Microbial physiology in genomic era

Microbial Physiology
in Genomic Era
A revolutionary Tale

•Manahil Khanum Durrani
•Ariba Nameen
•Aneeqa Sadiq
•Afra Ejaz
•Minahil Khalid
•Hafiza Iqra Malik
Group Members

Contents
• Genomic & proteomic tools
• Cloning a Genome
• DNA sequencing
• Internet Tools for DNA
sequencing
• Gene Replacement
• Gene Arrays
• Proteomics
• Traditional tools
• Reporter genes
• DNA mobility Shifts
• Primer Extension
• Detecting DNA ,RNA proteins by
Southern & western Blots
• Two-Hybrid Analysis

Introduction
• The field of microbial physiology is undergoing a
dramatic revolution. This reformation is the direct
result of three technological achievements:
• The personal computer,
• The Internet
• rapid DNA sequencing techniques

Advantages
• The personal computer and Internet have fueled this
renaissance of microbial physiology by allowing
unfettered sharing of this information among scientists
around the world.
• Rapid DNA techniques, are useful in synthetic biology as in
metabolic engineering in plants & Microbes
• They are also useful in forensic techniques, as increase
performance of human identification processes,

Genomics &
Proteomics
• Genomics provides an overview of
the complete set of genetic
instructions provided by the DNA,
while transcriptomics looks into gene
expression patterns.
• Proteomics studies dynamic protein
products and their interactions, while
metabolomics is also an intermediate
step in understanding organism's
entire metabolism.

Why Genomics is important??
• Genetics involves scientific studies of genes and their effects.
... Genomics includes the scientific study of complex diseases
such as heart disease, asthma, diabetes, and cancer because
these diseases are typically caused more by a combination of
genetic and environmental factors than by individual genes

Use of proteomics
• Proteomics is used to detect protein expression
patterns at a given time in response to a specific
stimulus, but also to determine functional protein
networks that exist at the level of the cell, tissue, or
whole organism

Genomic & Proteomic Tools & Techniques
Genomic tools contains;-
• Gene cloning
• Sequencing
• Micro Array etc
While proteomics techniques includes;-
• SDS page
• Isoelectric focusing
• Polyacrylamide Gel Electrophoresis

Gene Cloning: The insertion of a fragment of DNA carrying a
gene into a cloning vector and subsequent propagation of
recombinant DNA molecules into many copies is known as
gene cloning.

BASIC STEPS OF GENE CLONING
 Construction of recombinant DNA molecule
 Transport of the recombinant DNA to the host cell
 Multiplication of recombinant DNA molecule
 Division of the host cell
 Numerous cell division resulting in a clone

Gene cloning requires specialized tools and
techniques:
Vehicles: The central component of a gene cloning
experiment is the vehicle, which transport the gene into the
host cell and is responsible for its replication. To act as a
cloning vehicle a DNA molecule must be capable of entering
a host cell and, once inside, replicating to produce multiple
copies of itself.
Vector: A DNA molecule, capable of replication in a host
organism, into which a gene is inserted to construct a
recombinant DNA molecule.

Different types of cloning
1. Recombinant DNA technology or DNA
cloning
2. Reproductive cloning and
3. Therapeutic cloning

How can cloning technologies be used?
• Gene therapy can be used to treat certain genetic conditions by
introducing virus vectors that carry corrected copies of faulty genes
into the cells of a host organism.
• Genes from different organisms that improve taste and nutritional
value or provide resistance to particular types of disease can be used
to genetically engineered food crops.
•Reproductive cloning also could be used to repopulate endangered
animals or animals that are difficult to breed.

Can organs be cloned for use in transplants?
 Scientists hope that one day therapeutic cloning can be used to
generate tissues and organs for transplants.
 The stem cells would be used to generate an organ or tissue that
is a genetic match to the recipient.
 In theory, the cloned organ could then be transplanted into the
patient without the risk of tissue rejection.

Objectives
• Compare and contrast the chemical (Maxim/Gilbert) and chain
termination (Sanger) sequencing methods.
• List the components and molecular reactions that occur in chain
termination sequencing.
• Discuss the advantages of dye primer and dye terminator
sequencing.
• Derive a text DNA sequence from raw sequencing data.
• Describe examples of alternative sequencing methods, such as
bisulfite sequencing and pyrosequencing.

