LABORATORY MANUAL

BTY430

SYNTHETIC AND SYSTEMS BIOLOGY

(For private circulation only)

Name of the Student:

Registration Number/Roll No.

Section and Group

School of Bioengineering and Biosciences

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 General guidelines for the students

General Guidelines for the students:

First practical session and first practical session after MTE is for introduction to all the
experiments before and after MTE respectively.

Students are expected to follow the instructions attentively and, before coming to the subsequent
labs they should browse through the database and tools.

Student should attend theory class so that they can clearly understand the theory behind the
experiments. They should keenly observe and understand the database structure which is
demonstrated in class.

Compulsory things to be carried by the students in lab: Lab manual.

Do’s:

1. Do write all the points discussed in lab.

2. Lab manual should contain all experiments; complete lab manual along with front page,
table of content should be brought.

3. Keep your cell phone in bags in switched off/silent mode.

4. Always be punctual in lab otherwise no attendance will be awarded.

Don’ts:

1. Don’t Share lab manuals/ worksheets.

2. Don’t talk among yourselves.

3. Don’t copy the result.

4. Don’t roam around in lab.

5. Don’t open restricted sites in lab.

LMB TY588

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 TABLE OF CONTENTS

S. No. Title of the Experiment Page No.

1. ENCODE: Encyclopedia of DNA Elements at UCSC 3-7

2. MGI: Mouse Genome Informatics 7-10

3. RGD: Rat Genome Database 11-13

4. FlyBase: A Database of Drosophila Genes & Genomes 14-17

5. WormBase: Explore Worm Biology 18-21

6. TAIR: The Arabidopsis Information Resource 22-24

7. EcoCyc: E. coli Database 25-27

8. SGD: Saccharomyces Genome Database 28-30

9. Design and construction of a biosensor 31-34

10. Crispr-Cas9 based genome editing 35-38

Reference Book(s):
1. DNA MICROARRAYS:METHODS EXPRESS by SCHENA M.

4 LMB T

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 Experiment 1

1. Experiment: Animal Databases - Encyclopedia of DNA Elements at UCSC

Equipment Required: Computer with internet connection.

2. Learning Objectives:
The student will know the importance of ENCODE.
The student will learn to classify databases on the basis of the information stored.
The students will also learn to select database to be used on the basis of problem stated.

3. Theory/Principle/Background of the topic:

The ENCODE (Encyclopedia of DNA Elements) project is collaborative project funded by the
National Human Genome Research Institute (NHGRI). The main aim of the ENCODE project
is to explore the non protein coding regions of DNA and uncover biomedically relevant
information useful for future research endeavors. Biochemical and computational approaches
were used in the project to determine functional parts of the non protein coding regions.
Although the ENCODE project has been received favorably by most of the scientific
community, there are still some criticisms about the project. These include claims by some of
the researchers involved in the project that 80% of the non protein coding region is 'functional',
the cost of the project so far (about 400 million dollars), no return of investment from the
project so far and the lack of a concrete end goal for the project. Despite these criticisms, the
project has been seen as a widespread success due to the enormous data generated from it and
is expected to enter it's next phase in 2017.

4. Outline of Procedure:
1) Determine the problem whose results you want to search for (e.g. Effects of siRNA
mediated RNAi on CTCF RNA)
2) Filter results to arrive at the result of interest (e.g. For the aforementioned problem,
set filter choices as Assay category = Transcription, Assay = siRNA RNA-seq, project
= ENCODE3, Organism = Homo sapiens, Target of Assay = RNA binding protein)
3) From the result(s), choose result of interest.
4) Explore the data set of the choice and record relevant information.

5. General calculations: Difference in number of records after each refinement. (Q1-Q2) hits.

6. Results:
i. Number of records obtained after performing search.
ii. Number of records obtained after search refinement.

7. Scope of result: Students will know annotations of relevant record like molecule type,
source, length, functions and features including sequence details.

8. Results required: Parameter: Limits advanced.

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9. Cautions: Refined search to be performed.

