicals  on  the  environment  and  human  health.  Might  there  be  a  
solution  to  both  of  these  problems?  The  biotechnology  industry  thinks  so.  Its  proponents  
believe  genetically  modified  organisms  (GMOs),  particularly  genetically  modified  (GM)  crop  
plants,  can  solve  both  problems.  This  proposed  solution,  however,  has  met  with  great  
opposition  throughout  the  world.  Dubbed  "frankenfoods"  by  opponents  and  restricted  in  most  
European  countries,  GMOs  are  widely  produced  and  sold  in  the  United  States.  Currently  in  the  
US,  foods  that  contain  GMOs  do  not  have  to  be  labeled  as  such.  
 
Genetic  manipulation  of  crop  plants  is  not  new.  Farmers  have  been  genetically  modifying  crops  
for  centuries  and  crop  breeding  to  encourage  specific  traits,  such  as  high  yield,  is  still  an  
important  part  of  agriculture  today.  However,  there  is  now  the  option  to  place  genes  for  
selected  traits  directly  into  crop  plants.  These  genes  do  not  have  to  originate  from  the  same  
plant  species—in  fact,  they  do  not  have  to  come  from  plants  at  all.  One  popular  class  of  GM  
crops  has  a  gene  from  the  soil  bacterium  Bacillus  thuringiensis  (Bt)  inserted  into  their  genomes.  
Bt  crops  produce  a  protein  called  delta-­‐endotoxin  that  is  lethal  to  European  corn  borers,  a  
common  pest  on  corn  plants.  Farmers  who  plant  Bt  crops  do  not  have  to  apply  pesticide  
because  the  plants  produce  the  toxic  protein  inside  their  cells.  When  the  corn  borers  feed  on  
the  genetically  modified  plant,  they  die.  Other  GMOs  include  those  that  are  herbicide-­‐resistant  
delayed  for  fruit  ripening,  are  resistant  to  fungi  or  drought,  have  increased  crop  yield,  or  bear  
improved  fruits.  
 
Many  people  object  to  the  use  of  GM  crop  plants.  They  argue  that  there  is  a  potential  to  create  
super-­‐weeds  through  cross-­‐pollination  with  herbicide-­‐resistant  crops  or  that  super-­‐  bugs  will  
evolve  that  are  no  longer  resistant  to  the  toxins  in  pest-­‐resistant  crops.  Many  are  concerned  
with  potential  allergic  reactions  to  the  novel  proteins  or  antibiotic  resistance  arising  from  the  
selectable  markers  used  to  develop  the  crops  or  other  unforeseen  effects  on  public  health.  
Proponents  of  GM  foods  argue  these  crops  are  actually  better  for  the  environment.  Fewer  toxic  
chemicals  are  put  into  the  environment  and  thus  fewer  toxic  chemicals  can  harm  the  
environment  and  human  health.  In  addition,  these  crops  can  preserve  arable  land  by  reducing  
stresses  on  the  land,  improve  the  nutritional  value  of  food  in  developing  countries,  and  allow  
crops  to  be  grown  on  previously  unfarmable  land.  
 
Whatever  position  one  takes  in  the  GMO  debate,  it  would  be  beneficial  to  be  able  to  test  foods  
found  in  the  grocery  store  for  the  presence  of  GMO-­‐derived  products.  This  can  be  done  in  
several  ways.  One  would  be  to  use  an  antibody-­‐based  test  such  as  the  enzyme-­‐linked  
immunosorbent  assay  (ELISA),  which  can  detect  the  proteins  that  are  produced  specifically  by  
GM  crops.  However,  the  ELISA  is  not  useful  for  testing  foods  that  have  been  highly  processed,  
because  the  proteins  have  most  likely  been  destroyed  and  different  GM  foods  produce  different  
proteins.  Another  method  is  to  use  the  polymerase  chain  reaction  (PCR)  to  look  for  a  DNA  
sequence  common  to  GM  foods.  DNA  is  more  resistant  than  proteins  to  processing  and  can  be  

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extracted  from  even  highly  processed  foods.  It  is  these  GMO  DNA  sequences  that  we  will  be  
testing  for  in  this  laboratory.  
In  this  lab  you  will  extract  genomic  DNA  from  food  samples,  you  will  run  PCR  reactions  to  
amplify  GMO  and  natural  plant  sequences  from  the  DNA,  and  finally  you  will  electrophorese  the  
amplified  samples  to  visualize  the  DNA.  

 
Fig.  1.  Detecting  GM  foods  by  PCR.  Genomic  DNA  is  extracted  from  test  foods  (Lesson  1)  and  
then  two  PCR  reactions  are  performed  on  each  test  food  genomic  DNA  sample  (Lesson  2).  One  
PCR  reaction  uses  primers  specific  to  a  common  plant  gene  (plant  primers)  to  verify  that  viable  
DNA  was  successfully  extracted  from  the  food.  No  matter  whether  the  food  is  GM  or  not,  this  
PCR  reaction  should  always  amplify  DNA  (See  lanes  1  and  3  of  the  gel  above).  The  other  PCR  
reaction  uses  primers  specific  to  sequences  commonly  found  in  GM  crops  (GMO  primers).  This  
PCR  reaction  will  only  amplify  DNA  if  the  test  food  is  GM  (See  lane  4).  If  the  test  food  is  non-­‐GM,  
then  the  GMO  primers  will  not  be  complementary  to  any  sequence  within  the  test  food  genomic  
DNA  and  will  not  anneal,  so  no  DNA  will  be  amplified  (see  lane  2).  To  find  out  whether  DNA  has  
been  amplified  or  not,  the  PCR  products  are  electrophoresed  on  a  gel  and  stained  to  visualize  
DNA  as  bands  (Lesson  3).  A  molecular  weight  ruler  (lane  5)  is  electrophoresed  with  the  samples  
to  allow  the  sizes  of  the  DNA  bands  to  be  determined.

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PCR  Review  
 
PCR  is  such  a  powerful  tool  because  of  its  simplicity  and  specificity.  All  that  is  required  are  
minute  quantities  of  the  DNA  template  you  want  to  amplify,  DNA  polymerase,  two  DNA  primers,  
four  DNA  base  pair  subunits  (deoxyribonucleotide  triphosphates  of  adenine,  guanine,  thymine,  
and  cytosine)  and  buffers.  
 
Because  PCR  identifies  a  specific  sequence  of  DNA  and  makes  millions  of  copies  of  (or  amplifies)  
that  sequence,  it  is  one  of  the  most  useful  tools  of  molecular  biology.  Scientists  use  PCR  to  
obtain  the  large  amounts  of  a  specific  sequence  of  DNA  that  are  necessary  for  such  techniques  
as  gene  cloning,  where  DNA  is  physically  moved  from  one  genome  to  another.  You  are  using  the  
property  of  PCR  that  allows  identification  of  a  specific  sequence,  namely,  the  ability  of  PCR  to  
search  out  a  single  sequence  of  a  few  hundred  base  pairs  in  a  background  of  billions  of  base  
pairs.  For  example,  the  corn  genome  has  2.5  billion  base  pairs  of  DNA.  This  sequence  is  then  
amplified  so  that  there  are  millions  of  copies  of  it  so  that  it  stands  out  from  the  few  copies  of  
the  original  template  DNA.  
 
PCR  locates  specific  DNA  sequences  using  primers  that  are  complementary  to  the  DNA  
template.  Primers  are  short  strands  of  DNA  (usually  between  6  and  30  base  pairs  long)  called  
oligonucleotides.  Two  primers  are  needed  to  amplify  a  sequence  of  DNA,  a  forward  primer  and  a  
reverse  primer.  The  two  primers  are  designed  and  synthesized  in  the  laboratory  with  a  specific  
sequence  of  nucleotides  such  that  they  can  anneal  (bind)  at  opposite  ends  of  the  target  DNA  
sequence  on  the  complementary  strands  of  the  target  DNA  template.  The  target  DNA  sequence  
is  copied  by  the  DNA  polymerase  reading  the  complementary  strand  of  template  DNA  and  
adding  nucleotides  to  the  3'  ends  of  the  primers  (see  fig  2).  Primers  give  the  specificity  to  the  
PCR,  but  they  are  also  necessary  because  DNA  polymerase  can  only  add  nucleotides  to  double-­‐
stranded  DNA.  
 
During  PCR,  double-­‐stranded  DNA  template  is  separated  by  heating  it,  then  each  primer  binds  
(anneals)  to  its  complementary  sequence  on  each  of  the  separated  DNA  strands,  and  DNA  
polymerase  elongates  each  primer  by  adding  nucleotides  to  make  a  new  double-­‐stranded  DNA  
(see  fig  2).  
 
The  DNA  polymerase  used  in  PCR  must  be  a  thermally  stable  enzyme  because  the  PCR  reaction  
is  heated  to  94°C,  which  would  destroy  the  biological  activity  of  most  enzymes.  The  most  
commonly  used  thermostable  DNA  polymerase  is  Taq  DNA  polymerase.  This  was  isolated  from  a  
thermophillic  bacterium,  Thermus  aquaticus,  which  lives  in  high-­‐  temperature  steam  vents  such  
as  those  in  Yellowstone  National  Park.  
 
 
 
 
 
 
 
 
 

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Figure  2:  A  complete  PCR  cycle  

 
 
Usually,  thermal  cycling  continues  for  about  40  cycles.  After  each  thermal  cycle,  the  number  of  
template  strands  doubles,  resulting  in  an  exponential  increase  in  the  number  of  template  DNA  
strands.  After  40  cycles  there  will  be  1.1  x  1012  more  copies  of  the  original  number  of  template  
DNA  molecules  

 
 
PCR  generates  DNA  of  a  precise  length  and  sequence.  On  the  first  cycle,  the  two  primers  anneal  
to  the  original  genomic  template  DNA  strands  at  opposite  ends  and  on  opposite  strands.  After  
the  first  complete  temperature  cycle,  two  new  strands  are  generated  that  are  shorter  than  the  
original  template  strands  but  still  longer  than  the  length  of  the  DNA  that  the  researcher  wants  
to  amplify.  It  isn't  until  the  third  thermal  cycle  that  fragments  of  the  precise  length  are  
generated.  

 
 
 

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Figure  3:  Generation of precise-length fragments

 
 
PCR  Step  by  Step  
 
PCR  has  three  steps,  a  denaturing  step,  an  annealing  step,  and  an  elongation  step.    
 
During  the  denaturing  step,  the  DNA  template  is  heated  to  94°C  to  separate  (or  denature)  the  
double-­‐stranded  DNA  molecule  into  two  single  strands.  The  DNA  is  then  cooled  to  59°C  to  allow  
the  primers  to  locate  and  anneal  (bind)  to  the  DNA.  Because  the  primers  are  so  much  shorter  
than  the  template  DNA,  they  will  anneal  much  more  quickly  than  the  long  template  DNA  strands  
at  this  temperature.  The  final  step  is  to  increase  the  temperature  of  the  PCR  reaction  to  72°C,  
which  is  the  optimal  temperature  for  the  DNA  polymerase  to  function.  In  this  step  the  DNA  
polymerase  adds  nucleotides  (A,  T,  G,  or  a  C)  one  at  a  time  at  the  3’  end  of  the  primer  to  create  

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a  complementary  copy  of  the  original  DNA  template.  These  three  steps  form  one  cycle  of  PCR.  A  
complete  PCR  amplification  undergoes  multiple  cycles  of  PCR,  in  this  case  40  cycles.  
 
The  entire  40  cycle  reaction  is  carried  out  in  a  test  tube  that  has  been  placed  in  a  thermal  cycler  
or  PCR  machine.  This  is  a  machine  that  contains  an  aluminum  block  that  can  be  rapidly  heated  
and  cooled.  The  rapid  heating  and  cooling  of  this  thermal  block  is  known  as  thermal  cycling.  
 
Two  new  template  strands  are  created  from  the  original  double-­‐stranded  template  during  each  
complete  cycle  of  PCR.  This  causes  exponential  growth  of  the  number  of  target  DNA  molecules,  
i.e.,  the  number  of  target  DNA  molecules  doubles  at  each  cycle;  this  is  why  it  is  called  a  chain  
reaction.  Therefore,  after  40  cycles  there  will  be  240,  or  over  1,100,000,000,000  times  more  
copies  than  at  the  beginning.  Once  the  target  DNA  sequences  of  interest  have  been  sufficiently  
amplified,  they  can  be  visualized  using  gel  electrophoresis.  This  allows  researchers  to  determine  
the  presence  or  absence  of  the  PCR  products  of  interest.    
 
 
 
Outline  GMO  Lab  
 
DAY  1    
• Extraction  of  DNA  from  food  samples    
• Setting  up  the  PCR  reaction  
 
DAY  2    
• Electrophoresis  of  PCR  products  and  data  analysis  
• Class  Discussion  Pro/Con  of  GMOs  
 
 
 
   

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DAY  1  Extraction  of  DNA  from  food  samples  and  setting  up  the  PCR  reaction  
 
Extraction  of  DNA  from  food  samples  
In  this  lab,  you  will  analyze  DNA  extracted  from  a  control  non-­‐GMO  food  and  from  grocery  store  
food  item  that  you  will  test  for  the  presence  of  GMOs.  The  most  commonly  modified  foods  are  
corn  and  soy-­‐based,  and  so  the  test  food  could  be  fresh  corn  or  soybeans,  or  a  prepared  or  
processed  food  such  as  cornmeal,  cheese  puffs,  veggie  sausage,  etc.    

The  non-­‐GMO  control  will  be  provided  and  students  will  extract  DNA  from  food  sample.  First  
you  will  weigh  your  food  sample,  then  grind  it  with  water  to  make  a  slurry.  You  will  then  add  a  
tiny  amount  of  the  slurry  to  a  screwcap  tube  containing  InstaGene  matrix  and  boil  it  for  5  
minutes.  

The  cellular  contents  you  are  releasing  from  the  ground-­‐up  sample  contain  enzymes  (DNases)  
that  can  degrade  the  DNA  you  are  attempting  to  extract.  The  InstaGene  matrix  is  made  of  
negatively  charged  microscopic  beads  that  “chelate”  or  grab  metal  ions  out  of  solution.  It  
chelates  metal  ions  such  as  Mg2+,  which  is  required  as  a  cofactor  in  enzymatic  reactions.  When  
DNA  is  released  from  your  sample  in  the  presence  of  the  InstaGene  matrix,  the  charged  beads  
grab  the  Mg2+  and  make  it  unavailable  to  the  enzymes  that  would  degrade  the  DNA  you  are  
trying  to  extract.  This  allows  you  to  extract  DNA  without  degradation.  Boiling  the  samples  
destroys  these  enzymes.  After  you  centrifuge  the  samples  to  remove  the  InstaGene  matrix  and  
debris,  the  supernatant  will  contain  intact  extracted  DNA.  This  extracted  DNA  will  be  used  to  set  
up  a  PCR  reaction  

 
Set  Up  PCR  Reactions  
After  you  has  been  extracted  DNA  from  a  certified  non-­‐GMO  food  sample  and  a  test  food  
sample  you  will  do  a  PCR  reaction  to  determine  whether  there  are  GMO  DNA  sequences  in  the  
test  food.    
 
PCR  is  DNA  replication  in  a  test  tube.  PCR  allows  you  to  amplify  specific  sections  of  DNA  and  
make  millions  of  copies  of  the  target  sequence.  Your  experiment  is  to  determine  whether  or  not  
the  DNA  you  extracted  from  food  in  Lesson  1  contains  or  does  not  contain  the  target  sequences  
of  interest  typically  found  in  genetically  modified  (GM)  foods.  

 
 
 
 
 
 
 
 
 
 
 
 

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PROCEDURE  
 
REAGENTS  and  MATERIALS  
• Mortar  and  pestle  
• Scale  and  weigh  boats  
• Thermo  block  set  to  95–100°C  
• Beakers  with  distilled  water  
• InstaGeneTM  matrix  
• Disposable  plastic  transfer  pipets  (DPTPs)  
• Screwcap  tubes    
• Food  
• Marker  
• GMO+  DNA  
• GMO-­‐  DNA        
     
 
Procedure  to  extract  DNA  from  the  GMO+control  and  the  test  food  

1.  Find  2  screwcap  tubes  containing  500  μl  of  InstaGene  matrix  and  label  one  of  the  tubes  “GMO+”  and  the    
other  one    “test”.  
 

 
2.  Using  the  transfer  pipet,  add  5  ml  of  distilled  water  for  every  gram  of  food  using  the  graduations    
on  the  transfer  pipet.    
To  calculate  the  volume  of  water  you  need,  mulitply  the  mass  in  grams  of  the  food  weighed  out    
by  5  and  add  that  many  millimeters.      
                                                                                     Mass  of  Food  =   g  x  5  =   ml    
 

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3.  Grind  with  pestle  for  at  least  2  min  until  a  slurry  is  formed.    
4.  Add  5  volumes  of  water  again  and  mix  or  grind  further  with  pestle  until  the  slurry  is  smooth  
enough  to  pipet.    
5.  Add  50  μl  of  ground  slurry  to  the  screwcap  tube  containing  500  μl  of  InstaGene  matrix  labeled  
test  using  a  transfer  pipet.    