Sequencing Methods
• Maxam/Gilbert chemical sequencing
• Sanger chain termination sequencing
• Pyrosequencing
• Array sequencing

Maxam-Gilbert sequencing is performed by chain breakage
at specific nucleotides.
DMS
G
G
G
G
FA
G
A
G
G
A
G
A
A
H
C
T
T
C
T
C
C
T
H+S
C
C
C
C
Maxam-Gilbert Sequencing

Sequencing gels are read from bottom to top (5′ to 3′).
G G+A T+C C
3′
A
A
G
C
A
A
C
G
T
G
C
A
G
5′
Longer fragments
Shortest fragments
G
A
Maxam-Gilbert Sequencing

Chain Termination (Sanger) Sequencing
• A modified DNA replication
reaction.
• Growing chains are
terminated by
dideoxynucleotides.

Chain terminates
at ddG
The 3′-OH group necessary for formation of the phosphodiester bond is
missing in ddNTPs.

Template area to be sequenced
-3′ OH
TCGACGGGC…
5′OP-
Primer
Template
• A sequencing reaction mix includes labeled primer and
template.
• Dideoxynucleotides are added separately to each of the
four tubes.

ddATP + ddA
four dNTPs dAdGdCdTdGdCdCdCdG
ddCTP + dAdGddC
four dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC
dAdGdCdTdGdCdCddC
ddGTP + dAddG
four dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdCddG
ddTTP + dAdGdCddT
four dNTPs dAdGdCdTdGdCdCdCdG
A
C
G
T

• With addition of enzyme (DNA polymerase), the primer
is extended until a ddNTP is encountered.
• The chain will end with the incorporation of the ddNTP.
• With the proper dNTP:ddNTP ratio, the chain will
terminate throughout the length of the template.
• All terminated chains will end in the ddNTP added to
that reaction.

• The collection of fragments is a sequencing ladder.
• The resulting terminated chains are resolved by
electrophoresis.
• Fragments from each of the four tubes are placed in
four separate gel lanes.

Sequencing gels are read from bottom to top (5′ to 3′).
G A T C
3′
G
G
T
A
A
A
T
C
A
T
G
5′
Longer fragments
Shorter fragments
ddG
ddG

Cycle Sequencing
• Cycle sequencing is chain termination sequencing
performed in a thermal cycler.
• Cycle sequencing requires a heat-stable DNA
polymerase.

Alternative Sequencing Methods:
Pyrosequencing
• Pyrosequencing is based on the generation of light
signal through release of pyrophosphate (PPi) on
nucleotide addition.
• DNAn + dNTP  DNAn+1 + PPI
• PPi is used to generate ATP from adenosine
phosphosulfate (APS).
• APS + PPI  ATP
• ATP and luciferase generate light by conversion of
luciferin to oxyluciferin.

DNA sequence: A T C A GG CC T
Nucleotide added : A T C A G C T
Pyrosequencing
• Each nucleotide is added in turn.
• Only one of four will generate a light signal.
• The remaining nucleotides are removed enzymatically.
• The light signal is recorded on a pyrogram.

Bisulfite Sequencing
• Bisulfite sequencing is used to detect
methylation in DNA.
• Bisulfite deaminates cytosine, making uracil.
• Methylated cytosine is not changed by
bisulfite treatment.
• The bisulfite-treated template is then
sequenced.

Annotation
• DNA annotation or genome annotation is the
process of identifying the locations of genes and
all of the coding regions in a genome and
determining what those genes do. ... Once
a genome is sequenced, it needs to
be annotated to make sense of it

Web Science: Internet
Tools for DNA
Sequence Analysis

How to handle the large amount of
information?
• Answer: bioinformatics and Internet
Drew Sheneman, New Jersey--The Newark Star Ledger

Introduction: What is bioinformatics?
• Bioinformatics is a scientific discipline created from the interaction of
biology and computer science.
• Can be defined as the body of tools, algorithms needed to
handle large and complex biological information.
• The NCBI defines bioinformatics as:
"Bioinformatics is the field of science in which biology, computer
science, and information technology merge into a single discipline”

• There is a need for computers and algorithms that
allow:
Access, processing, storing, sharing,
retrieving, visualizing, annotating…
• But!!!!
•Why do we need the Internet?

