Julia Sevco

Ms. Williams
3 June 2016
Academic Biology
Academic Biology Transformation Lab
Introduction
Bacterial transformation is the process where foreign DNA is introduced into a bacterium
which can then clone the DNA. Transformation occurs naturally in some species of bacteria, but
scientists found ways to make transformation occur artificially. This lab uses heat shock as a
form of artificial transformation. The type of DNA most commonly used in bacterial
transformation is a plasmid. A plasmid is a small circular piece of DNA that contains important
genetic information for the growth of bacteria. A plasmid can reduce its size by supercoiling so it
can easily pass through pores in a cell membrane. A plasmid is a gene that is resistant to
antibiotics. Recombinant DNA or rDNA is an artificially made DNA strand that is formed by the
combination of two or more gene sequences (Smith 1).
The purpose of this lab was to understand the transformation procedure by artificially
transforming DNA into bacterial cells to see what effect this would have on the growth of the
cells. Since DNA is a very hydrophilic molecule, it wont normally pass through a bacterial cells
membrane. In order to make bacteria take in the plasmid, they must first be made competent to
take up the DNA. This is accomplished by treating the bacteria with calcium chloride which
causes water to enter into the cells and make them swell. These swollen bacteria are competent
bacteria.
The hypothesis for this lab was that the transformed E.coli with the ampicillin resistance
gene would grow in the ampicillin plates, but the non-transformed E.coli would not grow. Also,

the E. coli would grow in the plates with or without Luria broth because there is no antibiotic
present on those plates.
Natural transformation is useful to bacteria because bacteria grow in the same habitat as
molds and fungi which make toxins that kill the bacteria. As a result, bacteria make proteins to
inactivate the toxins (Bacterial Transformation 1). Natural transformation helps bacteria
survive.
Artificial transformation is useful to humans. For example, artificial transformation can
be used to make large amounts of certain human proteins, such as insulin, which can be used to
treat people with Type I diabetes. Millions of diabetics use synthetic insulin to control their blood
sugar.
Materials

2 15-mL test tubes

Microtube rack
Ice
Sterile plastic inoculating loops
500 L of ice cold .05 M Calcium chloride (CaCl2) solution
2 Sterile micropipets
Water bath
Glass beads
Tape
Timer
Streak plates of E.coli
4 media plates
P-GREEN plasmid
500 L of Luria broth
Spreading rod
Incubator

Procedures
1. Mark one sterile 15 mL tube + plasmid (experimental) and one - plasmid (control)
2. Using a sterile transfer pipet, add 250 L of ice cold CaCl2 (Calcium Chloride) to each
tube
3. Put both tubes in ice

4. Use a sterile plastic inoculating loop to transfer isolated colonies of E.coli from the starter
plate to the + plasmid tube.
4a. Do not pick up any agar as it may inhibit the transformation process.
4b. Immerse the cells on the loop in the CaCl2 solution in the + tube and
vigorously spin the loop to dislodge the cell mass. Hold the tube up to the light to
observe that the cell mass has fallen off the loop.
5. Immediately suspend the cells by repeatedly pipetting in and out with a sterile transfer
pipet. Examine the tube against the light to confirm that no visible clumps of cells remain
in the tube or are lost in the bulb on the transfer pipet. The suspension should appear
milky white.
6. Return the + tube to the ice.
7. Repeat steps 4 and 5 using the - tube.
8. Return the - tube to the ice.
9. Use a sterile plastic inoculating loop to add one loopful of plasmid DNA to the + plasmid
tube. (When the DNA solution forms a bubble across the loop opening, its volume is 10
L). Immerse the loopful of plasmid DNA directly into the cell suspension and spin the
loop to mix the DNA with the cells.
10. Return the + plasmid tube to ice. Incubate both tubes on ice for 15 minutes.
11. Label the bottom of media plates as follows:
a. LB/Amp plate - + plasmid This is an experimental plate
b. LB/Amp plate - - plasmid This is a negative control
c. LB plate + plasmid
d. LB plate - plasmid
12. Heat shock the cells. Remove both tubes directly from the ice and immediately immerse
them in 42 C water bath for 90 seconds. Gently agitate the tubes while they are in the
water bath. Return both tubes directly to the ice for 1 or more minutes.
13. Use a sterile transfer pipet to add 250 L Luria broth (LB) to each tube. Gently tap the
tubes with your finger to mix the LB with the cell suspension. Place the tubes in the test
tube rack at room temperature from a 5 to 15 minute recovery.
14. Use a sterile transfer pipet to add 100 L of cell suspension from the - plasmid
transformation tube to each appropriate plate. Immediately spread the cells over the
surface of the plate as follows:
a. Slightly open (clam shell) the lids and carefully pour 4 6 glass
beads onto each plate
b. Use a back and forth shaking motion to move the glass beads across
the entire surface of the plate, evenly spreading the cell suspension all
over the agar surface
c. Let the plates rest for a several minutes to allow the cell suspension to
be absorbed into the agar.
d. To remove the glass beads, hold each plate vertically over a container,
clam shell the lower part of the plate, and tap out the glass beads into
the container.

15. Use another sterile transfer pipet to add 100 L of cell suspension form the + plasmid
tube to each appropriate plate
16. Immediately spread the cell suspension as detailed in step 14
17. Wrap the plates together with tape and place the plates upside down either in the
incubator or at room temperature. Incubate them for approximately 24 36 hours in a 37
C incubator or 48-72 hours at room temperature.

Results
The LB plasmid and the LBAmp - plasmid plates did not show any growth. The
observed result on the LB + plasmid and LBAmp + plates showed no growth as well. The
LB - plasmid and the LB + plasmid plates are positive controls. The LBAmp - is a
negative control. The LBAmp + plasmid has the transformed bacteria.
Discussion
The LB - plasmid plate should have had bacterial growth because there was no
antibiotic. The LB + plasmid plate should have bacterial growth because the transformed
bacteria will grow since there is no antibiotic to inhibit growth. The LBAmp - plasmid plate
should have no bacterial growth because the untransformed bacteria is unable to grow in the
presence of bacteria. The LBAmp + plasmid plate the transformed bacteria is able to grow
even in the presence of the antibiotic because the plasmid contains the gene allowing for
antibiotic resistance.
My hypothesis was correct, but my results were incorrect except for the LBAmp -
plasmid plate. Some sources of error could have included the accidental picking up of agar when
transferring the E. coli from the starter plate to the + plasmid tube. (See Step 4) This would
inhibit the transformation process. Another source of error would include having visible clumps
of cells remain (See Step 5). Also, it was necessary to add the Luria broth to - first to avoid
cross contamination of plasmid. Not doing this correctly would be a source of error. Errors could
also have happened if the temperature and incubation periods used were not as directed.

Works Cited

Bacterial Transformation. Biotech learn. n.d. Web. 29 May 2016.

Rapoza, M. & Kreuzer, H. (2012). Transformations: A Teachers Manual. Burlington: Carolina
Biological Supply Company
Smith, Yolanda. What is recombinant DNA? News-medical. n.d. Web. 29 May 2016.