 
 
7.  Recap  tube  and  shake  well.    

 
8.  Repeat  same  procedure  from  step  2  using  the  provided  grinded  GMO+  control  
 
 

 
3.  After  you  centrifuge  the  samples  to  remove  the  InstaGene  matrix  and  debris,  the  supernatant  
will  contain  intact  extracted  DNA.  
4.  Transfer  the  supernatant  to  a  clean  tube  with  your  initials.    
 
Now  that  you  have  extracted  DNA  from  the  test  food  sample  you  will  analyze  for  the  presence  
of  GMO  DNA  sequences  using  a  PCR  reaction.  PCR  is  DNA  replication  in  a  test  tube.  PCR  allows  
you  to  amplify  specific  sections  of  DNA  and  make  millions  of  copies  of  the  target  sequence.  Your  
experiment  is  to  determine  whether  or  not  the  DNA  you  extracted  from  food  contains  or  does  
not  contain  the  target  sequences  of  interest  typically  found  in  genetically  modified  (GM)  foods  

Setting  up  the  PCR  reaction  

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For  this  experiment  you  will  set  up  two  PCR  reactions  for  each  DNA  sample,  which  makes  6  PCR  
reactions  in  total.  One  PCR  reaction,  using  the  plant  master  mix  (PMM),  is  a  control  to  
determine  whether  or  not  you  have  successfully  extracted  plant  DNA  from  your  test  food.  This  is  
done  by  identifying  a  DNA  sequence  that  is  common  to  all  plants  by  using  primers  (colored  
green  in  the  kit)  that  specifically  amplify  a  section  of  a  chloroplast  gene  used  in  the  light  reaction  
(photosystem  II).    
Why  is  this  necessary?  What  if  you  do  not  amplify  DNA  using  the  GMO  primers?  Can  you  
conclude  that  your  test  food  is  not  GM  or  might  it  just  be  that  your  DNA  extraction  was  
unsuccessful?    
 
 
 
 
 
 
 
The  second  PCR  reaction  you  carry  out  will  determine  whether  or  not  your  DNA  sample  contains  
GM  DNA  sequences.  This  is  done  by  identifying  DNA  sequences  that  are  common  to  most  
(~85%)  of  all  GM  plants  using  primers  specific  to  these  sequences.  These  primers  are  colored  red  
and  are  in  the  GMO  master  mix  (GMM).  
Why  do  you  have  to  set  up  a  PCR  reaction  with  DNA  from  certified  non-­‐GMO  food?    
 
 
 
 
 
 
 
 
Setting  the  PCR  reaction  
PCR  Amplification  and  Sterile  Technique  
PCR  is  a  powerful  and  sensitive  technique  that  enables  researchers  to  produce  large  quantities  
of  specific  DNA  from  very  small  amounts  of  starting  material.  Because  of  this  sensitivity,  
contamination  of  PCR  reactions  with  unwanted  DNA  is  always  a  possible  problem.  Therefore,  
utmost  care  must  be  taken  to  prevent  cross-­‐contamination  of  samples.  Steps  to  be  taken  to  
prevent  contamination  and  failed  experiments  include:  
 
o Change  pipet  tips.  Always  use  a  new  pipet  tip  when  entering  a  reagent  tube  for  the  first  
time.  If  a  pipet  tip  is  used  repeatedly,  contaminating  DNA  molecules  on  the  outside  of  the  
tip  will  be  transferred  to  other  solutions,  resulting  in  contaminated  PCR  reactions.  If  you  are  
at  all  unsure  if  your  pipet  tip  is  clean,  err  on  the  safe  side  and  discard  the  tip  and  get  a  new  
one.    
 
o When  opening  tubes  or  pipetting  reagents,  leave  the  tubes  open  for  as  little  time  as  
possible.  Tubes  that  are  open  and  exposed  to  the  air  can  easily  become  contaminated  by  
aerosolized  DNA  molecules.  Go  into  reagent  tubes  efficiently,  and  close  them  as  soon  as  you  

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are  finished  pipetting.  Also,  try  not  to  pick  tubes  up  by  the  rim  or  cap,  as  you  can  easily  
introduce  contaminants  from  your  fingertips.    
 
Bleach  at  a  concentration  of  10%  destroys  DNA,  so  wiping  down  surfaces  and  rinsing  plastic  
pipet  barrels,  mortars,  and  pestles  with  10%  bleach  can  get  rid  of  any  surface  DNA  
contamination  that  may  arise.    
 
PCR  procedure    

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This  lab  utilizes  a  three-­‐step  cycle:  the  DNA  undergoes  denaturation  at  94°C  for  1  minute,  
annealing  at  59°C  for  1  minute,  and  extension  at  72°C  for  2  minutes.  This  cycle  is  repeated  40  
times  during  the  course  of  PCR  amplification.  The  PCR  reaction  will  take  approximately  3.5  hours  
to  complete.  
 
Day  2:  Electrophoresis  of  PCR  products  and  data  analysis  
You  have  completed  your  PCR  amplification.  You  cannot,  however,  at  this  point  determine  
whether  or  not  you  have  PCR  products.  To  do  this,  you  must  visualize  your  products.  You  will  do  
this  using  gel  electrophoresis.  
The  product  band  sizes  in  this  lab  are  455  bp  for  the  plant  primers  and  200  bp  for  the  GMO  
these  bands  will  be  separated  using  a  3%  agarose  gel..  
 
1.  Obtain  your  PCR  tubes  and  Pulse-­‐spin  the  tubes  for  ~3  seconds.    
2.  Using  a  fresh  tip  each  time,  add  10  μl  of  Orange  G  loading  dye  (LD)  to  each  sample  and  mix  
well.  
3.  Load  20  μl  of  each  sample  in  the  agarose  gel.  
4.  Run  the  agarose  gel  for  30  min  at  100  V.  
the  gel  should  be  loaded  with  a  molecular  weight  ruler  (DNA  standard)  so  that  you  have  a  
reference  to  determine  your  product  bands'  sizes.    
 
References  
TM   TM  
Biotechnology  Explorer GMO  Investigator Kit.  BioRad  Catalog.    
 

 
 
 
 

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Worksheet  6a:  Genetically  Modified  Organisms  

 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  
   

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1.    (5pts)  What  are  the  two  methods  that  can  be  used  to  test  a  food  to  find  out  if  it  contains  
material  derived  from  a  genetically  modified  organism  (GMO)?    Briefly  describe  what  each  
method  determines.  
 
 
 
 
 
2.  (10pts)  Besides  the  nucleus,  in  which  organelles  is  plant  DNA  localized?    Explain  your  answer  
based  on  your  knowledge  of  the  endosymbiotic  theory.    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3.  (2.5pts)  Many  foods  containing  GM  crops  are  highly  processed.  Can  you  suggest  how  DNA  
from  whole  plants  may  differ  from  that  extracted  from  processed  foods,  e.g.,  corn  chips,  
cornmeal,  etc.?      
 
 
 
   
4.  (2.5pts)    What  molecules  are  present  in  the  cell  that  might  interfere  with  DNA  extraction?      
 
 
 
 
 
 
 
5.  (20pts)  What  is  the  function  of  each  of  the  following  components  used  in  a  PCR  reaction  
component?  
Taq  DNA  polymerase  
 
   
 

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Deoxynucleotide  triphosphates    
 
 
 
 
 
 
Primers    
 
 
 
 
 
Buffers  and  cofactors    
 
 
 
 
 
5.  Pro/Con  Data  Sheet    (60pts)  

Include  at  least  three  reasons  why  we  should  use  GM  crops.  Each  of  those  reasons  should  be  
supported  by  references  from  a  valid  source  and  should  include  at  least  one  primary  paper  

   

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Include  at  least  three  reasons  why  we  should  not  use  GM  crops.  Each  of  those  reasons  should  be  
supported  by  references  from  a  valid  source  and  should  include  at  least  one  primary  paper  

   

   

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Worksheet  6b:  Genetically  Modified  Organisms.  Data  Analysis.  

 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  
   

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1.  (5pts)  During  DNA  extraction,  why  was  the  non-­‐GMO  food  control  prepared  prior  to  your  test  
food  sample?      
 
 
 
 
 
 
 
2.  (Total  15pts)  During  the  PCR  reaction  you  set  up  6  different  tubes,  only  two  tubes  contained  
your  test  food.    
2a  (5pts)  Why  were  you  performing  two  PCR  reactions  on  each  DNA  sample?      
 
 
 
 
 
 
 
2b.  (5pts)  Why  did  you  perform  the  analysis  on  food  that  is  known  to  be  a  non-­‐GMO  control?      
 
 
 
 
 
 
 
2c.(5pts)  What  was  the  purpose  of  the  GMO  positive  control  DNA?      
 
 
 
 
 
 
 
3.    (5pts)  Explain  why  DNA  fragments  separate  according  to  size  in  an  electrophoresis  gel.      
 
 
 
 
 
 
 
 
 
 
 

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4.  (2.5pts)  Why  do  you  need  a  molecular  mass  ruler  alongside  your  samples?      
 
 
 
 
 
 
5.  (Total  30pts)  Make  a  figure  in  a  separate  page  using  the  picture  of  the  agarose  gel  containing  
your  samples.  
Figure  should  have  a  title  and  legend  (10pts).  
Label  the  DNA  markers  (5pts).  
 
6.    (Total  18pts)  Data  analysis  
6a.  (1pts)  What  was  your  test  food?  
 
 
 
6b.  (5ps)  Did  your  test  food  generate  a  200  bp  band  with  GMO  primer?  Yes  or  no.    
What  does  this  tell  you  about  the  GMO  status  of  your  food?      
 
 
 
 
 
 
 
6c.  (10pts)  How  do  the  results  of  your  other  five  PCR  reactions  help  support  or  undermine  your  
result  for  your  test  food?      
 
 
 
 
 
 
 
 
 
 
 
 6d.(2pts)  Do  your  results  match  our  predictions?  If  not,  explain  any  possible  sources  of  error.  
 
 
 
 
 
 
 
 

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7.  (20pts)  Examine  the  150  base  sequence  below.  
 
5'  TAGAAAAGGA  AGGTGGCTCC  TACAAATGCC  ATCATTGCGA  TAAAGGAAAG    GTATCATTC    

AAGATGCCTC  TGCCGACAGT  GGTCCCAAAG  ATGGACCCCC    ACCCACGAGG  AGC  ATCGTGG    

AAAAAGAAGA  CGTTCCAACC  ACGTCT  TCAA  3'    

o Write  in  the  sequence  of  the  complementary  strand  and  mark  the  3'  and  5'  ends  of  the  
complementary  strand  .  (5pts)  
o Remembering  that  DNA  polymerases  can  only  add  nucleotides  to  the  3'  end  of  DNA,  design  
a  forward  primer  and  a  reverse  primer,  each  10  bases  long,  to  amplify  a  target  sequence  of  
the  DNA  that  is  at  least  100  bp  long.  Write  the  sequence  of  the  primers  below,  with  their  3'  
and  5'  ends  indicated.  (10pts)  
o Also  indicate  on  the  sequence  above  which  strand  they  are  complementary  to  (will  anneal  
to).  (5pts)  
 
 
 
8  (5pts).  You  had  a  class  discussion  about  the  pros  and  cons  of  the  use  of  GMOs.  Explain  your  
position.      

 
 
 
 

   

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Lab  7    Fungi  Kingdom  

 
Before  coming  to  the  lab,  write  the  purpose  of  the  lab  activities.    
 
Background  
Fungi  are  one  of  the  most  diverse  groups  of  eukaryotic  organisms.  Fungi  share  a  number  of  
features  with  animals  because  these  two  groups  have  a  common  single-­‐celled  ancestor  that  
lived  in  an  aquatic  environment.  Then,  multicellularity  evolved  separately  in  fungi  and  animals.  
Fungi  are  heterotrophs,  and  because  they  cannot  move,  they  secrete  proteolytic  enzymes  to  
break  down  diverse  complex  organic  molecules.  Some  fungi  feed  on  dead  plants  and  animals,  
and  others  form  symbiotic  associations  with  plants  and  animals.  Most  fungi  are  multicellular,  
consisting  of  branched  filaments  named  hyphae.  However,  not  all  fungi  are  multicellular.  Yeasts  
are  single-­‐celled  fungi  that  have  lost  the  hyphal  development  during  evolution.    

   
 

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Budding  yeast,  Saccharomyces  cerevisiae.  A)  Mitotically  dividing  yeast.  B)  Haploid  yeast  spores  
in  an  ascus.  In  the  above  images,  part  A  on  the  left  shows  a  scanning  electron  microscope  image  
of  a  population  of  budding  yeast  cells  dividing  by  mitosis  via  bud  formation.  In  part  B  on  the  
right,  a  single  ascus  containing  four  spores  (haploid  meiotic  progeny)  is  shown.  Note  that  the  
magnification  on  the  image  to  the  right  is  about  3  times  the  magnification  of  the  image  on  the  
left.    
 
Today,  you  will  finish  the  GMO  lab  and  you  will  set  up  an  experiment  using  yeast  cells.  
 
Experiment:  What  are  the  conditions  that  favor  spore  formation  in  budding  yeast  cells?  
You  will  observe  two  strains,  A  and  B,  each  grown  in  two  different  media.    
After  observing  the  yeasts  growing  in  the  two  different  media  and  using  the  figures  above  to  
distinguish  between  mitotically  budding  yeast  and  haploid  spores  you  will  be  able  to  answer:  
 
1)  What  are  the  conditions  that  favor  spore  formation  in  budding  yeast  cells?    
2)  Which  strain,  A  or  B,  is  the  haploid  strain  and  which  is  the  diploid  strain?    

Materials    
-­‐Strain  A  and  Strain  B  patched  onto:  
o rich  media  plate  (YPD)  
o low-­‐nitrogen  minimal  media  plate    
-­‐Light  microscope    
-­‐Glass  slides  and  cover  slips  
-­‐Sterile  water  
-­‐Toothpicks  
 
Procedure  
Students  will  work  in  groups  of  two.  Each  student  will  examine  a  few  cells  from  each  strain  
growing  on  each  plate  (total  of  4  samples  to  be  examined  by  each  pair).  To  prepare  a  slide  
containing  each  one  of  the  yeast  samples:  
1.  Pipet  3  ul  of  sterile  water  in  a  spot  in  the  middle  of  the  slide.    
2.  Using  a  sterile  toothpick,  very  lightly  touch  a  small  part  of  one  of  the  yeast  cell  samples,  
noting  the  strain  and  the  plate  type.  (You  only  want  a  VERY  small  sample  of  cells!)  
3.  Touch  the  toothpick  containing  the  yeast  cells  to  the  water  spot  on  the  slide  and  swirl  around  
to  spread  the  yeast  throughout  the  spot.  
4.  Discard  the  toothpick  and  carefully  place  a  cover  slip  on  top  of  the  water  spot.    

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5.  Examine  your  yeast  cells  under  10x,  40x,  and  100x  magnification  to  determine  whether  the  
yeast  cells  in  the  sample  are  growing  mitotically  via  budding  (like  in  A  above)  or  whether  some  
of  the  yeast  cells  have  undergone  sporulation  to  form  spores  via  meiosis  (like  in  B).    
[NOTE:  Spore  formation  is  not  a  100%  efficient  process.  Even  under  conditions  where  
sporulation  is  favored,  all  of  the  cells  may  not  have  formed  spores.  However,  under  conditions  
where  sporulation  is  NOT  favored,  you  should  not  observe  ANY  spores  in  an  ascus  and  all  the  
cells  should  be  growing  mitotically].  
 
D6-­‐  Draw  on  worksheet  examples  of  yeast  cells  growing  in  the  two  different  media  under  100x  
magnification.    
.    
 

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Worksheet  7:  Domain  Eukarya:  Fungi  Kingdom  
 
 
 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  
 
   

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Fungi  Kingdom  
 
1.  (10pts)  Write  a  short  paragraph  describing  the  most  common  fungal  body  structures  and  
explain  how  the  morphology  of  multicellular  fungi  affects  the  efficiency  of  nutrient  
absorption.    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.   (5pts)   Compare   and   contrast   the   nutritional   mode   of   a   fungus   with   your   own   nutritional  
mode.    
 
 
 
 
 
 
 
 
 
 
 
 
3.  (10pts)  Many  fungi  form  symbiotic  associations  such  as  lichens  and  mycorrhiza.  Describe  
the  organisms  present  in  both  symbiotic  relationships  and  their  roles.    
 
 
 
 
 
 
 
 
 

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EXPERIMENT  WITH  Saccharomyces  cerevisiae    
 
4.  (10pts)  Describe  some  of  the  advantages  of  using  Saccharomyces  cerevisiae  as  experimental  
system.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
D6-­‐  (10pts)  Draw  examples  of  yeast  cells  growing  in  the  three  different  media.    
 
Strain  A  
                         rich  media  (YPD)                                                                                                                  nitrogen  minimal  media    
 
 

 
 
                   
 
 
 
 
 
 
 
Strain  B  

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                             Rich  media  (YPD)                                                                                                                      Nitrogen  minimal  media    

 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
5.  (10pts)  What  is  the  purpose  of  spore  formation  in  fungi?  
 