Suggested Reading:
Websites:
https://genome.ucsc.edu/ENCODE/

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Date of Performance Worksheet of the student Registration Number:

Aim:

Observations:

Calculations:

Result and Discussion:

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Learning Outcome

To be filled in by Faculty
S. No. Parameter Marks Max. Marks
obtained

1 Understanding of the student about the 20

Procedure/apparatus.
2 Observations and analysis including learning 20
Outcomes.
3 Completion of experiment, Discipline and 10
Cleanliness.
4 Signature of Faculty.

LMBTY430 Page | 7
 Experiment 2

1. Experiment: Animal Databases – Mouse Genome Informatics

Equipment Required: Computer with internet connection.

2. Learning Objectives:
The student will know the importance of the MGI database.
The student will learn to classify the database on the basis of the information stored.
The students will also learn to select database to be used on the basis of problem stated.

3. Theory/Principle/Background of the topic: MGI is the international database resource

for the laboratory mouse, providing integrated genetic, genomic, and biological data to
facilitate the study of human health and disease.

4. Outline of Procedure:
1. Visit the homepage of Wikipathways.
2. Select 'Browse'.
3. Select organism as Mus musculus.
4. Select pathway of interest.
5. Select gene coding for enzyme catalyzing reaction of interest.
6. Go to MGI homepage.
7. Enter gene name into search box.
8. Refine search until required result is arrived at.

1. General calculations: Difference in number of records after each refinement. (Q1-Q2) hits.

2. Results:
i. Number of records obtained after performing search.
ii. Number of records obtained after search refinement.

3. Scope of result: Students will know annotations of relevant record like molecule type,
source, length, functions and features including sequence details.

4. Results required: Parameter: Limits advanced.

5. Cautions: Refined search to be performed.

Suggested Reading:
Websites:
http://www.informatics.jax.org/

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Date of Performance Worksheet of the student Registration Number:

Aim:

Observations:

Calculations:

Result and Discussion:

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Learning Outcome

To be filled in by Faculty
S. No. Parameter Marks Max. Marks
obtained

1 Understanding of the student about the 20

Procedure/apparatus.
2 Observations and analysis including learning 20
Outcomes.
3 Completion of experiment, Discipline and 10
Cleanliness.
4 Signature of Faculty.

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 Experiment 3

1. Experiment: Animal Databases - Rat Genome Database

Equipment Required: Computer with internet connection.

2. Learning Objectives:
The student will know the importance of biological database.
The student will learn to classify database on the basis of the information stored.
The students will also learn to select database to be used on the basis of problem stated.

3. Theory/Principle/Background of the topic:

The Rat Genome Database (RGD) was established in 1999 and is the premier site for genetic,
genomic, phenotype, and disease data generated from rat research. In addition, it provides easy
access to corresponding human and mouse data for cross-species comparisons. RGD's
comprehensive data and innovative software tools make it a valuable resource for researchers
worldwide.

4. Outline of Procedure:
1. Go to RGD homepage.
2. Search for pathway of interest in the pathways section.
3. Select gene of interest from pathway.
4. Go to the 'Genes' section on homepage.
5. Enter gene name into the search box.
6. Refine on the basis of organism by choosing Rat as the organism of interest.

5. General calculations: Difference in number of records after each refinement. (Q1-Q2) hits.

6. Results:
i. Number of records obtained after performing search.
ii. Number of records obtained after search refinement.

7. Scope of result: Students will know annotations of relevant record like molecule type,
source, length, functions and features including sequence details.

8. Results required: Parameter: Limits advanced.

9. Cautions: Refined search to be performed.

Suggested Reading:
Websites:
http://rgd.mcw.edu/

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Date of Performance Worksheet of the student Registration Number:

Aim:

Observations:

Calculations:

Result and Discussion:

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Learning Outcome

To be filled in by Faculty
S. No. Parameter Marks Max. Marks
obtained

1 Understanding of the student about the 20

Procedure/apparatus.
2 Observations and analysis including learning 20
Outcomes.
3 Completion of experiment, Discipline and 10
Cleanliness.
4 Signature of Faculty.