• “omics” projects and the information associated
with involve a huge amount of data that is stored
on computers all over the world.
• Because it is impossible to maintain up-to-date
copies of all relevant databases within the lab.
Access to the data is via the internet.

DNA (nucleotide sequences) databases
• They are big databases and searching either one should produce similar
results because they exchange information routinely.
• -GenBank (NCBI): http://www.ncbi.nlm.nih.gov
• -Ensembl: http://useast.ensembl.org/index.html
•
• -DDBJ (DNA Database of Japan): http://www.ddbj.nig.ac.jp
• -TIGR: http://tigr.org/tdb/tgi
• -Yeast: http://yeastgenome.org
• -Microbes: http://img.jgi.doe.gov/cgi-bin/pub/main.cgi

Protein (amino acid) databases
• Known proteins:
-Swiss-Prot (very high level of annotation)
http://au.expasy.org/
-PIR (protein identification resource) the world's most
comprehensive catalog of information on proteins http://www.pir.uniprot.org/
• Translated databases:
-TREMBL (translated EMBL): includes entries that have not been
annotated yet into
Swiss-Prot. http://www.ebi.ac.uk/trembl/access.html
-GenPept (translation of coding regions in GenBank)
-pdb (sequences derived from the 3D structure
• Brookhaven PDB) http://www.rcsb.org/pdb/

Which algorithm to use for database similarity
search?
• Speed:
• BLAST > FASTA > Smith-Waterman (It is very slow and uses a lot of
computer power)
• Sensitivity/statistics:
• FASTA is more sensitive, misses less homologues
• Smith-Waterman is even more sensitive.
• BLAST calculates probabilities
• FASTA more accurate for DNA-DNA search then BLAST

Reverse Genetics
• From gene to phenotype - using genetic tools to
identify the function of a gene without prior
knowledge of its function.
• Knockout - screen for phenotype
• Overexpression
• Ectopic expression

Suicide Mutagenesis
• It involves deleting
an internal portion
of the gene you
wish to mutate and
placing the deleted
gene into a
suicide vector

Gene Array
• A snapshot that captures the activity pattern of
thousands of genes at once

Basics of microarrays
• DNA attached to solid
support
Glass, plastic, or nylon
• RNA is labeled
Usually indirectly
• Bound DNA is the probe
Labeled RNA is the “target”

proteomics
The terms proteome and proteomics were coined by
Mark wilkins and colleagues in the early 1990’s

Definition:
That’s just not a protein biochemistry !
Proteome :
It is the complement protein found in a single cell in a
particular environment./ is complete collection of proteins encoded by
genome of an organism.
Proteomics :
It is the study of composition, structure, function and
interaction of the proteins directing the activities of each living cell.

Role of Proteomics
• To study the structure and function of protein.
• To study the 3D structure of protein.
• Study of qualitative and quantitative analysis of proteins.

Structural proteomics
• Helps to identify newly discovered genes and drug interaction

Expression proteomics
• Helps to identify the main gene in a particular sample.

Interaction proteomics
• It the pathway in which proteins combined in large
complexes.

Introduction:
•Two-dimensional gel electrophoresis
is considered a powerful tool for
proteomics work.
• It is used for separation and
fractionation of complex protein
mixtures from biological samples.
Two dimensional Gel Electrophoresis

The first one is called isoelectric
focusing (IEF) which separates
proteins according to
isoelectric points (pI)
The second step is SDS-
polyacrylamide gel
electrophoresis (SDSPAGE) which
separates proteins based on the
molecular weights
•Thus, thousands of proteins
can be separated, and the
information about IEF and
molecular weights can be
obtained
Steps

•Proteins are amphoteric
molecules and the positive,
negative, or zero net charge they
carry depending on the pH of the
surroundings.
•The isoelectric point (pI) is
defined as the pH of a solution at
which the net charge of the
protein becomes zero.
Isoelectric Focusing (IEF)

• A protein with a positive net charge will migrate toward the
cathode, becoming less positively charged until reaching its
pI. While a protein with a negative net charge will migrate
toward the anode, becoming less negatively charged until it
also reaches its pI.