 
 
 
 
 
 
 
 
 
 
6.  (10pts)  Fungi  used  different  mechanisms  to  enhance  spore  dispersal.    Describe  two  of  those  
mechanisms.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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7.  (10pts)  Based  on  your  experiment,  which  condition(s)  trigger  formation  of  spores?  Why?    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 8.  (10pts)  What  would  happen  if  the  spores  were  transferred  to  a  rich-­‐media  plate?    
 
 
 
 
 
 
 
 
 
 
9.  (10pts)  Which  of  the  strains  you  examined  was  haploid  and  which  one  was  diploid?  How  do  
you  know?    
 
 
 
 
 
 
 
 
 
 
 
 
 
References  (5pts)  
 
 
 
 
   

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Lab  8:  Animal  Kingdom:  Sponges,  Cnidarians  and  Bilaterians.    

           
All  animals  are  multicellular  heterotrophs.  
Their  anatomy  varies  from  very  simple  
(sponges)  to  very  complex  (fish,  humans).  
The  phylogenetic  tree  on  the  right  shows  
the  increasing  animal  complexity  from  
choanoflagellates  (closest  protists  relatives)  
to  animals  with  bilateral  symmetry.    

                           

Before  coming  to  the  lab,  write  the  purpose  of  the  lab  activities.  Make  a  list  of  the  species  
that  you  will  observe.  Write  any  protocol  that  you  will  use.  
 
PART  I    
Sponges  
The  sponges  are  an  extremely  simple  form  of  animal  life.    Their  bodies  are  NOT  composed  of  
tissue   layers   but   of   three   cell   types:   epithelial,   amoebocyte,   and   collar   cells,     each   with  
specific  tasks  to  perform.    The  general  body  plan  of  a  sponge  is  that  of  a  sac  with  many  small  
pores   (ostia)   in   the   walls   and   a   large   pore   (osculum)   at   the   top.     Water   passes   through   the  
central   cavity   (spongocoel),   through   the   ostia   and   out   through   the   osculum.     The   epithelial  
cells  are  found  on  the  outer  surface,  the  collar  cells  line  the  spongocel  and  the  amoebocytes  
are  located  between  the  epithelial  and  collar  cell  layers.      
 
A  cross-­‐section  through  a  sponge  reveals  that  each  ostium  opens  into  an  incurrent  canal  that  
is  lined  with  epithelial  cells.  Tiny  pores  connect  the  incurrent  canal  with  the  spongocoel.  You  
may   also   be   able   to   see   the   spicules   mingled   with   the   amoebocytes.   The   spicules   are   spiky,  
star-­‐shaped  structures  that  make  up  the  skeleton  on  the  sponge.    
 
1.  Examine  a  microscope  slide  of  the  sponge  Grantia  
• Draw  a  longitudinal-­‐section  of  Grantia  labeling  the  cells  (epithelial,  amoebocyte,  and  
collar  cells),  and  the  spicules.  
•  Trace  the  path  of  water  into,  through  and  out  of  the  sponge.  D1  
 
Cnidarians  
Cnidarians   are   also   called   the   coelenterates   and   include   familiar   animals   such   as   the   Hydra,  
jellyfish  and  corals.  Members  of  this  phylum  are  slightly  more  complex  than  the  sponges.  They  
have  two  true  tissues:  an  outer  ectoderm  and  an  inner  endoderm  separated  my  non-­‐cellular  
mesoglea.  Cnidarians  are  also  characterized  by  polymorphism,  or  the  existence  of  two  forms.  
One   form   is   the   sessile   polyp   and   the   other   is   the   swimming   medusa.   The   presence   of  
cnidocytes,  or  special  stinging  cells,  in  the  epidermis  of  the  tentacles  is  typical  of  these  animals  

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also.  Stimulation  of  the  cnidocyte  triggers  the  release  of  a  threadlike  stinger  (nematocyst)  that  
aids  in  the  capture  of  prey.    
 
Class  Hydrozoa  –  the  polyp  is  the  dominant  form  of  the  members  of  this  class.    
2.  Observation  of  Living  Hydra  
• Using   a   dropper,   very   carefully   transfer   a   Hydra   from   the   stock   bottle   to   a   watch   glass  
containing   several   drops   of   spring   water.   It   is   important   to  avoid   the   use   of   tap   water  
for  aquatic  organisms  because  the  chlorine  in  the  water  will  kill  then.  
• Examine  the  Hydra  with  the  dissecting  microscope.  Note  that  the  animal  attaches  to  
the  dish  by  its  base,  with  the  body  extending  upward  to  the  mouth.  The  mouth  is  
surrounded  by  tentacles.    
• Make   a   labeled   drawing   of   the   living   Hydra.   These   animals   reproduce   asexually   by  
budding.  If  there  are  any  buds  on  the  animal  include  them  in  the  drawings.    
How  many  tentacles  are  there  and  what  is  their  function?    D2  
 
Make  note  of  the  Hydra’s  behavior  and  record  all  of  your  observations  in  your  worksheet.    
• Touch  -­‐  Describe  what  happens  when  you  touch  the  Hydra  (gently)  with  a  toothpick.  
What  happens  when  you  shake  the  watch  glass?  
• Movement-­‐  How  does  the  Hydra  move?    
o Look  for  tumbling  or  inchworm  movements.  Use  the  microscope  lamp  to  
determine  whether  the  Hydra  moves  toward  or  away  from  the  strong  light.    
o What  happens  if  you  change  the  intensity  of  the  light?  
• Feeding  some  Daphnia  near  the  mouth  of  your  Hydra  and  record  what  happens.  
 
3.   Observation   of   Extrusion   of   Nematocysts   –   since   this   procedure   kills   the   animals,   do   not  
perform  it  until  you  have  completed  all  the  previously  described  exercises.    
 
• Transfer  the  Hydra  to  a  drop  of  water  on  a  slide  and  cover  it  with  a  cover  slip.    
• Observe   under   low,   then   high   power   to   locate   the   wart-­‐like   clumps   of   cnidocytes   in  
the  tentacles.    
• Without   removing   the   slide   from   the   microscope,   place   a   drop   of   safranin   stain   at   the  
edge  of  the  cover  slip.  As  the  stain  seeps  under  the  cover  slip  it  will  touch  the  Hydra  
and  stimulate  the  release  of  the  nematocysts  from  the  cnidocytes.    
• Write  your  observations  in  the  notebook.  
 
Bilaterians  
This  part  of  the  laboratory  exercise  is  designed  to  introduce  you  to  living  and  parasitic  forms    
of  “flatworms”    
 
Platyhelminthes  
Members   of   this   phylum   exhibit   bilateral   symmetry,   a   characteristic   found   in   most   active  
animals.   The   flatworms   are   composed   of   three   true   tissue   layers,   the   ectoderm,   mesoderm  
and   endoderm.   There   is   no   body   cavity   hence   these   organisms   are   called   acoelomates.   The  
tissues   of   the   flatworms   are   organized   into   organs   and   organ   systems   that   perform   specific  
functions.   A   single   opening   in   the   gut   serves   the   purpose   of   both   mouth   and   anus;  
consequently  the  flat  worms  possess  a  two-­‐way  digestive  tract.  
Some  flatworms  are  free-­‐living  and  others  are  parasitic.    

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Class  Turbellaria  –  free-­‐living  flatworms  

4.  Observation  of  Living  Planaria  
• Obtain  a  living  Planaria  and  transfer  it  with  a  drop  of  spring  water  (no  tap  water!!)  to  a  
watch  glass.  
• Observe   the   morphology   and   movement   of   the   flatworm   as   it   swims   about   in   the  
water.    
• Locate  the  eyes  and  the  two  lobe-­‐shaped  auricles  on  either  side  of  the  head.  Identify  
the  dorsal  and  ventral  surfaces  of  the  worm.  Note  the  pigment  on  the  dorsal  surface.    
• Locate   the   pharynx   (a   long   tube   extending   from   the   ventral   surface)   and   the  mouth   at  
the  free  end.  
• Behavior  –  note  how  the  Planaria  moves,  what  happens  if  you:  
o Turn  it  over  onto  its  dorsal  side  
o Poke  it  gently  with  a  toothpick  
o Give   it   a   choice   of   lighted   or   shaded   area   (Cover   half   of   the   area   with  
aluminum  foil  and  illuminate  it  with  the  microscope  lamp).    
o Record  your  observations  in  your  notebook  
 
5.  Examine  a  microscope  slide  of  Planaria    
• Examine   a   prepared   slide   of   the   Planaria   (whole-­‐mount).   Note   any   features   that   you  
may  have  missed  in  the  living  specimen.  
D-­‐3  Make  drawings  of  both  living  and  preserved  specimens  
In  the  drawing  of  the  preserved  Planaria,  label  auricles,  dorsal  and  ventral  surfaces,  mouth,  
pharynx.  
 
  Class  Cestoidea  –  parasitic  forms  
6.    Examine  a  prepared  slide  of  the  tapeworm  Taenia  pisiformis.    
This   worm   is   parasite   that   has   no   digestive   system   of   its   own.   It   attaches   itself   to   the   inner  
wall  of  the  digestive  tract  of  the  host  animal  (humans  and  other  vertebrates)  and  absorbs  
nutrients  through  its  body  wall.  
 
• Locate  the  scolex  or  head  region.    It  is  equipped  with  suckers  (how  many?)  and  hooks  
which  allow  the  worm  to  attach  itself  to  the  host.  
• Just   posterior   to   the   scolex   find   the   proglottids   or   segments   that   are   concerned   with  
reproduction.   Notice   that   the   proglottids   nearest   the   scolex   are   the   smallest   and   most  
recently   formed.   As   the   proglottids   mature,   they   are   pushed   posteriorly   by   newly  
forming   proglottids.   Mature   segments   towards   the   mid-­‐section   of   the   worm   are   the  
characterized   by   a   lateral   genital   pore   that   leads   anteriorly   to   the   sperm   duct   and  
posteriorly  to  the  vagina.    
• The   most   posterior   proglottids   are   the   largest   and   are   filled   with   the   fertilized   eggs  
(gravid).  The  gravid  segments  fall  off  and  pass  out  of  the  host’s  feces.    
D4.   Prepare   three   drawings   of   the   tapeworm,   one   of   the   scolex,   one   of   the   mature  
proglottid  and  one  of  a  gravid  (ripe)  proglottid.  Label  all  the  observed  structures.  
 
 
 
 
 

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PART  II  
Design  an  experiment  on  Planaria  Regeneration  
 
In   this   part   of   the   lab,   you   will   set   up   an   experiment   that   will   illustrate   the   powers   of  
regeneration  in  flatworms.  Flatworms  are  fresh  water  animals  and  generally  do  well  in  spring  
or  pond  water  but  will  die  in  chlorinated  tap  water.  Be  sure  that  you  use  spring  water  for  all  
phases   of   this   exercise,   DO   NOT   USE   TAP   WATER!!   If   no   spring   water   is   available   in   the   lab,  
notify  the  lab  instructor.  
 
Planaria  are  used  as  a  model  organism  by  researchers  studying  stem  cells,  because  of  their  
ability  to  regenerate.  Despite  their  simple  appearance,  these  organisms  are  quite  complex  and  
capable  of  regenerating  a  wide  range  of  tissue  structures.  The  only  dividing  cell  in  Planaria  is  
the  neoblast.  This  means  that  the  neoblast  has  the  capability  of  differentiate  into  any  cell  
type.    

Students  working  in  groups  of  two  will  set  up  an  experiment  to  try  to  answer  the  following  
questions:    
Is  there  any  difference  in  the  regenerating  power  between  the  head  and  the  tail  sections?  
 
Each  pair  of  students  will  receive  4  Planaria.  Cut  them  in  half  across  the  mid-­‐section  and  care  
for  the  sections  during  the  period  of  regeneration.  The  experiment  will  be  followed  for  three  
weeks.  Water  needs  to  be  changed  EVERY  WEEK.  
Write  down  in  your  notebook  your  hypothesis  and  predictions.    
 
Procedure:    
1. Obtain  4  Planaria  by  gently  sucking  them  up  in  a  medicine  dropper  and  transferring  them  
to  spring  water  in  a  Petri  dish.  Make  a  drawing  of  one  intact  flatworm.  
2. Transfer   one   flatworm   to   a   piece   of   moistened   paper   towel   and   when   it   has   stretched   out  
to  full  length,  use  a  clean  razor  blade  to  make  a  transverse  cut  about  halfway  between  the  
head  and  the  tail.  
 

 
                                                             Figure  1:  Illustration  of  flatworm  before  and  after  cutting  
 
3. Transfer   the   head   to   a   cup   of   spring   water  labeled   “Heads”  and   the   tail   to   a   similar   cup  
labeled  “Tails”.  The  cups  should  be  about  one  third  full  of  SPRING  WATER.    
4. Repeat  steps  2  and  3  for  the  remaining  flatworms,  collecting  all  3  heads  in  one  cup  and  all  
3  tails  in  another.    
5. Planaria  should  be  KEPT  IN  THE  DARK.  

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6. For  the  fourth  Planaria  you  should  make  a  cut  of  your  choosing.    
7. Observations:  
• Make  a  drawing  of  the  severed  head  and  tail  sections.  
• Every   week   look   at   the   segments   and   record   your   observations   in   written   form   and   as  
drawings.   Be   sure   to   record   the   date   and   number   of   animals   exhibiting   particular  
changes.  
• After   you   have   made   your   observations,   change   the   spring   water   by   pouring   off   the  
old  and  adding  new  up  to  the  original  level.  
• After   a   week,   observe   the   head   and   tail   fragments   under   the   dissecting   microscope.  
Does  the  tail  develop  eyes?  Make  drawings  and  take  notes  of  your  observations    
• REPEAT  THE  OBSERVATIONS  DURING  WEEKS  2  and  3.    
• These  data  will  be  the  base  of  the  last  lab  report  
 
Lab  Report  3:  Planaria  Regeneration  Experiment  
Guidelines  
Introduction    (Total:  25pts)  
(Total  1.5  pages  double-­‐spaced)  
Context    
Introduce  the  reader  to  the  subject  matter  
Some  suggestions:  What  is  regeneration?  What  are  stem  cells?  What  animals  can  undergo  
regeneration?  Do  humans  can  regenerate  parts  of  their  bodies?  Why  are  Planaria  used  as  an  
experimental  system  to  study  regeneration?  Do  they  have  genes  in  common  with  humans  
important  for  regeneration?    
Outline  your  experiments  in  your  last  paragraph:  be  brief!!  
1)  Describe  what  you  were  interested  in  determining      
2)  Describe  how  the  experiment  was  done  and  what  the  results  were  (briefly!)  
3)  Describe  your  hypothesis/predictions    
 
Materials  and  Methods  (Total:  10pts)  
Do  not  copy  the  handout  instructions.  Write  a  paragraph  (in  past  tense)  describing  how  the  
experiment  was  done,  including  in  your  description  all  the  materials  used  (do  not  list  them  
separately).  Describe  what  conditions  are  kept  constant  during  the  experiment.  
 
Results  (Total:  35pts)    
This  section  should  be  written  in  paragraph  format  and  include  a  figure    
 
o Start   with   a   brief   description   of   the   purpose   of   the   experiments   (rationale,   why   the  
experiments  were  done)  and  how  they  were  done.    
o Briefly  describe  the  experiment  without  repeating  experimental  details  already  
described  in  the  Materials  and  Methods  section.  
o Finish  with  ONE  sentence  with  the  conclusion  of  the  experiment.  Then,  in  the  
Discussion  section  the  conclusion  will  be  expanded  in  a  more  general  context.  
o Include  a  Figure  with  drawings  showing  changes  observed    in  weeks  1  and  3.  The  
figure  should  be  numbered,  have  a  title,  and  a  legend.  
 
Conclusions  and  Discussion  (Total:  25pts)    
DO  NOT  repeat  the  information  presented  in  the  Results  section.  Analyze  your  data.      

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Was  your  hypothesis  supported  or  rejected?  Was  the  degree  of  regeneration  in  the  heads  and  
tails  the  same?  Why  or  why  not?    Why  genes  are  important  in  planaria  regeneration?  Explain  
the   importance   of   understanding   regeneration.   If   applicable,   discuss   any   possible   sources   of  
error  in  your  experiment.  What  other  factors  might  have  influenced  your  results  What  would  
your  next  experiment  be  to  better  understand  the  phenomenon  further?  (3pts)  
 
References  (Total  5pts)  
Include  at  least  three  references  (make  sure  to  use  at  least  one  book  and  one  primary  article)  
List  the  references  at  the  end  of  the  paper  but  also  include  them  in  the  text.    
For  more  detailed  information  referred  to  instructions  outlined  in  Lab  Report  #1.  
 
 
   

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Lab  Report  #3:  Planaria  Regeneration  Experiment  

 
 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  
 
 
YELLOW  PAGES  OF  THE  NOTEBOOK  INCLUDED_____  
   

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Worksheet  8:  Animal  Kingdom  I  

PART  I:  Sponges,  Cnidarians,  and  Bilaterians.  
 