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 Experiment 4

1. Experiment: Animal Databases – A Database of Drosophila Genes & Genomes

Equipment Required: Computer with internet connection.

2. Learning Objectives:
The student will know the importance of biological database.
The student will learn to classify database on the basis of the information stored.
The students will also learn to select database to be used on the basis of problem stated.

3. Theory/Principle/Background of the topic:

The FlyBase project is carried out by a consortium of Drosophila researchers and computer
scientists at: Harvard University, University of Cambridge (UK), Indiana University and the
University of New Mexico.

4. Outline of Procedure:
1. Visit the homepage of FlyBase.
2. Enter the name of the gene of interest in the search box in 'simple' category in the
quick search section.
3. Refine your search by selecting 'Gene' option under the data class column in the table
on the subsequent page.
4. Refine your search further on basis of criteria such as 'Cellular Compartment' after
clicking on the 'Results Analysis/Refinement' button.

5. General calculations: Difference in number of records after each refinement. (Q1-Q2) hits.

6. Results:
i. Number of records obtained after performing search.
ii. Number of records obtained after search refinement.

7. Scope of result: Students will know annotations of relevant record like molecule type,
source, length, functions and features including sequence details.

8. Results required: Parameter: Limits advanced.

9. Cautions: Refined search to be performed.

Suggested Reading:
Websites:
http://flybase.org/

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Date of Performance Worksheet of the student Registration Number:

Aim:

Observations:

Calculations:

Result and Discussion:

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Learning Outcome

To be filled in by Faculty
S. No. Parameter Marks Max. Marks
obtained

1 Understanding of the student about the 20

Procedure/apparatus.
2 Observations and analysis including learning 20
Outcomes.
3 Completion of experiment, Discipline and 10
Cleanliness.
4 Signature of Faculty.

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S. No. Parameter Marks Max. Marks
obtained

1 Understanding of the student about the 20

Procedure/apparatus.
2 Observations and analysis including learning 20
Outcomes.
3 Completion of experiment, Discipline and 10
Cleanliness.
4 Signature of Faculty.

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 Experiment 5

1. Experiment: Animal Databases - Explore Worm Biology

Equipment Required: Computer with internet connection.

2. Learning Objectives:
The student will know the importance of biological database.
The student will learn to classify database on the basis of the information stored.
The students will also learn to select database to be used on the basis of problem stated.

3. Theory/Principle/Background of the topic:

WormBase is an international consortium of biologists and computer scientists dedicated to
providing the research community with accurate, current, accessible information concerning
the genetics, genomics and biology of C. elegans and related nematodes. Founded in 2000, the
WormBase Consortium is led by Paul Sternberg of CalTech, Paul Kersey of the EBI, Matt
Berriman of the Wellcome Trust Sanger Institute, and Lincoln Stein of the Ontario Institute for
Cancer Research.

4. Outline of Procedure:
1) Visit the homepage of WormBase.
2) Click on the search box on the top right corner of the home page and enter the gene of
interest into the search box.
3) Select 'for a gene' from the drop down menu on the right of the search query and press
the search buttion.
4) Refine your search results by clicking on more options followed by selecting 'C.
elegans' as the species of interest.
5) Press the search button.

5. General calculations: Difference in number of records after each refinement. (Q1-Q2)

hits.

6. Results:
i. Number of records obtained after performing search.
ii. Number of records obtained after search refinement.

7. Scope of result: Students will know annotations of relevant record like molecule type,
source, length, functions and features including sequence details.

8. Results required: Parameter: Limits advanced.

9. Cautions: Refined search to be performed.

Suggested Reading:
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Websites:
http://www.wormbase.org/

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Date of Performance Worksheet of the student Registration Number:

Aim:

Observations:

Calculations:

Result and Discussion:

LMBTY430 Page | 20
Learning Outcome

To be filled in by Faculty
S. No. Parameter Marks Max. Marks
obtained

1 Understanding of the student about the 20

Procedure/apparatus.
2 Observations and analysis including learning 20
Outcomes.
3 Completion of experiment, Discipline and 10
Cleanliness.
4 Signature of Faculty.