Isoelectric Focusing (IEF)
• A protein mixture is loaded at
the basic end of the pH
gradient gel.
• After applying an electric
field, the proteins are
separated depending on
charges, focusing at positions
where the pl value is
equivalent to the surrounding
pH.
• Larger proteins will move
more slowly through the gel,
but with sufficient time will
catch up with small proteins of
equal charge

• Be performed on flatbed or vertical systems on a slab
gel.
• The second dimension is often performed by SDS-
PAGE), which is an electrophoretic method.
• This technique is used to separate proteins by their
molecular weight.
• SDS negatively charged detergent used to denature
proteins.
SDS-PAGE
(SDS-polyacrylamide gel electrophoresis)

How does an SDS-PAGE Seperation Work?
• Negatively charged proteins move to positive Electrode.
• SDS coated larger proteins migrate slowly through the Gel
Matrix.
• SDS coated smaller proteins
migrate quickly through the Gel Matrix.

Results:
Silver staining:
• Sensitive and non-radioactive method.
• Silver staining is suitable for low protein levels
because of its sensitivity.

Coomassie Blue staining
• A relatively simple method and more quantitative than silver
staining.
• It is suitable to detect protein bands containing about 0.2μg
or more proteins.

MASS SPECTROMETRY
• While 2D- gel electrophoresis separates proteins, it doesn’t
identify them.
• MS is used to identify them which separates charged
particles or ions according to mass.

3 Major Parts
• Source ionized the sample.
• Analyzer separate the ions on m/z ratio.
• Detector sees the ions and analyzed the result.

How does a Mass Spectrometer
work?
• Samples easier to manipulate if ionised.
• Separation in analyser according to mass-to-charge ratios
(m/z).
• Detection of separated ions and their relative abundance.
• Signals sent to data system and formatted in a m/z spectrum .

•The study of proteins is very complex because the
concentration of protein is different in each organism
and in each cell of the organism.
Complexities in proteomics

• Shows that genetic alterations are not the
reason for all types of diseases.
• Helps in determining the proper treatment of
diseases.
• With the help of three dimensional analysis of
proteins we have found that HIV protease is the
enzyme which is responsible for AIDS.
Advantages of study of proteomics

Advanced screening for disease.
• One of the most important use of
proteomics in diagnosis is the
identification of biomarkers.
• The study of drugs in proteomics is
called pharmacoproteomics

Biomarkers
• Biomarkers are molecules that indicate normal or
abnormal process taking place in your body and may
be a sign of an underlying condition or disease.
• DNA (genes), proteins or hormones, can serve as
biomarkers, since they all indicate something about
your health.

TRADITIONAL TOOLS
• Mutant Hunts:
Used to expose the details of a biochemical pathway
requires the presence of a selectable phenotype.
Mutants that have lost this phenotype are then sought.
Once a mutant is found:
1. Encoding gene can be mapped.
2. Identiﬁed.
3. Cloned.
4. Sequenced.

Positive Selection
• Only mutants that fail to transport or use
the amino acid analog will grow and form
colonies on a plate containing that analog.
• In looking for mutants defective in
carbohydrate fermentation, mutant colonies
that fail to ferment the test sugar will
appear white among many no mutated red
colonies.

Negative selection
• The phenotype of an E. coli
mutant that cannot make the
amino acid alanine will only grow
on a deﬁned medium if that
medium contains alanine.

Transcriptional and
Translational Gene
Fusions
(Reporter Genes)

• Very powerful tool for analyzing various
aspects of gene expression involves
fusing easily assayed reporter genes such
as lacZ (ß-galactosidase) or gfp (green
fluorescent protein) to host target gene
promoters.

Reporter genes
• Reporter gene is defined as a gene
whose products detects or marks the
cells, tissues, organisms that express
the gene from those that do not.

• Reporter genes isolated from prokaryotes, E.coli,
are used in fishes-lacZ gene,
Cat(Chloramphenicol Acetyl Transferase gene).
• Recently luciferase and green fluorescent protein
are used.

Functions
• Encode for the products that can
be easily quantified in
transformed tissues by simple
assays.

• Also help to detect transformation
events during gene transfer
experiment.
• Encode for the products that are
themselves fluorescent or give
fluorescence reactions upon
treatment with other substrates.

Types of reporter genes
Scorable reporter genes:
Expression of this result in
quantified phenotype.
Easily detected by highly
sensitive enzyme assays.
Selectable reporter
genes:
Expression of resistance to
Toxin.
Selection of transformants
in growth media containing
selective agent.