 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  

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PART  I  
SPONGES  
Q1.  What  are  the  functions  of  epithelial,  amoebocyte,  and  collar  cells  in  sponges?  (9pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
D1.  Draw  a  longitudinal-­‐section  of  Grantia  labeling  epithelial,  amoebocyte,  and  collar  cells  and  
tracing  the  path  of  water  into,  through  and  out  of  the  sponge.  Include  labels.  (6pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Q2.   What   do   sponges   eat?   Do   they   have   intracellular   or   extracellular   digestion?   Does   this  
limit  the  type  of  food  they  can  ingest  and  digest?  (5pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
CNIDARIANS  
Q3.  Explain  what  are  the  main  differences  between  sponges  and  cnidarians?  (5pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
             D2.  Make  a  labeled  drawing  of  the  living  Hydra  (5pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Q4.  How  many  tentacles  are  there  and  what  is  their  function?  (2pts)  
 
 
 
 
 
Q5.  Describe  your  observations  of  Hydra’s  behavior:  Touch,  Movement,  Feeding.  (7.5pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Q6.  What  role  is  played  by  the  stinging  cells?  (2.5pts)  
 
 
 
 
 
 
 
 
 
 
 
 

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Q7.   What   is   the   function   of   epidermis,   gastrodermis,   and   mesoglea   in   the   hydra’s   body?  
(9pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Q8.   What   are   the   differences   and   similarities   between   the   skeletons   of   the   sponges   and  
those  of  corals  (cnidarians)?  (5pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
Q9.  Flatworms  have  bilateral  symmetry  and  as  all  bilaterally  symmetrical  animals  are  
tripoblastic.    What  does  it  mean  that  they  are  tripoblastic?  Explain.  (7.5pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Platyhelminthes  
D3.  Make  a  drawing  of  living  and  preserved  Planaria.  (7.5pts)  
In  the  drawing  of  the  preserved   Planaria,  label  auricles,  dorsal  and  ventral  surfaces,  mouth,  
and  pharynx.  
 
                   Living  Planaria                        Preserved   Planaria  
       
 
 
 
 

 
 
 
 
 
 
Q10.  Describe  your  observations  of    
 
Planaria’s  behavior  (7.5pts)  
What  happened  if  you:    
• Turn  it  over  onto  its  dorsal  side?    
• Poke  it  gently  with  a  toothpick?    
• Give  it  a  choice  of  lighted  or  shaded  area?    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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D4.  Taenia  pisiformis    (10pts)  
 
                                 Scolex                                                                                                                                            Mature  proglottid/gravid  proglottid  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Q-­‐11  How  many  suckers  and  hooks  allow  the  worm  to  attach  itself  to  the  host?  (2.5pts)  
 
 
 
 
 
 
Q-­‐12   Explain   how   tapeworms   can   survive   without   a   coelom,   a   mouth,   a   digestive   system,   or  
an  excretory  system.  (5pts)  
 
 
 
 
 
 
 
 
 
 
 
 
 
References.  (5pts)  
 

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Lab  9  Animal  Kingdom  II:  Insect  Life  Cycle  

         
 
Background  
The  painted  lady  butterfly  (Vanessa  cardui)  provides  an  example  of  the  complete  metamorphic  
life  cycle  typical  of  most  (90  %)  insects.  You  will  have  the  opportunity  to  follow  the  entire  
sequence  of  events  from  larva  to  egg  laying  adults.  The  painted  lady  butterfly  is  found  
throughout  the  world.  There  is  no  danger  of  upsetting  the  ecological  balance  by  releasing  these  
insects  in  New  England  because  they  are  quite  common  locally.  It  is  a  migratory  species  that  
lives  for  about  two  weeks.  After  hatching  from  a  pale  green  egg,  the  larva  begins  feeding  on  
the  leaves  of  specific  plants  (mallow  or  hollyhock).  As  it  eats  and  grows  it  will  build  a  webbed  
nest  from  the  leaves.  This  is  a  period  of  rapid  and  impressive  growth  during  which  the  
caterpillar  goes  through  five  molts.  When  large  enough  the  larva  hangs  upside  down  and  
prepares  to  pupate

At  this  time  the  quiescent  larva  develops  

into  a  chrysalis  (pupa).  After  24  hours,  the  
caterpillar’s  skin  splits  and  the  chrysalis,  
which  has  formed  under  the  skin,  wiggles  
free.  Within  about  4  hours  the  chrysalis  
hardens.  The  adult  butterfly  emerges  in  7  
to  10  days.  After  emergence,  the  adult  
must  expand  its  wings  and  allow  them  to  
harden  for  a  couple  of  hours  before  flying.  
Shortly  thereafter  males  and  females  match  
up  and  mate  and  the  females  complete  the  
cycle  by  laying  eggs.    
 

.  

 
 
 
 
 
 
 
 
 
 

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Procedure:  
1.    From  the  lab  instructor  obtain  a  clear  plastic  vial  with  lid  and  a  small  square  of  tissue  paper.  
2.    After  carefully  washing  your  hands,  pack  about  2  cm  of  culture  medium  (malva  is  a  mixture  
of  ground  up  mallow  leaves  and  other  nutrients  that  caterpillars  like)  into  the  bottom  of  the  
plastic  vial.  Use  your  finger  to  make  a  smooth  surface  of  the  medium.  As  the  caterpillars  eat,  
they  will  grow,  molt  and  produce  pellets  of  fecal  matter  (frass).  Each  time  they  molt  they  ingest  
their  own  skin  but  not  the  head  capsule.  If  you  can  locate  the  head  capsules  on  the  bottom  of  
the  vial  and  count  them  you  will  be  able  to  keep  a  record  of  the  number  of  molts.  
3.  With  a  camel’s  hair  brush  very  gently  transfer  two  larvae  to  the  medium  in  the  vial.  Be  very  
careful  in  the  transfer  because  the  larvae  are  easily  injured.  This  is  sufficient  food  to  support  
growth  of  the  larvae  to  the  chrysalis  stage.    
4.  Cover  the  vial  with  a  small  square  of  tissue  paper  and  then  put  the  cap  on  the  vial.  
5.  Put  your  initials  and  today’s  date  on  the  vial.    
6.  Measure  the  length  and  width  of  each  of  the  caterpillars  and  record  the  measurements  in  
your  lab  notebook.  
7.  Place  the  vial  in  a  well-­‐lighted  area  where  the  temperature  remains  between  22  and  25º  C  
(72  and  77º  F).  DO  NOT  set  the  vial  in  direct  sunlight.      
8.  When  larval  growth  is  complete  (5-­‐10  days)  it  will  hang,  head  down,  from  the  tissue  paper  
under  the  lid.  When  this  happens  the  larva  is  preparing  to  form  a  chrysalis  and  should  not  be  
disturbed  for  1  or  2  days.  An  indication  that  the  transition  from  larva  to  chrysalis  is  about  to  
occur  is  the  curling  of  the  larva  into  the  J-­‐shape  (see  diagrams).  If  you  watch  closely  you  should  
be  able  to  see  and  record  all  of  the  stages  of  the  transformation.  Although  the  chrysalis  appears  
to  be  non-­‐  living  it  is  undergoing  tremendous  changes  inside  (metamorphosis)  and  may  wiggle  
or  spin  from  time  to  time.    
Note:  It  is  very  important  that  you  not  leave  the  chrysalis  in  the  vial  beyond  this  point  
because  if  the  butterfly  emerges  from  the  chrysalis  while  it  is  still  in  the  vial  it  will  not  be  able  
to  expand  its  wings  and  will  be  unable  to  fly  
 
9.  At  this  point  transfer  the  paper  disc  (with  attached  chrysalis)  to  the  flight  cage  where  the  final  
stages  will  be  completed.  Emergence  of  the  adult  is  quite  spectacular  and  catching  the  event  
takes  a  considerable  amount  of  patience.  When  the  adult  butterfly  emerges  from  its  chrysalis  
(usually  in  the  morning)  it  moves  slowly  and  the  first  obvious  event  is  the  expansion  of  the  
wings.  Often  some  red  liquid  oozes  out  of  the  tail  end  of  the  butterfly  after  emergence;  this  is  a  
normal  event  and  this  waste  liquid  is  called  meconium.  
 
While  the  adults  are  in  the  flight  cage  we  will  feed  them  with  a  5  %  sugar  solution.    Within  a  few  
days  the  male  and  female  butterflies  will  begin  to  pair  off  and  mate.  The  male  positions  himself  
next  to  the  female  and  curls  his  abdomen  to  attach  the  tip  of  his  abdomen  to  the  tip  of  hers  and  
deposit  the  sperm.  Shortly  thereafter,  if  host  plants  are  available,  the  female  will  deposit  
fertilized  eggs  on  the  leaves  of  the  plant.  Within  3-­‐4  days  tiny  larvae  will  begin  to  emerge  from  
the  eggs  and  the  life  cycle  will  be  complete.  At  this  point  we  will  release  the  adult  butterflies.  
 
 
 
 
 
 
 

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Activity:  
Your  task  is  to  make  daily  (sometimes  hourly)  observations  in  order  to  follow  the  larvae  
to  the  adult  stage.    
• Keep  a  complete  record  of  measurements  and  drawings  of  all  the  changes  in  both  
animals.  Since  they  will  not  be  coordinated  in  their  development,  if  you  miss  a  stage  or  
event  in  one  you  should  be  able  to  catch  it  in  the  other.    
• Keep  track  of  how  long  each  stage  lasts  and  make  drawings  of  significant  changes.  
 
 
• The  Worksheet  should  be  in  the  form  of  a  dated  log  or  diary  that  keeps  accurate  track  
of  the  all  of  the  changes  in  the  animal  from  the  beginning  to  the  end  of  its  life  cycle.  
This  should  include  drawings,  measurements  and  observations.  (70pts)  
• Include  some  research  about  the  role  of  the  endocrine  system  in  growth  and  
development  of  insects.  Explain  how  molting  and  metamorphosis  are  regulated.  
Include  your  references(  20pts)  
• Go  to  PubMed  or  Google  Scholar  and  look  for  a  recent  scientific  paper  (no  more  than  
5  years  old)  addressing  some  interesting  question  about  insects’  development.  Briefly  
summarize  the  question  that  the  authors  wanted  to  address  and  the  main  
conclusions.  Include  complete  reference:    title  of  the  article,  authors  and  journal  
(name,  year,  volume,  pages)  (10pts)  
• Include  cover  page    
 
 
 

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Worksheet  10.  Insect  Life  Cycle  

 
 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  
 
 
 
 
 
 
   

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References:  
 
Campbell,  N.  A  and  Reece  J.  B.  Biology,  Ninth  Edition.  The  Pearson-­‐Benjamin/Cummings  
Publishing  Company,  New  York.  2011.  
 
Morris  J.R,  Hartl  D.L,  Knoll  A.H,  Lue  R.A,  Berry  A,  Biewener  A,Farrell  B,  Holbroo,  N.M,  Pierce  N,  
and  Viel,  A.  Biology:  How  Life  Works,  First  Edition.  MacMillan  W.H.Freeman  and  Company,  
New  York.  2013.  
 
AlbertsB;  Bray  D,  Hopkin  K,  Johnson  A,  Lewis  J,  Raff  M,  Rober  K.  Essential  Cell  Biology,  Fourth  
Edition.  Garland  Science.  2013  
 
Strete  and  Vodopich.  Photo  Atlas  for  General  Biology.  McGraw-­‐Hill,  New  York.  2002.      
 
   

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Lab  10  Myosin  Comparative  Proteomics    

Myosin  Western  Blot  Experiment  
 
Before  coming  to  the  lab,  write  in  your  notebook:    
• Goal/purpose  of  lab  activity    
• Make  a  flow  chart  indicating  what  will  be  the  main  activities  in  weeks  1,  2  and  3  
• Write  the  protocol  corresponding  to:  Preparation  of  protein  extracts  for  SDS-­‐PAGE  
analysis.  
BRING  YOUR  COMPUTER  TO  THE  LAB    
 
Background  

Myosin  is  a  motor  protein  that  is  important  for  muscle  contraction  in  all  animal  cells.  In  this  
set  of  labs,  you  will  examine  the  myosin  light  chain  protein  from  different  fish  samples  to  get  a  
sense  of  the  relatedness  of  these  organisms  to  each  other  and  to  birds.  To  do  this,  you  will  use  
SDS-­‐PAGE  (polyacrylamide  gel  electrophoresis)  and  western  blot  analysis.  
 
SDS-­‐PAGE  allows  proteins  to  be  separated  by  size.  You  will  prepare  protein  extracts  from  
different  fish  samples  and  analyze  the  proteins  contained  in  these  samples  by  western  
blotting.  In  this  technique,  proteins  separated  by  SDS-­‐PAGE  are  transferred  to  a  nitrocellulose  
membrane,  which  is  then  probed  with  an  antibody  that  is  specific  to  the  protein  of  interest  (in  
our  case,  the  myosin  light  chain  protein).  Since  this  antibody  was  made  using  the  myosin  light  
chain  from  chickens,  only  fish  proteins  that  are  similar  to  chicken  myosin  light  chain  will  be  
identified  using  this  antibody.  This  procedure  will  allow  you  to  determine  1)  whether  a  protein  
similar  to  the  chicken  myosin  light  chain  is  present  in  each  fish  sample;  and  2)  the  size  of  the  
myosin  light  chain  protein  in  each  fish.

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Muscle Proteins
All animal activity is dependent upon muscle proteins. From swimming and running to
breathing and digestion, all movement is driven by interactions between specialized proteins
comprising muscle fibers. Illustrated below are the basic contractile elements that comprise
animal muscle cells. Functional units called “myofibrils” are bundled to form muscle fibers.
Each myofibril consists of a linear series of contractile units called "sarcomeres".

STUDENT MANUAL
BACKGROUND

Fig. 6. Telescopic view of muscle structure: Thick myosin filaments and thin actin filaments form myofibrils, which are
bundled together to make muscle fibers. (Figure modified from Campbell 1996 with permission.)

Student Manual
35

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 BIOL1106    

Fig. 7. Hydrolysis of ATP causes myosin and actin filaments to slide past one another, shortening the sarcomere and
contracting the muscle. (Figure modified from Campbell 1996 with permission.)

Western
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quantify filaments
a single proteininteract with thin
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accuracy.
This procedure will be performed in this laboratory! the neck of each myosin globular head region and
help to regulate the contraction of the myosin protein.
This procedure
Western blotting can will categorically
be performed in this
identify laboratory!
a specific protein among hundreds or thousands
of other
Western proteins
The antibodyblotting within
in this biological
can experiment
categorically samples.
specifically This
identify a binds surefire
specific themethod
to protein myosin
amongof identifying
light chain proteins.
hundreds proteins
or thousandsis
STUDENT MANUAL
BACKGROUND

based on two distinguishing features of proteins: molecular

of other proteins within biological samples. This surefire method of identifying proteins is mass and antibody binding
specificity.
based on two Bioscience researchers
distinguishing features useofwestern
proteins:blotting
molecular to identify
mass proteins,
and antibody quantifybindingprotein
expression levels, and determine whether proteins have undergone
specificity. Bioscience researchers use western blotting to identify proteins, quantify protein posttranslational
modification.
expression levels, Because anditdetermine
is so accurate, whether western
proteins blotting
haveisundergone
the methodposttranslational
of choice ATP used to
confirm positive test results for HIV, lupus, or bovine spongiform
modification. Because it is so accurate, western blotting is the method of choice used encephalopathy (BSE;to
mad cow disease).
confirm positive test results for HIV, lupus, or bovine spongiform encephalopathy (BSE;
Myosin light chain 1
mad lab
This cowmoves
disease). beyond DNA and into the new frontier of proteomics to explore evolution at
Myosin light chain 2
the molecular level. You will generate protein fingerprint profiles from distantly and closely
This lab moves beyond DNA and into the new frontier of proteomics to explore evolution at
related species of fish and use western blotting to test the hypothesis that proteins can be
the molecular level. You will generate protein fingerprint profiles from distantly and closely
indicators of genetic and evolutionary relatedness. Myosin is a major muscle protein
related species of fish and use western blotting to test the hypothesis that proteins can be
essential for locomotion and survival in all animals. As such, the essential structure and
indicators of genetic and evolutionary relatedness. Myosin is a major muscleMyosin protein
function of myosin has remained relatively stable or “conserved" in all animals over
heavy chain
essential for locomotion and survival in all animals. As such, the essential structure and
evolutionary time.
function of myosin has remained relatively stable or “conserved" in all animals over
STUDENT
BACKGROUND

evolutionary
Protein time.
gel electrophoresis and western blotting will be used to specifically identify a subunit
of a myosin light chain from the many thousands of proteins comprising the muscle tissues
STUDENT
BACKGROUND