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 Experiment 6
1. Experiment: Plant Databases - The Arabidopsis Information Resource
Equipment Required: Computer with internet connection.

2. Learning Objectives:
The student will learn to classify database on the basis of the information stored.

3. Theory/Principle/Background of the topic:

The Arabidopsis Information Resource (TAIR) maintains a database of genetic and molecular
biology data for the model higher plant Arabidopsis thaliana . Data available from TAIR
includes the complete genome sequence along with gene structure, gene product information,
gene expression, DNA and seed stocks, genome maps, genetic and physical markers,
publications, and information about the Arabidopsis research community. Gene product
function data is updated every week from the latest published research literature and
community data submissions. TAIR also provides extensive linkouts from our data pages to
other Arabidopsis resources.

4. Outline of Procedure:
1. Visit the homepage of TAIR.
2. In the search drop down menu, select 'genes' option.
3. In 'Search by name or phenotype' section, enter the name or part of the name of the
gene you want to search for. Select the species as Arabidopsis thaliana in this section.
4. Refine your search by entering a keyword term in the 'Search by Associated Keyword'
section.
5. Press submit query.

5. General calculations: Difference in number of records after each refinement. (Q1-Q2) hits.

6. Results:
i. Number of records obtained after performing search.
ii. Number of records obtained after search refinement.

7. Scope of result: Students will know annotations of relevant record like molecule type,
source, length, functions and features including sequence details.

8. Results required: Parameter: Limits advanced.

9. Cautions: Refined search to be performed.

Suggested Reading:
Websites:
https://www.arabidopsis.org/

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Date of Performance Worksheet of the student Registration Number:

Aim:

Observations:

Calculations:

Result and Discussion:

LMBTY430 Page | 23
Learning Outcome

To be filled in by Faculty
S. No. Parameter Marks Max. Marks
obtained

1 Understanding of the student about the 20

Procedure/apparatus.
2 Observations and analysis including learning 20
Outcomes.
3 Completion of experiment, Discipline and 10
Cleanliness.
4 Signature of Faculty.

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 Experiment 7

1. Experiment: Microbial Databases – E. coli Database

Equipment Required: Computer with internet connection.

2. Learning Objectives:
The student will know the importance of biological database.
The student will learn to classify database on the basis of the information stored.
The students will also learn to select database to be used on the basis of problem stated.

3. Theory/Principle/Background of the topic: EcoCyc is a scientific database for the

bacterium Escherichia coli K-12 MG1655. The EcoCyc project performs literature-based
curation of the entire genome, and of transcriptional regulation, transporters, and metabolic
pathways.

4. Outline of Procedure:
1. Go to EcoCyc homepage.
2. Select 'Genes, Proteins or RNAs' from the search drop down menu.
3. Select 'Search by Gene name or database identifier' tick box and enter the gene name
into the same section's corresponding search box.
4. Refine your search by selecting appropriate tick boxes and selecting the desired option
(eg. Selecting the 'Search/Filter by Cellular compartment' search box and selecting the
cytoplasm as the refinement criteria.)
5. Click on 'Submit Query' and choose the desired option.

5. General calculations: Difference in number of records after each refinement. (Q1-Q2) hits.

6. Results:
i. Number of records obtained after performing search.
ii. Number of records obtained after search refinement.

7. Scope of result: Students will know annotations of relevant record like molecule type,
source, length, functions and features including sequence details.

8. Results required: Parameter: Limits advanced.

9. Cautions: Refined search to be performed.

Suggested Reading:
Websites:
http://ecocyc.org/

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Date of Performance Worksheet of the student Registration Number:

Aim:

Observations:

Calculations:

Result and Discussion:

LMBTY430 Page | 26
Learning Outcome

To be filled in by Faculty
S. No. Parameter Marks Max. Marks
obtained

1 Understanding of the student about the 20

Procedure/apparatus.
2 Observations and analysis including learning 20
Outcomes.
3 Completion of experiment, Discipline and 10
Cleanliness.
4 Signature of Faculty.