How does lacZ REPORTER GENE
WORK?
• lacZ gene encodes the beta-galactosidase,
which catalyzes the cleavage of lactose to
form galactose and glucose.
• Reporter gene can be used under the
control of promoter or enhancer.

Types of Fusion
• There are two types of fusion
• 1st type;
Operon fusion
• 2nd type;
Gene fusion

Operon fusion
• Operon or transcriptional fusion are used when a
promoter less lacZ gene are inserted within a
target gene.
• This occurs in an orientation that places lacZ under
the control of target gene promoter.

• Factors control expression from the
target gene also control the
production of ß-galactosidase..
• Since lacZ message resulting from
fusion still contains its ribosome
binding site.
• Observe regulation is typical, but not
always due to transcriptional control.

Gene fusion
• 2nd type known as gene or protein fusion.
• Involves inserting a reporter gene lacZ into
target gene that is missing its both ribosome
binding site and promoter.

• Messages from the target and reporter gene fused.
• Truncated target gene peptide and reporter gene peptide
also fused when inserted in a proper reading frame
In this case, anything controlling the transcription and
translation of target gene will also control the ß-galactosidase
levels.

• Construction of these fusions sometimes
involves using a genetically engineered µ phage
to randomly insert reporter lacZ gene into E.coli
or Salmonella genome.

• Alternatively, the fusion can be constructed in vitro and
transferred into recipient cell.
• Once constructed these fusions, allow for monitoring of
how different growth conditions or known regulators
influence transcription/translation of the gene.

DNA mobility shifts
• Once a gene encoding a putative transcriptional regulatory
protein is identified whether the protein directly binds to
target DNA sequence.
• One approach is to take purified protein
• Add it to putative target DNA fragment
• See if the protein cause of shift in the electrophoretic
mobility of fragment.

• Linear DNA undergoing electrophoresis in an agarose
or polysaccharide gel matrix will travel at the rate
based on size of DNA fragment.
• Larger molecules travel slower than the smaller
molecules.

• If protein is bind to DNA fragment complex
will be large and travel slowly.
• This is called mobility shift or GEL shift.

EMSA
• Electrophoretic mobility shift assay
• Method used for analyzing protein DNA interaction.
• Simple, efficient and sensitive technique
• DNA moves through the gel faster when not bound to protein.
• Used to identify DNA binding proteins.

Super shift
• A super shift of the target fragment will occur
when an antibody directed against putative DNA
binding protein into binding reaction.
• Complex of anti DNA binding protein , DNA binding
protein and DNA is even larger than binding protein
DNA complex causing greater gel shift.

HISTORY
• As is the photo copier a basic requirement in
an office ,so is the PCR machine in a
molecular biology Laboratory !!!!!!!!!
• PCR is DNA replication in a test
Tube…….
Great mind behind this PCR :Kary Banks
Mullis
Developed PCR in 1985 and was awarded
Nobel prize in 1993.
PCR machine otherwise called Thermo
cycler.

OLD PCR APPARATUSNEW PCR APPARTUS

Polymerase Chain Reaction(PCR)
• PCR targets and
amplifies a specific
region of a DNA
strand.
• It is an in vitro
technique to generate
large quantities of a
specified DNA

SAMPLE OF DNA
• Often, only a small amount of DNA is available e.g. drop of blood, Semen strains,
• Single hair, vaginal swabs etc.
• Two methods currently exist for amplifying the DNA or making Copies.
• Cloning—takes a long time for
enough clones to reach maturity.
• PCR—works on even a
single molecule quickly

Requirements of
PCR
• DNA Template
• Primers
• Taq polymerase
• Deoxynucleoside
• Triphosphates(dNTPs)
• Buffer solution
• Divalent cations(eg.Mg2+ )

Factors for Optimal PCR
1.PCR Primers
-correctly designed pair of primers is
required
-primer dimer , hairpin formation should be
prevented
-length of primer

2.DNA Polymerase
-Thermus aquaticus-170° F
-Taq polymerase is heat resistant
-It lacks proof reading exonuclease activity
-Other polymerases can be used .eg:
Tma DNA Polymerase from Thermotoga maritama,
Pfu DNA Polymerase from Pyrococcus furiosus