Protein gel electrophoresis and western blotting will be used to specifically identify a subunit
of different fish. Myosin light chain proteins will be compared from different species for
ofFig.
a 8.
myosin light chain from the many thousands of proteins comprising the muscle tissues
variation, commonality,
Myosin protein structure.or evolutionary divergence.
of different fish. Myosin light chain proteins will be compared from different species for
Are
Thethere
variation, discernible
commonality, differences
orcontains between
evolutionary the site
myosin
divergence. and proteins extracteddomain.from the species
MANUAL

myosin head region a catalytic an actin-binding The head

you are
region investigating?
binds to actin What
and are
flexes they?
at the How
neck might
region these
to pull variations
the ends occur,
of the and
sarcomere why? How
together.
Are there discernible differences between the myosin proteins extracted from the species
MANUAL

might
Myosin variations
obtains in themyosin
energy between species be used to determine theirconversion
evolutionary
you are investigating? Whatfor aremuscle
they? How contraction
might thesethrough enzymatic
variations occur, and why? of How
relationships?
adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The combined
might variations in myosin between species be used to determine their evolutionary
mini-contractions of the countless sarcomeres composing a muscle fiber causes the
relationships?
macro-contraction of the entire muscle.
Student Manual
36

127
BIOL1106    

Sample Preparation

Sample Preparation
In order to study a particular muscle protein, muscle tissue must first be broken down to
release proteins from within the cells and the proteins must be denatured to their linear
In order to study a particular muscle protein, muscle tissue must first be broken down to
forms. This is because linear molecules move through the pores of a sieving gel matrix
release proteins from within the cells and the proteins must be denatured to their linear
more efficiently than 3-D ones.
forms. This is because linear molecules move through the pores of a sieving gel matrix
more
Youefficiently
will beginthan
this3-D ones. by extracting proteins from the muscle tissues of different fish
laboratory
species.
You The
will begin cell
this membranes
laboratory of all animals
by extracting proteinsarefrom
composed
the muscle mainly
tissuesof lipid bilayer.fish
of different The lysis
buffer The
species. usedcell
to membranes
break open of orall
lyse the muscle
animals cells contains
are composed mainly the ionic
of lipid detergent
bilayer. sodium
The lysis
dodecyl
buffer usedsulfate
to break (SDS)
open and a strong
or lyse reducing
the muscle cells agent
contains called dithiothreitol
the ionic detergent(DTT).
sodiumSDS
effectively coats all the proteins in the sample with negative
dodecyl sulfate (SDS) and a strong reducing agent called dithiothreitol (DTT). SDS charge and DTT breaks the
disulfide bridges that contribute to protein secondary, tertiary, and
effectively coats all the proteins in the sample with negative charge and DTT breaks the quaternary structure.
disulfide
SDS and bridges
DTTthat
arecontribute
containedtoinprotein
the lysissecondary, tertiary, and
buffer (Laemmli quaternary
sample buffer).structure.
Heating to 95°C
SDS and DTT
further are contained
denatures proteins.in Once
the lysis buffer (Laemmli
extraction sample
is complete, allbuffer). Heating
the proteins into 95°C
the sample are
further denatures
uniformly coatedproteins. Once
with SDS extraction
and is complete,
carry equivalent all the proteins
negative charge in the sample
density. are
SDS-PAGE
uniformly coated with
electrophoresis can SDS
thenand
becarry
usedequivalent
to separate negative
protein charge density.
subunits, SDS-PAGE based on
or polypeptides,
electrophoresis
their sizes. The canLaemmli
then be sample
used to separate
buffer also protein subunits,
contains Tris –ora polypeptides, based ona constant
buffer that maintains
their sizes. The Laemmli sample buffer also contains Tris – a buffer that maintains a constant
pH, glycerol to add density to samples so they sink into the wells when loading the gel, and
pH, glycerol to add density to samples so they sink into the wells when loading the gel, and
the dye Bromophenol Blue, to help visualize sample loading and to allow for tracking protein
the dye Bromophenol Blue, to help visualize sample loading and to allow for tracking protein
migration during electrophoresis.
migration during electrophoresis.
Proteins
Proteins migrate
migrate through
through the sieving
the sieving gel matrix
gel matrix of theof the
gel gel according
according to theirto their
size, size,iswhich is
which
determined by the number and kind of amino acids composing
determined by the number and kind of amino acids composing the primary structure of the primary structure of
each
each polypeptide.
polypeptide. TheThe smaller
smaller the peptide,
the peptide, the more
the more rapidlyrapidly it migrates
it migrates throughthrough
the gel the gel
towards
towards thethe positive
positive electrode;
electrode; larger
larger peptides
peptides take longer
take longer to navigate
to navigate throughthrough
the gel. the gel.
Similarly,
Similarly, denatured
denatured (linear)
(linear) peptides
peptides can
can be be more
more readilyreadily
analyzedanalyzed
via gel via gel electrophoresis
electrophoresis
than
than large
large 3-D3-D complexes
complexes of proteins.
of proteins. The sieving
The sieving properties
properties of mostofgels
mostaregels
not are not capable
capable
of of
separating fullyfully
separating native (non-denatured)
native (non-denatured) protein molecules.
protein molecules.

Fig. 10. The combination of heat and the detergent SDS denatures proteins for SDS-PAGE analysis.
Fig. 10. The combination of heat and the detergent SDS denatures proteins for SDS-PAGE analysis.
STUDENT MANUAL

STUDENT MANUAL
LESSON 1

LESSON 1

Student Manual
41
Student Manual
41

128
 BIOL1106    

STUDENT MANUAL
Lesson 2: Protein Gel Electrophoresis
Separating Proteins Using SDS-PAGE

LESSON 2
In this investigation, polyacrylamide gel electrophoresis (PAGE) is used to separate proteins
from the muscle tissue of different species. Using an electric current, proteins coated in
SDS-containing sample buffer are separated in a sieving gel matrix that separates proteins
by their size. A polyacrylamide gel is positioned in a buffer-filled chamber between two
electrodes and muscle extracts are loaded into wells at the top of the gel. Then the
electrodes are connected to a power supply that generates a voltage gradient from negative
to positive down the gel. The SDS-coated, negatively charged proteins migrate through the
STUDENT MANUAL
LESSON 2

gel toward the positively charged anode with the larger proteins migrating more slowly than
the smaller proteins.

Once the electric current is applied, the SDS-coated proteins begin their race toward the
positive electrode. Smaller proteins move through the gel more quickly than the larger ones
and over time proteins will be separated according to size.
Protein size is quantified in Daltons, a measure of molecular mass. One Dalton is defined
as the mass of a hydrogen atom, which is 1.66 x 10-24 grams (g). Most proteins have masses
of thousands of Daltons, therefore the term kiloDalton (kD) is often used to describe
protein molecular mass. Given that the average mass of an amino acid is 110 Daltons, the
predicted mass of a protein can be approximated from the number of amino acids it contains.
• Average amino acid = 110 Daltons
• Approximate molecular mass of protein = number of amino acids x 110 Daltons

Monitoring Invisible Proteins During Electrophoresis

While it is not possible to visualize the proteins in the muscle extracts while the gel is running,
Precision Plus Protein Kaleidoscope prestained protein standards are designed to be
watched. These genetically engineered proteins have dyes covalently bound to them and
resolve into multi-colored bands that move down the gel during electrophoresis. The blue
tracking dye in the sample buffer can also be used to monitor the progress of the run. The
blue dye is negatively charged and smaller than most known proteins, so it is drawn toward
the positive electrode slightly ahead of the proteins. If the electric current is left on for too
long, the standards, the dye, and the proteins will eventually run off the bottom of the gel.
Keep an eye on the progress of the tracking dye and the protein standards to monitor the
extent of electrophoresis.
6 7
Experimental Controls
There are two types of controls used in this lab. The visible prestained standards are used
to monitor the progress of proteins during the electrophoresis and blotting procedures.
These standards are run through the gel and are transferred along with the unknown samples
during the blotting procedure. The prestained standards are finally used to determine the
molecular weights of the myosin light chain proteins on the western blots.
The molecular weights (sizes) of prestained standard protein sizes are as follows:

Color Size, kD
Fig. 11. ABlue 250 denatured with reducing agents, heat, and SDS, can be separated into individual
quaternary protein complex
Purple
proteins and 150 SDS-PAGE.
resolved by size using
Blue 100
Pink 75 Student Manual
Blue 50 45
Green 37
Pink 25
Blue 20
Blue 15 129
Yellow 10
 electrodes and muscle extracts are loaded into wells at the top of the gel. Then the
electrodes are connected to a power supply that generates a voltage gradient from negative
to positive down the gel. The SDS-coated, negatively charged proteins migrate through the
BIOL1106    
gel toward the positively charged anode with the larger proteins migrating more slowly than
the smaller proteins.

6 7

Fig. 11. A quaternary protein complex denatured with reducing agents, heat, and SDS, can be separated into individual
proteins and resolved by size using SDS-PAGE.

Student Manual
45

130
 tracking dye in the sample buffer can also be used to monitor the progress of the run. The
blue dye is negatively charged and smaller than most known proteins, so it is drawn toward
the positive electrode slightly ahead of the proteins. If the electric current is left on for too
long, the standards, the dye, and the proteins will eventually run off the bottom of the gel.
BIOL1106  
Keep an eye on the progress of the tracking dye  and the protein standards to monitor the
extent of electrophoresis.

Experimental Controls
There are two types of controls used in this lab. The visible prestained standards are used
to monitor the progress of proteins during the electrophoresis and blotting procedures.
These standards are run through the gel and are transferred along with the unknown samples
during the blotting procedure. The prestained standards are finally used to determine the
molecular weights of the myosin light chain proteins on the western blots.
The molecular weights (sizes) of prestained standard protein sizes are as follows:

Color Size, kD
Blue 250
Purple 150
Blue 100
Pink 75
Blue 50
Green 37
Pink 25
Blue 20
Blue 15
Yellow 10

The actin & myosin standard is a mixture of rabbit myofibrils and contains actin, myosin,
tropomyosin, and trace amounts of other muscle filament proteins. The primary antibody in
this kit is designed to detect myosin light chain. This control sample serves as a positive
experimental
Lesson control for Western
3: Perform the immunodetection
Blotting procedure.
Lesson 3: Perform Western Blotting
Overview
Overview of
of Blotting
Blotting
Student Manual
In
In the previous two
the previous two steps,
steps, proteins
proteins werewere extracted
46 from
extracted from muscle
muscle tissue,
tissue, then
then separated
separated
according
according to their sizes via electrophoresis. The rest of the laboratory focuses on
to their sizes via electrophoresis. The rest of the laboratory focuses on using
using
antibodies to identify myosin light chain proteins in the muscle extracts.
antibodies to identify myosin light chain proteins in the muscle extracts. The separated The separated
muscle
muscle proteins
proteins are
are currently
currently embedded
embedded within
within a
a flimsy
flimsy and
and fragile
fragile gel.
gel. To
To probe
probe the
the samples
samples
with
with the myosin-specific antibody, proteins must first be transferred or "blotted" from
the myosin-specific antibody, proteins must first be transferred or "blotted" from within
within
the
the gel
gel onto
onto the
the surface
surface of
of aa membrane.
membrane. A A membrane
membrane is is more
more stable
stable and
and longer
longer lasting
lasting
than
than aa gel
gel and
and proteins
proteins bound
bound to to the
the surface
surface of
of a
a membrane
membrane are are more
more accessible
accessible to to
antibodies. This procedure is called western
antibodies. This procedure is called western blotting. blotting.
Proteins
Proteins are
are electrophoretically
electrophoretically transferred
transferred from
from the
the gel
gel onto
onto a a nitrocellulose
nitrocellulose membrane.
membrane.
Proteins,
Proteins, still negatively charged from the SDS, migrate out of the gel
still negatively charged from the SDS, migrate out of the gel and
and bind
bind to
to the
the surface
surface
of the membrane, creating a mirror image of proteins separated in the original
of the membrane, creating a mirror image of proteins separated in the original gel. gel. MANUAL
STUDENTMANUAL
Once
Once proteins
proteins are
are transferred
transferred to to the
the nitrocellulose
nitrocellulose membrane
membrane (the
(the 'blot'),
'blot'), the
the next
next step
step is
is to
to
probe
probe the
the blot
blot with
with anan antibody
antibody that
that has
has been
been specifically
specifically engineered
engineered toto detect
detect the
the protein
protein
of
of interest.
interest. But
But first,
first, the
the blot
blot must
must bebe incubated
incubated inin a
a protein-rich
protein-rich solution
solution such
such as
as one
one
LESSON33

derived
derived from
from powdered
powdered milk milk protein.
protein. Incubating
Incubating the
the blot
blot with
with milk
milk protein
protein effectively
effectively coats
STUDENT

coats
LESSON

the
the entire
entire surface
surface area
area ofof the
the membrane
membrane wherewhere no
no proteins
proteins have
have been
been blotted
blotted and
and blocks
blocks
nonspecific
nonspecific protein
protein binding
binding sites.
sites.
Next
Next the
the blot
blot is
is incubated
incubated withwith an
an antibody
antibody engineered
engineered toto bind
bind only
only to
to myosin
myosin light
light chain
chain
proteins
proteins (the primary antibody). Following a quick rinse, the membrane is incubated with
(the primary antibody). Following a quick rinse, the membrane is incubated with an
an
enzyme-linked secondary antibody that has been engineered to bind
enzyme-linked secondary antibody that has been engineered to bind specifically to thespecifically to the
primary
primary antibody.
antibody. Finally,
Finally, aa colorless
colorless colorimetric
colorimetric enzyme
enzyme substrate
substrate isis added
added toto the
the
membrane
membrane in solution. The enzyme that is linked to the secondary antibody oxidizes the
in solution. The enzyme that is linked to the secondary antibody oxidizes the
colorimetric
colorimetric substrate
substrate into
into anan insoluble
insoluble colored
colored precipitate,
precipitate, leaving
leaving aa visible
visible deposit
deposit onon the
the
membrane
membrane at at the
the precise
precise location
location of
of the
the blotted
blotted myosin
myosin light
light chain
chain proteins.
proteins.

131
 of the proteins
Once membrane, creating a mirror
are transferred to theimage of proteins
nitrocellulose separated
membrane (thein 'blot'),
the original gel.step is to
the next
probe the blot with an antibody that has been specifically engineered to detect the protein

MA
MANU
Once proteins are transferred to the nitrocellulose membrane (the 'blot'), the next step is to
of interest. But first, the blot must be incubated in a protein-rich solution such as one
probe the blot with an antibody that has been specifically engineered to detect the protein

3
STUDENT
derived from powdered milk protein. Incubating the blot with milk protein effectively coats
of interest. But first, the blot must be incubated in a protein-rich solution such as one
BIOL1106  
the entire surface area of the membrane where no   proteins have been blotted and blocks

LESSON
LESSON 3
STUDENT
derived from powdered milk protein. Incubating the blot with milk protein effectively coats
nonspecific protein binding sites.
the entire surface area of the membrane where no proteins have been blotted and blocks
nonspecific
Next the blotprotein bindingwith
is incubated sites.
an antibody engineered to bind only to myosin light chain
proteins (the primary antibody). Following a quick rinse, the membrane is incubated with an
Next the blot is incubated with an antibody engineered to bind only to myosin light chain
enzyme-linked secondary antibody that has been engineered Lid to bind specifically to the
Lid
proteins (the primary antibody). Following a quick rinse, the membrane
Lid is incubated with an
primary antibody. Finally, a colorless colorimetric enzyme substrate is added to the
enzyme-linked secondary antibody that has been engineered to bind specifically to the
membrane in solution. The enzyme that is linked to the secondary antibody oxidizes the
primary antibody. Finally, a colorless colorimetric enzyme substrate is added to the
colorimetric substrate into an insoluble colored precipitate, leaving
Fiber a visible deposit on the
Fiber pad
pad
membrane in solution. The enzyme that is linked to the secondary Fiber pad
Blotting
antibody
paper
oxidizes the
membrane at the precise location of the blotted myosin light chain Blottingproteins.
paper
colorimetric substrate into an insoluble colored precipitate, leaving
Blotting
Membrane
Membrane
Gel
Membrane
a visible
paper deposit on the
Gel
membrane at the precise location of the blotted myosin light chain Blotting
Gel
Blottingproteins.
paper
paper
Blotting paper Fiber
Fiber pad
STUDENT
LESSON

pad
STUDENT
LESSON

Fiber pad
STUDENTMANUAL
LESSON333

Gel
Gel holder
holder
cassette
Gel holder
cassette
cassette

Electrode
Electrode
Electrode
module Bio-Ice
MANUAL

module Bio-Ice
MANUAL

module Bio-Ice
cooling
cooling
cooling
unit
unit (keep
the (keep
Fig. 12. Overview of Immunodetection on the blot. The membrane is incubated withfrozen
unit primary
(keep
at antibody, followed by
frozen at –20°C)
–20°C)
incubation with the secondary antibody, and lastly the substrate is added. frozen at –20°C)
Fig. 12. Overview of Immunodetection on the blot. The membrane is incubated with the primary antibody, followed by
incubation with the secondary antibody, and lastly the substrate is added.
Western Blot Reagents and Equipment Buffer tank
Buffer tank
Buffer tank