LMBTY430 Page | 27
 Experiment 8

1. Experiment: Microbial Databases - Saccharomyces Genome Database

Equipment Required: Computer with internet connection.

2. Learning Objectives:
The student will know the importance of biological database.
The student will learn to classify database on the basis of the information stored.
The students will also learn to select database to be used on the basis of problem stated.

3. Theory/Principle/Background of the topic:

The Saccharomyces Genome Database (SGD) provides comprehensive integrated biological
information for the budding yeast Saccharomyces cerevisiae along with search and analysis
tools to explore these data, enabling the discovery of functional relationships between
sequence and gene products in fungi and higher organisms.

4. Outline of Procedure:
1. Visit the homepage of SGD.
2. Enter the name of the gene of interest in the top right search box.
3. Select the refinement category as 'Genes' in the category lest.
4. Refine your search further by refining on the basis of categories such as cellular
compartment, molecular function, Feature type and Biological process.
5. Select option of choice.

6. General calculations: Difference in number of records after each refinement. (Q1-Q2) hits.

7. Results:
i. Number of records obtained after performing search.
ii. Number of records obtained after search refinement.

8. Scope of result: Students will know annotations of relevant record like molecule type,
source, length, functions and features including sequence details.

9. Results required: Parameter: Limits advanced.

10. Cautions: Refined search to be performed.

Suggested Reading:
Websites:
http://www.yeastgenome.org/

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Date of Performance Worksheet of the student Registration Number:

Aim:

Observations:

Calculations:

Result and Discussion:

LMBTY430 Page | 29
Learning Outcome

To be filled in by Faculty
S. No. Parameter Marks Max. Marks
obtained

1 Understanding of the student about the 20

Procedure/apparatus.
2 Observations and analysis including learning 20
Outcomes.
3 Completion of experiment, Discipline and 10
Cleanliness.
4 Signature of Faculty.

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 Experiment 9

1. Experiment: Design and construction of a biosensor

Equipment Required: Computer with internet connection.

2. Learning Objectives:
The student will know the importance of Biosensor.
The student will learn that the biosensors continue to play a critical role across a myriad of fields
including biomedical diagnosis, monitoring of treatment and disease progression, drug discovery,
food control and environmental monitoring.

3. Theory/Principle/Background of the topic:

Biosensor technology exploits the unique properties of a biological recognition event on a
transducing device. In such an event, the interaction of the analyte with the bioreceptor is
converted into a suitable output that is easily readable by the user.

4. Outline of Procedure:

A typical biosensor consists of the following components.

 Analyte: A substance of interest that needs detection. For instance, glucose is an ‘analyte’
in a biosensor designed to detect glucose.
 Bioreceptor: A molecule that specifically recognises the analyte is known as a bioreceptor.
Enzymes, cells, aptamers, deoxyribonucleic acid (DNA) and antibodies are some examples
of bioreceptors. The process of signal generation (in the form of light, heat, pH, charge or
mass change, etc.) upon interaction of the bioreceptor with the analyte is termed bio-
recognition.
 Transducer: The transducer is an element that converts one form of energy into another. In
a biosensor the role of the transducer is to convert the bio-recognition event into a
measurable signal. This process of energy conversion is known as signalisation. Most
transducers produce either optical or electrical signals that are usually proportional to the
amount of analyte–bioreceptor interactions.
 Electronics: This is the part of a biosensor that processes the transduced signal and prepares
it for display. It consists of complex electronic circuitry that performs signal conditioning
such as amplification and conversion of signals from analogue into the digital form. The
processed signals are then quantified by the display unit of the biosensor.
 Display: The display consists of a user interpretation system such as the liquid crystal
display of a computer or a direct printer that generates numbers or curves understandable by
the user. This part often consists of a combination of hardware and software that generates
results of the biosensor in a user-friendly manner. The output signal on the display can be
numeric, graphic, tabular or an image, depending on the requirements of the end user.

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5. General calculations: Difference in number of records after each refinement.

6. Results:
i. Number of records obtained after performing search.

7. Scope of result: Students will l e a r n t h e i m p o r t a n c e o f b i o s e n s o r s i n

biomedical world.