3.Annealing Temperature
• Very important since the success and specificity of PCR depend on
it because DNA-DNA hybridization is a temperature dependent
process.
• If annealing temperature is too high ,pairing between primer and
template DNA will not take place then PCR will fail.
• Ideal Annealing temperature must be low enough to enable
hybridization between primer and template but high enough to
prevent amplification of no target sites.
• Should be usually 1-2° C or 5° C lower than melting temperature of
the template-primer duplex

4.Melting Temperature
• Temperature at which 2 strands of the duplex dissociate.
• It can be determined experimentally or calculated from
formula
• Tm = (4(G+C)) + (2(A+T))
G/C content
- ideally a primer should have a near random mix of
nucleotides, a 50% GC content
- there should be no PolyG or PolyC stretches that can
promote non-specific annealing

TYPES OF PCR
• Anchored PCR
• Reverse transcriptase PCR
• RACE PCR
• Quantitative real time PCR (Q-RT PCR)
• Asymmetric PCR
• Allele- Specific PCR

APPLICATION OF PCR
1.Molecular Identification
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• DNA fingerprinting
• Classification of organisms
Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens

APPLICATION OF PCR
2. Bioinformatics
• Genomic cloning
• Human Genome Project
3.Genetic Engineering
 Site-directed mutagenesis
• Gene expression studies

Limitations
• Sequence Information
• Amplicon size
• Error rate during amplification
• Sensitivity to inhibitors
• Contamination
• Artifacts

Transcription starts by primer
extension.

Primer extension.
1
2
3
• It is a method that is used
to map 5’ end of an RNA
• It determines the start site of transcription
• It provides the evidence
where the promoter is
located within a cloned
gene.

Primer extension .
RNA from genes have multiple
promtor that produce different-
sized primer extension
fragments.
The size of the primer extension
fragmets will be equal to one of
the “rungs”on the ladder.
Radioactive nucleotides are used
to radiolabelled the cDNA.
Reverse transcriptase+dNTPs
are added that extend the primer
at 5’ end of RNA
A DNA primer annels to the
mRNA that is complemtary to it
at 5’end.
The primer extension fragments
are separated by gel
electrophoreisis on
polyacrylamide gel.

Southern Blotting.
• A British biologist Edwin Southern developed this technique in 1975.
Definition.
“It is a technique that is used to detect DNA in a sample ,
and determine how much DNA is present”.

Mechanism.
.GEL ELECTROPHORESIS.
The fragments are separated according
to their molecular weight on agarose gel
by electrophoresis.
.BLOTTING.
The fragments of DNA are
transferred to a sheet of nylon
or nitrocellulose, which hold
the fragments immobile.
.WASHING.
.The unhybridized or unbound
probe is removed by washing with
buffer.
.PROBE LABELING.
.DNA/RNA EXTRACTION.
DNA which has been extracted from
cells is purified.
.AUTORADIOGRAPH.
After anneling the DNA fragments are visualized
by exposing the membrane to X-ray film.
.RESTRICTION ENZYME.
DNA is cleaved into fragments
by the enzyme knwn as
Restriction endonucleases.
.HYBRIDIZATION.
The labelled probe is incubated with the
complementary DNA sequence to form a
section of double stranded DNA, or to
promote hybridization of complemetary
sequences.
1
5
2
6
8
4
7 3
A nucleic acid probe with sequence homologous to the target
sequence is labelled with flourescent dye, biotin or enzyme
that generate chemiluminescent signal. .

Northern Blotting.
Definition.
“It is a technique that is used to identify RNA in a sample
on the basis of their size ,abundance, and processing”.
• This technique was developed in 1977 by James Alwine ,
David Kemp , and George Stark.

Mechanism.
NORTHERN
BLOTTING.
5
6
4
1
3
2
Washing.
Phosphorimaging.
The fragments are visualized
or autoradiographed
Probe labelling.
Radiolabeled DNA probe is
used to probed the membrane.
Gel electrophoresis.
The RNA is loaded on agarose
formaldehyde gel for
fractionation.
RNA isolation.
RNA is extracted from the cell
with RNases
Blotting.
The fragments are trasferred to nylon
or nitrocellulose membrane.
.The unhybridized or unbound
probe is removed by washing with
buffer.