Western
Mini Blot Reagents
Trans-Blot apparatus: andthe Equipment
Mini Trans-Blot is specifically designed to pass electric
current horizontally through
Mini Trans-Blot apparatus: the Mini the gel, forcing
Trans-Blotthe negatively
is specificallycharged proteins
designed to migrate
to pass electricout
of the
The
current gel
Mini onto
horizontallythe
Trans-Blot nitrocellulose
module
through is
the membrane.
designed
gel, forcing to fit into
the the
negatively Mini-PROTEAN
charged proteins 3 gel to electrophoresis
migrate out
The
The Mini
Mini Trans-Blot
Trans-Blot module
module is
is designed
designed to
to fit
fit into
into the
the Mini-PROTEAN
Mini-PROTEAN 3
3 gel
gel electrophoresis
electrophoresis
tank
of and
theand
tank lid.
gel lid. If
ontoIf thea
a Mini Trans-Blot
nitrocellulose
Mini Trans-Blot is not available,
membrane.
is not available, follow
follow the
the alternative
alternative protocol
protocol for transfer-
tankthe
ring andproteins
lid. If a Mini
using Trans-Blot
capillary is not available,
action described follow
in the alternative
Appendix B. protocol for transfer-
for transfer-
ring
ring the
the proteins
proteins usingusing capillary
capillary action
action described
described in in Appendix
Appendix B. B.
Nitrocellulose
Nitrocellulose membranes:
membranes: Nitrocellulose
Nitrocellulose acts
acts as a solid support for proteins bound to its
Nitrocellulose
positively charged membranes:
surface. Nitrocellulose
These durable acts as
membranes as aa solid
solid
can
support
support
undergo
for
for proteins
proteins
multiple
bound
bound
wash and
to
to its
its
positively
positively charged
charged surface.
surface. These
These durable
durable membranes
membranes can
can undergo
undergo multiple
multiple wash
wash and
and
incubation
incubation steps,
steps, and
and provide a white background on which to visualize the color development
incubation
at the site steps,
of the and provide
protein provide
of
a
a white
interestwhite background
background
only. Please
on
on which
avoid which
touching
to
to visualize
visualize
the
the
the color
membrane
development
colorwith
development
ungloved
at
at the
the site
site of
of the
the protein
protein of
of interest
interest only.
only. Please
Please avoid
avoid touching
touching the
the membrane
membrane with
with ungloved
ungloved
hands
hands as
as this
this may
may produce
produce protein-rich
protein-rich fingerprints!
fingerprints! Restrict
Restrict contact
contact with
with the
the membrane
membrane to
to
hands
outer as
edges this or may
use produce
forceps protein-rich
to handle. Eachfingerprints!
white Restrict
nitrocellulose contact
membranewith the ismembrane
packaged to
outer
outer edges
edges or
or use
use forceps
forceps to
to handle.
handle. Each
Each white
white nitrocellulose
nitrocellulose membrane
membrane is
is packaged
packaged
Student Manual
between
between two protective sheets of blue paper.
between two two protective
protective sheets sheets of of blue
blue paper.
paper. 51 Student Manual
Blotting
Blotting paper:
paper: Blotting
Blotting paper
paper is
is used
used to
to support
support the gel and nitrocellulose and to protect
Blotting paper: Blotting paper is used to 51 the
support the gel
gel and
and nitrocellulose
nitrocellulose and
and to
to protect
protect
them
them from
from the
the fiber
fiber pads
pads during
during assembly
assembly and
and electrophoresis.
electrophoresis. The
The blotting
blotting paper
paper also
also
them from
facilitates athe fiber
uniform flow pads during
of assembly
buffer and and
current through electrophoresis.
the The
gel. Blotting blotting
paper paper
is also
made of
facilitates
facilitates a
a uniform
uniform flow
flow of
of buffer
buffer and
and current
current through
through that the
the gel.
gel. Blotting
Blotting paper
paper is
is made
made of
of
100%
100% cotton
cotton fiber
fiber and
and does
does not
not contain
contain any
any additives
additives that may
may interfere
interfere with
with the
the blotting
blotting
100%
process. cotton fiber and does not contain any additives that may interfere with the blotting
process.
process.
Fiber
Fiber pads: Fiber pads press the gel and nitrocellulose together tightly and uniformly,
Fiber pads:
eliminatepads:air
Fiber
Fiber
bubbles,
pads
pads and
press
press
allow
the
the gel
gel and
efficient
nitrocellulose
andtransfer
nitrocellulose
of proteins
together
together
out of
tightly
tightly
the
and
and
gel and
uniformly,
uniformly,
onto the
eliminate
eliminate air
air bubbles,
bubbles, and
and allow
allow efficient
efficient transfer
transfer of
of proteins
proteins out
out of
of the
the gel
gel and
and onto
onto the
theuse
membrane.
membrane. The
The pads
pads must
must be
be thoroughly
thoroughly cleaned
cleaned and
and rinsed
rinsed in
in distilled
distilled water
water before
before use
membrane.
to remove The pads
contaminants. must be thoroughly cleaned and rinsed in distilled water before use
to remove contaminants.
to remove contaminants.
Blotting
Blotting buffer:
buffer: The
The 1x
1x blotting
blotting buffer
buffer is composed of 2.5 mM Tris, 19.2 mM glycine, and
Blotting
20% buffer:
ethanol and The
is pH1x8.3.
blotting
It buffer is
contains is composed
tris composed
to maintain
of 2.5
ofpH, mM
mM Tris,
2.5 glycine Tris, 19.2
19.2
ions to
mM
mM glycine,
glycine,
transmit
and
and
current,
20%
20% ethanol
ethanol and
and is
is pH
pH 8.3.
8.3. It
It contains
contains tris
tris to
to maintain
maintain pH,
pH, glycine
glycine ions
ions to
to transmit
transmit current,
current,
and
and ethanol to facilitate protein binding to the nitrocellulose.
and ethanol
ethanol to to facilitate
facilitate protein
protein binding
binding to to the
the nitrocellulose.
nitrocellulose.
Blocker:
Blocker: This
This solution
solution is
is 5%
5% nonfat
nonfat dried
dried milk
milk powder in phosphate buffered saline (PBS)
Blocker:
and 0.025% ThisTweensolution 20. isAll
5% nonfatarea
surface milk powder
driedunoccupied powder by
in phosphate
inproteins
phosphate buffered
bufferedfrom
transferred
saline
salinethe
(PBS)
(PBS)
gel
and
and 0.025%
0.025% Tween
Tween 20.
20. All
All surface
surface area
area unoccupied
unoccupied by
by proteins
proteins transferred
transferred from
from the
the gel
gel
needs
needs to
to be
be “blocked”
“blocked” prior
prior incubation
incubation with
with the
the primary
primary antibody
antibody by
by incubating
incubating with
with a
a blocking
blocking
needs to be “blocked” prior incubation with the primary antibody by incubating with a blocking
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agent such
agent such as
as this
this milk
milk solution.
solution. Without
Without this
this blocking
blocking step,
step, the
the primary
primary antibody
antibody can
can
randomly adhere to the membrane and obscure or weaken the specific antibody (anti-myosin)
randomly adhere to the membrane and obscure or weaken the specific antibody (anti-myosin)
signal. PBS
signal. PBS (1
(1 mM
mM sodium
sodium phosphate,
phosphate, 1515 mM
mM NaCl,
NaCl, pH
pH 7.4)
7.4) provides
provides the
the ideal
ideal pH
pH and
and salt
salt
conditions for maintaining milk protein binding integrity. Tween 20 is a detergent that
conditions for maintaining milk protein binding integrity. Tween 20 is a detergent that helps helps
keep nonspecifically
keep nonspecifically bound
bound antibody
antibody from
from adhering
adhering to
to the
the membrane.
membrane.

Setting Up
Setting Up for
for Protein
Protein Blotting
Blotting
After running
After running thethe polyacrylamide
polyacrylamide gel, gel, the
the gel
gel must
must bebe equilibrated
equilibrated in in blotting
blotting buffer
buffer to
to
remove excess SDS. Proteins can then be transferred from the gel
remove excess SDS. Proteins can then be transferred from the gel to a protein-binding to a protein-binding
nitrocellulose membrane.
nitrocellulose membrane. The The blot
blot isis set
set up
up as
as a
a sandwich
sandwich in in aa plastic
plastic cassette
cassette partially
partially
submerged in blotting buffer. The figure below illustrates the sandwich
submerged in blotting buffer. The figure below illustrates the sandwich construction construction
consisting of
consisting of a
a fiber
fiber pad
pad at
at the
the bottom
bottom followed
followed sequentially
sequentially by by aa layer
layer of
of blotting
blotting paper,
paper, the
the
gel, the membrane, another layer of blotting paper – and the final fiber pad.
gel, the membrane, another layer of blotting paper – and the final fiber pad. It is imperative It is imperative
that no
that no air
air bubbles
bubbles exist
exist between
between the the blotting
blotting paper,
paper, the
the gel,
gel, oror the
the membrane
membrane since since bubbles
bubbles

MANUAL
STUDENTMANUAL
will prevent proteins from being transferred. After adding each layer to the
will prevent proteins from being transferred. After adding each layer to the sandwich, a rollersandwich, a roller
is used to push out any air bubbles – starting at one end of the membrane/gel/paper
is used to push out any air bubbles – starting at one end of the membrane/gel/paper and and
rolling to the other. The sandwich is then clamped together in the
rolling to the other. The sandwich is then clamped together in the plastic cassette. plastic cassette.

LESSON33
Anode

STUDENT
Anode

LESSON
Gel holder
Gel holder
Fiber pad
Fiber pad
Filter paper
Filter paper

Membrane
Membrane
Gel
Gel
Filter paper
Filter paper
Fiber pad
Fiber pad

Gel holder
Gel holder

INSTRUCTOR'S MANUAL
Cathode
Cathode
Fig. 13.
Fig. 13. Schematic
Schematic of
of western
western blot.
blot. The
The current
current is
is conducted
conducted through
through the
the blotting
blotting buffer
buffer and
and n egatively charged
negatively charged proteins
proteins
migrate from the gel onto the protein binding membrane.
migrate fromcolorimetric
the gel onto the protein binding membrane.
colorless (color-producing) enzyme substrate is added to the membrane in

BACKGROUND
solution. The enzyme that is linked to the secondary antibody oxidizes the colorimetric
It is
It is important
important that
that the
the sandwich
sandwich be be oriented
oriented with
with the
the black
black edge
edge ofof the
the cassette
cassette facing
facing
substrate into an insoluble purple precipitate that leaves visible deposits on the membrane
down. The
down. The cassette
cassette is is then
then submerged
submerged in in blotting
blotting buffer
buffer in
in the
the transfer
transfer tank,
tank, aligning
aligning the
the
at the precise location of the blotted myosin light chain proteins. The combined blotting and
clear plastic
clear plastic side
side toto the
the red
red electrode
electrode and and black
black toto black
black with
with color-coded
color-coded electrodes
electrodes ofof the
the
immunodetection procedure is used to determine the exact position of myosin. The precise
blotting module.
blotting module. This
This orientation
orientation will
will ensure
ensure that
that the
the negative
negative current
current runs
runs from
from the
the gel
gel
molecular mass of myosin can then be determined for each sample by constructing a
toward the
toward the membrane.
membrane. Similarly to to running
running proteins
proteins vertically
vertically through
through the
the gel
gel during
during the
the
standard curve from theSimilarly
Precision Plus Protein Kaleidoscope prestained standards run
electrophoresis, here
electrophoresis, here the
the current
current will
will force
force the
the negatively
negatively charged
charged proteins
proteins horizontally
horizontally out
out
alongside the protein samples in the gel. (Please refer to Appendix D for detailed instructions
of the
of the gel
gel and
and onto
onto the
the surface
surface ofof the
the membrane.
membrane.
on generating standard curves for molecular weight determination of unknown proteins.)
Why is it necessary to transfer the proteins from the gel to the nitrocellulose membrane?
Why can't myosin be detected by applying antibodies directly on the gel? First, since the
proteins are contained within the gel and embedded within the polyacrylamide matrix,
antibodies would have difficulty reaching the proteins. Second, the gel is fragile and can
easily break during analysis (as some students may unfortunately discover while performing
this lab!) while a membrane is more stable and durable. Lastly, the membrane can be
stripped of antibodies and reprobed several times.
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Lesson 4: Immunodetection for Myosin Light Chains
Using Antibodies to Identify Proteins
Immunology is the study of the immune system and how the body protects itself against
disease. Over 100 years ago, biologists discovered that animals’ internal immune systems
respond to invasion from foreign entities by provoking an immune response that begins
with the production of proteins called antibodies. Any foreign invader that elicits antibody
production is called an antigen. Like magic bullets, antibodies seek out and attach themselves
to invading entities, flagging these foreigners for destruction by other cells of the immune
system. Antigenic invaders may consist of any molecule foreign to the body, including
components of infectious agents like bacteria, viruses, and fungi. Astonishingly, there are
between 106 and 1011 unique antibodies circulating in blood with each one recognizing a
different antigen. Antibodies comprise up to 15% of total blood serum protein!

Tapping Nature’s Tool Kit

Because of its accuracy, western blotting is used to confirm positive test results for HIV,
lupus, or bovine spongiform encephalopathy (BSE or mad cow disease) following initial
screening using high-throughput enzyme-linked immunosorbent assay (ELISA).
The human immune system generates antibodies that detect foreign invaders such as
viruses, bacteria, and allergens and tag them for destruction. The ability of antibodies to act
like magic bullets and home in on specific targets makes them ideal for bioscience
research, diagnostic tests, and medical therapies.
Western blotting can pinpoint a specific protein among hundreds or thousands of other
proteins within biological samples. This surefire method of identifying proteins is based on
two distinguishing features of proteins: molecular mass and antibody binding specificity.
Bioscience researchers use western blotting as a tool to investigate proteomes: to identify
proteins, quantify protein expression levels, and determine whether proteins have undergone
genetic or posttranslational modifications.

STUDENT MANUAL
Antibody Antigen

Antigen
Disulfide

LESSON 4
bonds

- -S
-S -S-S- -S
-S -
-S-S-

Heavy chain Light chain

A B

Fig. 14. A) Structure of IgG bound to the HIV capsid protein p24 as determined by X-ray crystallography (Harris et al. 1998,
Momany et al. 1996). These structures can be downloaded from the Protein Data Bank (www.pdb.ufmg.br, Berman et al. 2000)
using the PDB identification codes 1IGY and 1AFV and manipulated using free online software such as Rasmol and Protein
Explorer. B) A commonly used representation of an antibody bound to an antigen.

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The immune system's natural ability to generate unique antibodies is an invaluable

mechanism that has been taken advantage of to advance modern biological research and
drug discovery. Since a crucial component in biological research involves the ability to track
and identify proteins, antibodies have been used as flagging devices to identify and localize
whatever protein is being studied. For example, scientists design a specific antibody that
will recognize a disease-associated protein. These custom-made antibodies can then be
used in experiments to characterize the protein’s function. For example, antibodies may be
used to identify the presence and quantity of a protein involved in a disease state or they
can be used to determine whether drug treatments affect the disease-associated protein.

What Is Immunodetection?
Since antibodies seek out and bind to specific proteins, they are ideal tools for proteomic
research when proteins need to be identified and analyzed. Immunodetection is the term
used for laboratory methods that use antibodies to detect proteins. In this lesson, an
antibody that is specific for myosin light chain will be used to detect myosin from among the
thousands of proteins immobilized on the membrane, much like finding a needle in a
haystack.
Antigen: An antigen is by definition any substance that is recognized by an antibody. In this
experiment the antigen consists of two proteins: myosin light chain 1 (MLC1) and myosin
light chain 2 (MLC2). Both are recognized by the primary antibody. MLC1 is one of the
essential myosin light chains. MLC2 is known as the myosin regulatory light chain.
Although myosin light chain protein from fish muscle tissue is designated as the central
focus in this laboratory activity, the primary antibody in this kit will also detect myosin light
chain proteins in many other species including human, mouse, rabbit, chicken, and frog,
allowing students to run independent research projects investigating muscle proteins from
other species.
STUDENT MANUAL
LESSON 4

How Are Antibodies Made?

When exposed to a foreign entity (e.g., molecules, cells, or tissues) most animals generate
an immune response and produce antibodies. Each antibody recognizes only a single
antigen. The antigen in this experiment is myosin light chain, which is in the proteins
extracted from the fish muscle. Animals such as goats, rabbits, and mice can be injected
with an antigen to stimulate antibody production. Over time, antibodies will accumulate in
the blood serum and can be purified for use in the laboratory. In an immunoassay, the
antibodies produced in this way to identify antigens are called primary antibodies.
Secondary antibodies recognize and bind to primary antibodies in an immunoassay.
Secondary antibodies are prepared by injecting primary antibodies produced from one
species of animal into another species so that the foreign species will provoke an immune
response. For example, if the desired product is a secondary antibody that will recognize a
mouse-derived primary antibody, then mouse antibodies are injected into a different animal
such as a goat. Following the goat's immune response, its serum will contain antibodies
that recognize and bind to any mouse-derived antibodies. Secondary antibodies are fre-
quently tagged (or conjugated) so that they can be made visible. In this experiment, the
secondary antibody is conjugated to horseradish peroxidase (HRP), an enzyme that when
in the presence of its substrate, 4CN, produces a purple/gray precipitate that deposits color
on the membrane at the precise location where the antigen-primary antibody-secondary
antibody complex is bound.