8. Cautions: Refined search to be performed.

Suggested Reading:
Websites:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986445/

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Date of Performance Worksheet of the student Registration Number:

Aim:

Observations:

Calculations:

Result and Discussion:

LMBTY430 Page | 33
Learning Outcome

To be filled in by Faculty
S. No. Parameter Marks Max. Marks
obtained

1 Understanding of the student about the 20

Procedure/apparatus.
2 Observations and analysis including learning 20
Outcomes.
3 Completion of experiment, Discipline and 10
Cleanliness.
4 Signature of Faculty.

LMBTY430 Page | 34
 Experiment 10

1. Experiment: Crispr-Cas9 based genome editing

Equipment Required: Computer with internet connection.

2. Learning Objectives:
The student will know the importance of Crispr-Cas9 based genome editing
The student will learn that the CRISPR-Cas system functions as a prokaryotic immune system, in
that it confers resistance to exogenous genetic elements such as plasmids and phages and provides
a form of acquired immunity.

3. Theory/Principle/Background of the topic:

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an acronym for DNA loci
that contain multiple, short, direct repetitions of base sequences. Each repetition contains a series of
base pairs followed by the same or a similar series in reverse and then by 30 or so base pairs known
as "spacer DNA". CRISPRs are found in the genomes of approximately 40% of sequenced bacteria
and 90% of sequenced archaea. CRISPRs are often associated with cas genes which code for
proteins that perform various functions related to CRISPRs. CRISPR spacers recognize and silence
exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.

4. Outline of Procedure: The mechanism of CRISPR/Cas-9 genome editing can be generally

divided into three steps: recognition, cleavage, and repair. The designed sgRNA directs Cas-9 and
recognizes the target sequence in the gene of interest through its 5ʹcrRNA complementary base
pair component. CRISPR-P 2.0 provides web service for computer-aided design of highly
efficient sgRNA with minimal off-target effects. It has functions as follow:

1. It supports sgRNA design for 49 plant genomes. SgRNAs for 49 plant species with the latest
version of genome and annotation are provided.
2. Scoring system of sgRNA for on-target efficiency and off-target potential are improved.
CRISPR-P 2.0 uses a modified scoring system to rate the off-targeting potential and on-
targeting efficiency of sgRNAs for Streptococcus pyogenes Cas9 which is the widest used
CRISPR-Cas9 system. The scoring system in CRISPR-P 2.0 is based on the up-to-date
knowledge about SpCas9 genome editing.
3. It Supports various CRISPR-Cas systems. It supports to design guide sequences for various
CRISPR-Cas systems like Cpf1 and various Cas9 endonucleases.
4. A comprehensive analysis of the guide sequence is provided, including: GC content,
restriction endonuclease site, microhomology sequence flanking the targeting site
(microhomology score), and the secondary structure of sgRNA.
5. Identification of sgRNA from custom sequences is also provided. If user’s
genome/sequence is not listed in the selectable genomes, it allows users to upload custom
sequences and identify sgRNAs.

5. General calculations: Difference in number of records after each refinement.

LMBTY430 Page | 35
6. Results:
Number of records obtained after performing search.

7. Scope of result: Students will know that Clustered Regularly Interspaced Short Palindromic
Repeat (CRISPR) comprises of repetitive sequences that are separated by unique short DNA fragment
acquired from a previously invading organism.

8. Results required: Parameter: Limits advanced.

Suggested Reading:
Websites:
http://crispr.hzau.edu.cn/
CRIPR2/

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Date of Performance Worksheet of the student Registration Number:

Aim:

Observations:

Calculations:

Result and Discussion:

LMBTY430 Page | 37
Learning Outcome

To be filled in by Faculty
S. No. Parameter Marks Max. Marks
obtained

1 Understanding of the student about the 20

Procedure/apparatus.
2 Observations and analysis including learning 20
Outcomes.
3 Completion of experiment, Discipline and 10
Cleanliness.
4 Signature of Faculty

LMBTY430 Page | 38