Western Blotting.
Definition.
“A technique that is used for detecting specific proteins
separated by electrophoresis by the use of antibodies in
a given sample of tissue or extract”.
 This technique was also developed by Edwin Southern in 1977.

Mechanism.
WESTERN
BLOTTING.
5
6
4
1
3
2
Washing.
The unbound or unhybridized
probe is removed by washing
Autoradiography.
. The addition of hydrogen
peroxide and luminol creates a
luminescent light
Probe labelling.
Antibodies “sandwich” is used
to probe the membrane.
Gel electrophoresis.
The protein is loaded on
agarose gel for fractionation.
Protein extraction.
Proteins are extracted from the
tissue of the given cell
culture(MOUSE,RABBIT)
BLOTTING.
The fragments are transferred to
nylon or nitrocellulose
06
01
02
03
04
05

Detection of DNA-binding proteins.

Southwestern Blot.
Definition.
“It is a technique that identifies DNA binding proteins by
their ability to bind to specific oligonucleotide probe”.
It was first described by B. Bowen, Steinberg and
colleagues in 1980.

Mechanism.
Southwestern blot.
Protein bands bind to labeled
DNA is visualized by
Autoradiography.
The unbound or unhybridized
probe is remove by washing.
Radiolabeled Oligonucleotides
are used to probe the membrane
.
Proteins are separated using
standard SDS polyacrylamide gel
electrophoresis.
Protein that binds specific
DNA molecule are
extracted.
The fractionated protein bands
are transferred to a nylon or
nitrocellulose membrane.

Two-Hybrid Analysis
• Protein–protein interactions are an important part of many
regulatory circuits.
• Two-hybrid systems are cleverly designed tools that not
only measure in vivo protein–protein interaction
• Can be used to mine for unknown “prey” proteins that
interact with a known “bait” protein.

Using the Two-Hybrid System to Identify a
New Regulator
• Helicobacter pylori, a remarkable gram-
negative microbe that can inhabit the
human stomach and cause gastric
ulcers
• Provides an interesting example in
which a two-hybrid approach was used
in conjunction with proteomic and
genomic strategies to reveal a new
family of regulators

Common Methods to Analyze Protein-
Protein Interactions
• Biochemical and biophysical
approaches
• Affinity chromatography
• Co immunoprecipitation
• Fluorescence Resonance
Energy Transfer (FRET)
• Surface Plasma Resonance
• Atomic Force Microscopy
(AFM)
• X-ray Diffraction Molecular
genetic approaches
• Yeast Two-hybrid

Overall summary of Yeast two-hybrid
experiment
• Yeast two-hybrid experiments yield information on
protein protein interactions
• GAL4 Binding Domain
• GAL4 Activation Domain
• X and Y are two proteins of interest
•If X & Y interact then reporter gene is expressed

Major applications of classical
system
• Used to determine whether two known proteins
interact with one another.
• Used to identify unknown proteins, encoded by a
cDNA library, that interact with a protein of
interest.
• Powerful tool for investigating the network of
interactions that form between proteins involved in
particular biological processes

Reporter Genes
• LacZ reporter - Blue/White Screening
• HIS3 reporter - Screen on His+ media (usually need
to add 3AT to increase selectivity)
• LEU2 reporter - Screen on Leu+ media
• ADE2 reporter - Screen on Ade+ media
• URA3 reporter - Screen on Ura+ media (can do
negative selection by adding FOA

Modifications of the Yeast Two-
Hybrid system
• The hSos recruitment system
• Three protein system
• The dual-bait system
• The reverse two-hybrid system

Uses of the Yeast Two-Hybrid System
• Identification of caspase substrates
• Interaction of Calmodulin and L-Isoaspartyl
Methyltransferase
• Genetic characterization of mutations in E2F1
• Peptide hormone-receptor interactions
• Pha-4 interactions in C. elegans

Summary
• There are numerous commonly used molecular tools for
studying microbial physiology.
• With the help of above genomic & proteomic tools molecular
biologists use to examine ,DNA sequences, gene expression,
DNA–protein interactions, and protein–protein interactions
• Or when sequence of a complete genome is obtained and the
means by which that knowledge can be used to study microbial
physiology and evolution

Microbial physiology in genomic era

Microbial physiology in genomic era

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