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Immunodetection Step by Step

Primary antibody is added to the blot and incubated
to allow the antibody to bind to the myosin protein on
the membrane. The unbound antibody is then
washed away.
The primary antibody provided is a monoclonal
mouse anti-myosin light chain antibody. This antibody
was made by injecting purified chicken myosin protein
into mice and generating an immortalized antibody
producing cell line (a hybridoma) from one mouse
that constantly produces the same antibody.

Secondary antibody is added to the blot and incubated

to allow the secondary antibody to bind to the primary
antibody. The unbound secondary antibody is then
washed away.
The secondary antibody is a polyclonal goat anti-mouse
antibody conjugated to HRP. Secondary antibody
was produced by injecting goats with primary mouse
antibodies. The secondary goat anti-mouse antibodies
were purified from goat serum, and chemically linked
or conjugated to HRP. HRP is the enzyme that
catalyzes oxidation of the colorimetric substrate so
the protein of interest can be identified.

Colorimetric (color-producing) enzyme substrate

STUDENT MANUAL
is added to the membrane and incubated to allow
color to develop. Purple/gray bands will develop on
the membrane exactly where the myosin protein
bands are located.

LESSON 4
The colorimetric substrate in this kit is 4-chloro-1-
naphthol (4CN). When oxidized by HRP in the presence
of hydrogen peroxide, this colorless solution forms a
purple/gray precipitate that binds to the membrane at
the antigen location. Note: The HRP color detection
reagent is light sensitive and must be kept in the dark
at all times.

Antigen

Primary antibody

HRP conjugated
antibody

Enzyme substrate

 
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Procedure  Day  1  
 
Preparation  of  protein  extracts  for  SDS-­‐PAGE  analysis.  
1.  Each  pair:  Select  one  marine  animal  tissue  sample  to  prepare  protein  extract  from.  Record  
the  sample  name  in  your  notebook.  
 
2.  Label  one  1.5  ml  eppendorf  tube  with  your  initials  and  sample  name.    
 
3.  Add  250  ul  of  Bio-­‐Rad  Laemmli  SDS  Sample  Buffer  into  the  tube.  
 
4. Transfer  a  0.25cm3  x  0.25cm3  piece  of  the  sample  into  the  eppy  tube  and  close  the  lid.  
 
5.  Flick  the  eppy  tube  15  times  to  agitate  the  tissue  in  the  Laemmli  SDS  Sample  Buffer  and  
break  apart  the  tissue,  releasing  the  protein  from  the  tissue  sample.    
 
6.  Incubate  the  tube  for  5  minutes  at  room  temperature.  During  the  incubation,  obtain  a  
locking  eppendorf  tube  and  label  it  with  your  initials  and  sample  name.  
 
7.  Carefully  transfer  the  Sample  Buffer  by  pouring  from  the  normal  eppy  tube  into  the  locking  
eppy  tube.  Do  NOT  transfer  the  marine  animal  tissue!    
 
8.  Heat  the  sample  in  boiling  water  for  3  minutes.    
 
9.  Heat  the  sample  in  the  locking  eppy  tube  for  5  minutes  at  95°C  by  floating  the  tube  in  the  
beaker  with  hot  water.  (Make  sure  the  lock  is  on  the  lid  to  prevent  evaporation  of  the  
sample)!  
 
10.  Store  your  protein  extract  in  -­‐20°C  until  the  next  lab  period.  
   

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Procedure  Day  2  
SDS-­‐PAGE  analysis  of  protein  extracts  and  transfer  of  proteins  to  nitrocellulose  membrane  
to  prepare  for  Western  Blotting  
 
1. Each  section  will  run  two  gels.    
Choose  two  student  volunteers  to  help  set-­‐up  the  SDS-­‐PAGE  gels.  While  the  gels  are  
being  prepared  (instructions  2  –  5  below),  others  should  prepare  the  samples  and  
protein  ladder  for  loading.    
To  do  this,  heat  12  ul  of  protein  ladder,  and  12  ul  of  actin  and  myosin  standard  in  the  
locking  eppy  tubes  for  2  -­‐  5  minutes  at  95°C  by  floating  the  tube  in  a  beaker  with  hot  
water.  (Make  sure  the  lock  is  on  the  lid  to  prevent  evaporation  of  the  sample)!  
Thawed  at  RT  the  fish  samples  prepared  the  previous  class.  Make  sure  that  all  the  SDS  
is  in  solution  before  loading  your  sample  
 
 
2.  Prepare  two  10-­‐well  BioRad  Ready  Gel  4  –  20%  gradient  SDS-­‐PAGE  gels  by:  
a. removing  the  plastic  strip  on  the  bottom  of  each  gel  
b. placing  the  ready  gel  cassettes  with  short  plates  facing  inwards  (towards  each  
other)  into  the  electrode  assembly    
 
 
3.  Slide  the  electrode  assembly  containing  the  two  gels  into  the  clamping  frame  and  clamp  
closed  by  pressing  down  on  the  electrode  assembly  while  closing  the  two  cam  levers  of  the  
clamping  frame.  Lower  the  inner  chamber  into  the  mini  tank.  
 

 
4.  Completely  fill  the  inner  chamber  between  the  gels  with  1X  TGS  running  buffer,  making  sure  
that  the  buffer  covers  the  short  plate  (~150  ml).  
 
5.  Fill  mini  tank  up  to  the  “2  gels”  line  with  1X  TGS  running  buffer  (~200  ml).  
 
6.  Load  the  gels  as  follows.  RECORD  in  your  notebook  which  gel  you  have  loaded  your  sample  
onto.  Important  note:  the  protein  standards  should  be  loaded  in  different  locations  on  the  
gels  as  indicated  below  to  allow  for  ease  of  distinguishing  the  two  gels.  
   

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GEL  1  
Lane     Volume     Sample  
1&2     empty     Empty  
3     5  ul     Precision  Plus  Protein  Kaleidoscope  prestained  standards  
4     5  ul     Actin  and  myosin  standard  
5     5  ul       Group  1  marine  animal  protein  sample  
6     5  ul       Group  2  marine  animal  protein  sample  
7     5  ul       Group  3  marine  animal  protein  sample  
8     5  ul       Group  4  marine  animal  protein  sample  
9  &  10     empty     Empty  
 
GEL  2  
Lane     Volume     Sample  
1&2     empty     Empty  
3     5  ul     Actin  and  myosin  standard  
4     5  ul     Group  5  marine  animal  protein  sample  
5     5  ul       Group  6  marine  animal  protein  sample  
6     5  ul       Precision  Plus  Protein  Kaleidoscope  pre-­‐stained  standards  
7     5  ul       Group  7  marine  animal  protein  sample  
8     5  ul       Group  8  marine  animal  protein  sample  
9  &  10     empty     Empty  
 
7.  Electrophorese  for  30  minutes  at  200V.        

 
 

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8.  Disassemble  the  gel  apparatus  and  break  apart  the  plates  to  expose  the  gel.  Cut  off  the  
wells  and  the  bottom  of  the  gel  with  a  razor  blade.  
 

 
9.  CAREFULLY  and  wearing  clean  gloves,  transfer  the  gels  one  at  a  time  into  two  clean  
Tupperwares  containing  30  ml  of  gel  transfer  buffer.  Then,  into  each  of  the  Tupperwares,  
place  one  nitrocellulose  membrane  (use  gloves  when  handling  this  at  all  times);  two  sheets  of  
blotting  paper,  and  two  fiber  pads.    
 
10.  In  a  third  clean  Tupperware,  assemble  the  blotting  sandwiches  one  at  a  time:  
a. Add  15  ml  of  gel  transfer  buffer  to  the  container  and  insert  plastic  cassette  with  black  
side  down.  
b. Lay  a  wet  fiber  pad  on  the  black  side  of  the  cassette.  
c. Lay  one  wet  sheet  of  blotting  paper  on  top  of  the  fiber  pad  and  use  side  of  hand  to  
press  out  air  bubbles.  
d. Use  a  piece  of  parafilm  to  pick  up  the  gel.  Slide  the  gel  off  of  the  parafilm  and    onto  
the  sheet  of  blotting  paper.  Be  careful  not  to  leave  any  bubbles  between  the  gel  and  
the  blotting  paper.  
e. Lay  one  sheet  of  wet  nitrocellulose  membrane  onto  the  gel.  Smooth  out  any  air  
bubbles  gently  with  your  finger.  
f. Lay  one  wet  sheet  of  blotting  paper  on  top  of  the  nitrocellulose  membrane  and  use  
side  of  hand  to  gently  press  out  air  bubbles.  
g. Lay  a  wet  fiber  pad  on  top  of  the  blotting  paper.  

 
 
 

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11.  Close  the  cassette  and  clamp  the  sandwich  together  with  the  white  clip.    

 
 
12.  Repeat  steps  10  and  11  for  the  other  gel.    
 
13.  Set  up  the  Mini  Trans-­‐Blot  module  with  the  black  sides  of  the  cassettes  next  to  the  
black  side  of  the  Mini  Trans-­‐Blot  module  (insert  as  pictured  above).  Add  a  frozen  Bio-­‐
Ice  module.  
 
14.  Put  the  Mini  Trans-­‐Blot  module  with  the  cassettes  onto  a  stir  plate  and  put  a  stir  
bar  into  the  bottom  of  the  Trans-­‐Blot  module  tank.  Fill  with  transfer  buffer  up  to  the  
white  clip.  Do  not  overfill  or  the  module  will  leak.    
 

 
 
 
15.  Place  lid  on  tank,  matching  the  power  cords  red-­‐  to-­‐red  and  black-­‐to-­‐black  and  
turn  on  the  stir  plate.  Run  at  100V  for  1  hour.    
 
16.  Dismantle  the  sandwiches  and  using  a  pencil,  make  a  pencil  mark  on  each  of  the  
protein  standards.  Also,  label  the  bottom  of  the  membranes  with  pencil  with  the  lab  
section  number.    
 

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17.  Place  each  membrane  into  the  Ponceau  S  staining  solution  to  visualize  all  of  the  
proteins  that  have  been  transferred.  Take  a  picture  of  the  Ponceau-­‐stained  
membranes  for  your  records.  
 
18.  Put  each  membrane  into  20  ml  of  TBS-­‐T  solution  and  store  at  4°C  until  the  next  lab  
period.  
 
 

 
 
 
 
 
 
 
 
 
 
   

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Day  3:    
Western  Blotting  (Blocking  of  membranes,  incubation  with  primary  and  secondary  
antibodies,  detection  of  bound  secondary  antibody)  
 
1.  Block  membranes  overnight  in  10  ml  5%  milk/TBS-­‐T  solution  (done  prior  to  lab).  Alternative  
option:  block  for  15  minutes  prior  to  adding  primary  antibody  with  rocking.  
 
2.  Discard  milk  blocking  solution  and  incubate  membranes  in  10  ml  primary  antibody  solution  
for  15  minutes  with  rocking  to  ensure  constant  coverage  of  the  membrane.  
 
3.  Save  the  primary  antibody  in  a  Falcon  Tube.  Pour  50  ml  of  TBS-­‐T  wash  buffer  onto  
membranes,  swish  quickly,  and  discard.  
 
4.  Add  50  ml  of  TBS-­‐T  wash  buffer  onto  membranes  again,  incubate  with  rocking  for  3  
minutes,  and  discard.  
 
5.  Incubate  membranes  in  10  ml  of  secondary  antibody  solution  for  5  to  15  minutes  with  
rocking.    
 
6.  Pour  50  ml  of  TBS-­‐T  wash  buffer  onto  membranes,  swish  quickly,  and  discard.  
 
7.  Add  50  ml  of  TBS-­‐T  wash  buffer  onto  membranes  again,  incubate  with  rocking  for  3  
minutes,  and  discard.  
 
8.  Add  10  ml  of  HRP  color  detection  reagent  and  incubate  10  to  30  minutes  with  rocking  and  
watch  color  development.    
 
9.  Rinse  membrane  twice  with  distilled  water  and  blot  dry  with  paper  towel.  Then  air  dry  and  
photograph  results.  
 
 
 
   

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Worksheet  10a:  Myosin  Comparative  Proteomics    

 
 
 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  
 
   

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BACKGROUND    

Taxonomic  data  can  be  derived  from  many  sources:  DNA  sequences,  protein  sequences,  
morphology,  and  paleontology.  Classification  of  organisms  derives  from  these  sources.  
Inconsistencies  in  the  phylogenetic  trees  generated  between  molecular  and  taxonomic  data  
emphasize  why  data  from  different  sources  is  required  to  generate  phylogenetic  trees  and  
why  there  is  still  much  dispute  in  the  field  of  phylogenetics  on  the  correct  placement  of  
organisms  within  phylogenetic  trees.    

In  order  to  determine  how  closely  related  species  are,  scientists  often  will  study  amino  acid  
sequences  of  essential  proteins.  Any  difference  in  the  amino  acid  sequence  is  noted  and  a  
phylogenetic  tree  is  constructed  based  on  the  number  of  differences.  More  closely  related  
species  have  fewer  differences  (i.e.,  they  have  more  amino  acid  sequence  in  common)  than  
more  distantly  related  species.  

There  are  many  tools  scientists  can  use  to  compare  amino  acid  sequences  of  muscle  protein.  
One  such  tool  is  the  National  Center  for  Biotechnology  Information  protein  databases  
(http://www.ncbi.nlm.nih.gov/).  By  entering  the  amino  acid  sequence  of  a  protein  you  are  
interested  in,  the  BLAST  search  tool  compares  that  sequence  to  all  others  in  its  database.  

TERMINOLOGY  USED  IN  BLAST  

 'Query'  is  the  original  sequence  used  for  the  search  

 'Sbjct'  is  the  aligned  sequence  
 Value  for  'identities'  is  the  number  of  amino  acids  exactly  in  common  
 Value  for  'positives'  is  the  number  of  amino  acids  that  are  similar  to  each  other  (such  
as  serine  and  threonine)  
 Value  for  'gaps'  is  the  number  of  amino  acid  positions  that  are  absent  one  of  the  
sequences.  
 'Sbjct'  is  the  aligned  sequence  and  the  middle  sequence  shows  the  mismatches:  a  '+'  
indicates  a  positive  and  a  space  indicates  a  mismatch  that  is  not  a  positive.  

 
   

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1.  (10points)  Explain  why  scientists  compare  protein  sequences  instead  of  nucleotide  
sequences  to  determine  how  closely  related  species  are.  

2.  (2.5points)  Explain  the  difference  between  BLAST–n  and  BLAST-­‐p.  

3.  You  will  compare  the  chicken  myosin  heavy  chain  protein  sequence  against  the  tuna  myosin  
heavy  chain  protein  sequence.  

a) Go  to  http://www.ncbi.nlm.nih.gov/  and  choose  BLAST    

b) Choose  Protein-­‐Protein  BLAST  (BLAST-­‐p).    
c) Enter  the  chicken  myosin  heavy  chain  sequence  into  the  search  box    (Enter  Query  
Sequence)  and  tuna  myosin  heavy  chain  sequence  in  the  box  (Enter  Subject  
Sequence).  
d) You  can  enter  the  protein  sequence  (even  with  numbers)  or  the  ACCESSION  number  
e) Choose  the  option  ALIGN  TWO  OR  MORE  SEQUENCES  
f) Press  BLAST  
Myosin  heavy  chain  [Gallus  gallus](chicken)    ACCESSION      AAB47555  
 
   1  maspdaemaa  fgeaapylrk  sekerieaqn  kpfdakssvf  vvhpkesfvk  gtiqskeggk                61  vtvktegget  
ltvkedqvfs  mnppkydkie  dmammthlhe  pavlynlker  yaawmiytys              121  glfcvtvnpy  kwlpvynpev  
vlayrgkkrq  eapphifsis  dnayqfmltd  renqsilitg              181  esgagktvnt  krviqyfati  aasgekkkee  
qsgkmqgtle  dqiisanpll  eafgnaktvr              241  ndnssrfgkf  irihfgatgk  lasadietyl  leksrvtfql  paersyhify  
qimsnkkpel              301  idmllittnp  ydyhyvsqge  itvpsiddqe  elmatdsaid  ilgfsadekt  aiykltgavm              361  
hygnlkfkqk  qreeqaepdg  tevadkaayl  mglnsaellk  alcyprvkvg  nefvtkgqtv              421  sqvhnsvgal  
akavyekmfl  wmvirinqql  dtkqprqyfi  gvldiagfei  fdfnsfeqlc              481  inftneklqq  ffnhhmfvle  
qeeykkegie  wefidfgmdl  aacieliekp  mgifsileee              541  cmfpkatdts  fknklydqhl  gksnnfqkpk  
pakgkaeahf  slvhyagtvd  ynisgwlekn              601  kdplnetvig  lyqkssvktl  allfatygge  aeggggkkgg  
kkkgssfqtv  salfrenlnk              661  lmanlrsthp  hfvrciipne  tktpgamehe  lvlhqlrcng  vlegiricrk  gfpsrvlyad              
721  fkqryrvlna  saipegqfmd  skkasekllg  sidvdhtqyr  fghtkvffka  gllglleemr              781  ddklaeiitr  
tqarcrgflm  rveyrrmver  resifciqyn  vrsfmnvkhw  pwmklffkik              841  pllksaesek  emanmkeefe  
ktkeelakse  akrkeleekm  vvllqekndl  qlqvqaeads              901  ladaeercdq  liktkiqlea  kikevterae  
deeeinaelt  akkrkledec  selkkdiddl              961  eltlakveke  khatenkvkn  fteemavlde  tiakltkekk  
alqeahqqtl  ddlqveedkv            1021  ntltkaktkl  eqqvddlegs  leqekklrmd  lerakrkleg  dlklahdsim  
dlendkqqld            1081  eklkkkdfei  sqiqskiede  qalgmqlqkk  ikelqariee  leeeieaert  srakaekhra            1141  
dlsreleeis  erleeaggat  aaqiemnkkr  eaefqkmrrd  leeatlqhea  taaalrkkha            1201  dstaelgeqi  
dnlqrvkqkl  ekekselkme  iddlasnmes  vskakanlek  mcrtledqls            1261  eiktkeeqnq  rmindlntqr  
arlqtetgey  srqaeekdal  isqlsrgkqg  ftqqieelkr            1321  hleeeikakn  alahalqsar  hdcellreqy  
eeeqeakgel  qralskanse  vaqwrtkyet            1381  daiqrteele  eakkklaqrl  qdaeehveav  nakcaslekt  
kqrlqneved  lmvdversna            1441  acaaldkkqk  nfdkilaewk  qkyeetqtel  easqkesrsl  stelfkmkna  

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yeesldhlet            1501  lkrenknlqq  eiadlteqia  eggkavhele  kvkkhveqek  selqasleea  easleheegk            

1561  ilrlqlelnq  ikseidrkia  ekdeeidqlk  rnhlrivesm  qstldaeirs  rnealrlkkk            1621  megdlnemei  
qlshanrmaa  eaqknlrntq  gtlkdtqihl  ddalrtqedl  keqvamverr            1681  anllqaevee  lrgaleqter  
srkvaeqell  datervqllh  tqntslintk  kkletdivqi            1741  qsemedtiqe  arnaeekakk  aitdaammae  
elkkeqdtsa  hlermkknmd  qtvkdlhvrl            1801  deaeqlalkg  gkkqlqklea  rvrelegevd  seqkrsaeav  
kgvrkyerrv  keltyqceed            1861  rknilrlqdl  vdklqmkvks  ykrqaeeaee  lsnvnlskfr  kiqheleeae  
eradiaesqv            1921  nklrvksrei  hgkkieeee  
 
 
Myosin  heavy  chain  [  Thunnus  orientalis  myh-­‐1  mRNA  for  myosin  heavy  chain-­‐1,    
complete  cds  ACCESION  BAL27685.1  
 
MSTDAEMEAYGPAAIYLRKPEKERIEAQTAPFDAKTAFFVTDKE                                            
EMYLKGKLVKREGGKATVETDCGKTLTVKEDEIFPRNPPNFDKIEDMAMMTHLNEPCV                                            
LYNLKERFASWMIYTYSGLFCVVVNPYKWLPVYDAVVVGGYRGKKRIEAPPHIFSISD                                            
NAYQFMHTDRENQSILITGESGAGKTVNTKRVIQYFATIAAIGAKKAEPTPGKMQGSL                                            
EDQIVAANPLLESYGNAKTVRNDNSSRFGKFIRIHFGSTGKLASADIETYLLEKSRVT                                            
FQLSAERSYHIFYQLMTGHQPELLEGLLITTNPYDYPMVSQGEITVKSIDDVEEFIAT                                            
DTAIDILGFTAEEKMGIYKLTGAVMHHGNMKFKQKQREEQAEPDGTEVADKISYLLGL                                            
NSADMLKYLCYPRVKVGNEMVTKGQTVPQVNNAVSALCKSVYDREFLWMVIRINEMLD                                            
TKQPRQYFIGVLDIAGFEIFDFNSLEQLCINFTNEKLQQFFNHHMFVLEQEEYKKEGI                                            
VWEFIDFGMDLAACIELIEKPMGIFSILEEECMFPKASDTTFKNKLHDQHLGKTKAFE                                            
KPKPVKGKPEAHFSLVHYAGTVDYNITGWLDKNKDPLNDSVVQLYQKASNKLLAFLYA                                            
KHGAADEGGGGKKGKKKGGSFQTVSALFRENLGKLMTNLRSTHPHFVRCLIPNESKTP                                            
GLMENFLVIHQLRCNGVLEGIRICRKGFPSRILYGDFKQRYKVLNASVIPEGQFIDNK                                            
KAAEKLLGSIDVDHTQYKFGHTKVFFKAGLLGLLEEMRDEKLANLVPMTQALCRGFLM                                            
RLEFVKMMERREAVFSIQYNIRSFMNVKNWPWMNLYFKIKPLLKSAETEKELMNMKEN                                            
YEKMKTDLATALAKKKELEEKMVSLLQEKNDLQLEVASETENLSDAEERCEGLIKSKI                                            
QLEAKLKETTERLEDEEEINAELTAKKRKLEDECSELKKDIDDLELTLAKVEKEKHAT                                            
ENKVKNLTEEMASQDESIAKLTKEKKALQEAHQQTLDDLQAEEDKVNTLTKAKTKLEQ                                            
QVDDLEGSLEQEKKLRMDLERAKRKLEGDLKLAQESIMDLENDKQQSDEKIKKKEFET                                            
SQLLSKIEDEQSLGAQLQKKIKELQARIEELEEEIEAERAARAKVEKQRADLSRELEE                                            
ISERLEEAGGATAAQIEMNKKREAEFQKLRRDLEESTLQHEATSASLRKKQADSVAEL                                            
GEQIDNLQRVKQKLEKEKSEYKMEIDDLSSNMEAVAKSKGNLEKMCRTIEDQLSELKA                                            
KNDEHVRQLNDLNGQRARLQTENGEFSRQIEEKDALVSQLTRGKQAYTQQIEELKRHI                                            
EEEIKAKNALAHAVQSARHDCDLLREQYEEEQEAKGELQRGMSKANSEVAQWRTKYET                                            
DAIQRTEELEEAKKKLAQRLQDAEESIEAVNSKCASLEKTKQRLQGEVEDLMIDVERA                                            
NSLAANLDKKQRNFDKVLADWKQKYEEGQSELEGAQKEARSLSTELFKMKNSYEEALD                                            
HLETMKRENKNLQQEISDLTEQIGETGKSIHELEKAKKHVETEKTEIQTALEEAEGTL                                            
EHEEAKILRVQLELNQIKSEVDRKLAEKDEEMEQIKRNSQRVIDSMQSTLDAEVRSRN                                            
DALRIKKKMEGDLNEMEIQLSHANRQATESQKQLRNVQGQLKDAQLHLDDAVRGHEDM                                            
KEQVAMVERRNGLMLAEIEELRAALEQTERGRKVAEQELVDASERVGLLHSQNTSLIN                                            
TKKKLEADLVHIQGEVDDSIQEARNAEDKAKKAITDAAMMAEELKKEQDTSAHLERMK                                            
KNLEVSVKDLQHRLDEAEALAMKGGKKQLQKLESRVRELESEVDAESRRGADAIKGVR                                            
KYERRVKELTYQTEEDKKNVHRLQDLVDKLQLKVKSYKRQAEEAEEQANTHLSRYRKV                                            
QHEMEEAQERADIAESQVNKLRAKSRDHHHGKGEHAE  
 

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a.  Include  a  picture  of  the  Graphic  Summary:  Distribution  of  Blast  Hits  on  the  Query  
Sequence  (2.5points)  In  a  separate  page  
 
 
b.  Scroll  down  to  DESCRIPTIONS  and  indicate  (6points)  
 
• Total  score    
 
• What  is  the  meaning  of  the  score  number?  
 
 
 
 
 
• What  is  the  meaning  of  percentage  of  the  query  cover?  What  is  the  percentage  of  the  
query  cover  in  this  particular  search?    
 
 
 
 
 
 
 
 
 
c.  Scroll  down  to  ALIGNMENTS  and  indicate  (10points)  
 
• How  many  amino  acids  are  exactly  identical  between  the  two  sequences?    
 
 
• What  is  the  percentage  of  identity  between  the  two  sequences?    
 
 
• How  many  amino  acids  are  similar  (similar  amino  acids  include  the  identical  ones)  
between  the  two  sequences?    
 
 
• What  is  the  percentage  of  similarity  between  the  two  sequences?    
 
 
• Indicate  the  number  of  gaps  in  the  alignment    
 
 
 
d.  (10points)  In  position  55  of  the  chicken  myosin  sequence  there  is  the  amino  acid  S  and  in  
the  same  position  of  the  tuna  myosin  sequence  there  is  K.  Indicate  the  name  of  each  amino  

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acid  and  their  properties  (Non  polar,  Polar  (charged  positive,  charged  negative,  uncharged).  
Use  the  table  at  the  end  of  the  Worksheet.  Is  it  a  conserved  changed?  Explain.  
 
 
 
 
 
 
 
 
 
 
 
e.  (10points)  In  position  31  of  the  chicken  myosin  sequence  there  is  the  amino  acid  N  and  in  
the  same  position  of  the  tuna  myosin  sequence  there  is  T.  Indicate  the  name  of  each  amino  
acid  and  their  properties  (Non-­‐poplar,  Polar  (charged  positive,  charged  negative,  uncharged).  
Use  the  table  at  the  end  of  the  Worksheet).  Is  it  a  conserved  changed?  Explain.  
 
 
 
 
 
 
 
 
 
 
 
4.  You  will  compare  tuna  myosin  heavy  chain  protein  sequence  against  a  protein  database  of  
non-­‐redundant  protein  sequences  (nr).  Non-­‐redundant  means  that  entries  in  the  database  
with  absolutely  identical  sequences  have  been  merged.  
 
a) Choose  Protein-­‐Protein  BLAST  (BLAST-­‐p).    
b) Enter  the  tuna  myosin  heavy  chain  sequence  into  the  search  box    (Enter  Query  
Sequence)    
c) UNCHECKED  the  option  ALIGN  TWO  OR  MORE  SEQUENCES  
d) go  to  CHOOSE  SEARCH  SET      Database  Non-­‐redundant  protein  sequence  (nr).    
e) Press  BLAST  (It  can  take  a  few  minutes  until  you  receive  the  result  of  the  search.)  
 
a.  Include  a  picture  of  the  Graphic  Summary:  Distribution  of  Blast  Hits  on  the  Query  
Sequence  (2.5points)  
 
 
 
 
 
 

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b. (10  points)Complete  the  following  table  with  first  five  different  species  that  appear  in  the  
search  and  that  are  not  tuna.  
 
Scientific  Name   Common  Name   %  Coverage     %  Identity
 
       
 
       
 
       
 
       
 
       
 
 
c. (10  points)  Are  the  results  expected?  Explain.  
 
 
 
 
 
 
 
 
 
 
5.  Choose  the  option  ALIGN  TWO  OR  MORE  SEQUENCES  
Compare  the  myosin  heavy  chain  protein  sequences  of  tuna  and  shrimp  (Myosin  heavy  
chain  type  1  [Litopenaeus  vannamei]  Pacific  white  shrimp  ACCESSION      BAM65721)  
 
a.  (2.5  points)  Include  a  picture  of  the  Graphic  Summary:  Distribution  of  Blast  Hits  on  the  
Query  Sequence  
 
 
b.  (7  points)  Scroll  down  to  DESCRIPTIONS  and  indicate    
 
• Total  score  
 
• What  is  the  percentage  of  the  tuna  myosin  sequence  (the  query)  aligned  with  the  
shrimp  myosin  sequence?    
 
 
Scroll  down  to  ALIGNMENTS  and  indicate    
 
• How  many  amino  acids  identical  between  the  two  sequences?    
 

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• What  is  the  percentage  of  amino  acids  that  are  identical  between  two  sequences?    
 
 
 
• How  many  amino  acids  are  exactly  identical  between  the  two  sequences?    
 
 
• What  is  the  percentage  of  amino  acids  that  are  similar  between  the  two  sequences?  
 
 
• Indicate  the  number  of  gaps  in  the  alignment    
 
 
d. (10  points)  Are  the  results  expected?  Explain  based  on  the  numbers  of  this  BLAST  
search.  
 
 
 
 
 
 
 
 
6.  (7  points)  Based  on  the  previous  information  is  the  tuna  heavy  myosin  more  closely  related  
to  the  chicken  or  the  shrimp  heavy  myosin?  Explain  based  on  the  information  that  you  got  
from  the  Blast  searches  done  in  3  and  5.    

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Worksheet  10b:  Myosin  Western  Blot  Experiment    

 
 
 
STUDENT  NAME________________________________  
 
 
LAB  SECTION__________________________________  
 
 
DUE____________________  
 
 
TURNED  IN______________  
 
 
YELLOW  PAGES  INCLUDED  __________  
 
   

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Background    
 
1.  (9  points)  Describe  3  proteins  found  in  muscle.  What  do  they  do?  
 
 
 
 
 
 
 
 
 
 
2.  (5  points)  Why  has  the  structure  of  actin  and  myosin  been  conserved  over  millions  of  years?  
 
 
 
 
 
 
 
 
 
 
 
 
3.  (10  points)  How  do  variations  in  organisms  occur  in  nature,  and  why?  How  does  this  
contribute  to  biodiversity?  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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4.  (8  points)  How  might  variations  in  proteins  between  species  be  used  to  determine  their  
evolutionary  relationships?    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5.  (8  points)  Can  one  gene  encode  more  than  one  protein?  How  can  two  different  proteins  
derived  from  the  same  gene  have  different  sizes  and  different  functions?    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SDS-­‐PAGE  
 
6.  (6  points)  Based  on  what  property  does  SDS-­‐polyacrylamide  gel  electrophoresis  (SDS-­‐PAGE)  
separate  proteins  by?  
 
 
 
 
 
 
 
 
 
 
 

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7.  (6  points)  In  order  to  prepare  protein  extracts  from  the  fish  tissue  samples,  you  added  
Laemmeli  sample  buffer  containing  SDS  (sodium  dodecyl  sulfate)  and  the  reducing  agent  DTT.
What  are  the  purposes  of  the  SDS  and  the  DTT  during  the  sample  preparation  to  obtain  the  
desired  results?  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
8.  (8  points)  You  are  examining  the  levels  of  a  fish  protein  that  is  part  of  a  large  multi-­‐protein  
complex  in  muscle  cells  by  western  blot.  You  prepare  protein  extracts  by  heating  the  samples  
in  Laemmeli  sample  buffer,  but  you  forget  to  add  the  DTT!  You  run  an  SDS-­‐PAGE  gel,  transfer  
to  a  nitrocellulose  membrane,  and  incubate  your  blot  with  primary  and  HRP-­‐conjugated  
secondary  antibodies  after  an  overnight  blocking  step.  You  expect  to  see  a  protein  that  is  40  
kD,  but  upon  addition  of  the  HRP  colorimetric  substrate,  you  see  a  band  at  200  kD  instead.  
Explain  how  the  absence  of  DTT  in  your  sample  buffer  could  have  caused  this  result.  
 
 
 
 
 
 
 
 
 
 
 
Western-­‐blotting  
9.  (10  points)  After  transferring  the  proteins  on  the  SDS-­‐PAGE  gel  to  a  nitrocellulose  filter,  we  
performed  an  overnight  blocking  step  before  incubating  the  filters  in  primary  antibody.    
 
a.  (2  points)  What  is  the  key  ingredient  in  the  blocking  buffer?  
 
 
 
 
 

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b.  (8  points)  Describe  the  purpose  of  this  blocking  step  and  the  consequences  on  the  results  if  
we  did  not  perform  this  step.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
10.  (6  points)  Describe  why  antibodies  are  useful  in  detecting  the  presence  of  a  specific  
protein.  
 
 
 
 
 
 
 
 
 
 
 
 
11.  (8  points)  Outline  the  antibody  staining  procedure  briefly  and  describe  why  it  was  
advantageous  to  use  both  a  primary  and  secondary  antibody.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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12.  (10  points)  In  looking  at  your  results  and  the  results  of  your  labmates,  are  myosin  proteins  
the  same  or  different  across  species?  Fill  out  the  table  summarizing  the  results  below.  
   
Marine  animal  species   Myosin  protein  similar  to   Size  of  myosin  protein?  
chicken  myosin?  (Yes  or  No)  
     
     
     
     
     
     
     
     
 
 
 
13.  (6  points)  Based  on  the  data  you  have  described  in  question  12,  which  species  are  more  
closely  related  to  each  other?  Draw  a  simple  phylogenetic  tree  to  represent  the  relatedness  of  
the  species  that  you  investigated.  Uncertain  relationships  can  be  indicated  by  a  dotted  line.  
(Hint:  the  last  common  ancestor  between  all  of  the  species  you  investigated  will  be  a  marine  
invertebrate).  
 
   

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