Assignment-3

Analytical Techniques in
Biotechnology
Bt2062

ansh ruhela
Bs19b006
❖ The following assignment explains the below mentioned topics, according to my under-
standing of what has been taught in the class by Prof. S Mahalingam.

• Vectors (cloning and expression)

• Polymerase Chain Reactions (PCR)
• Molecular Cloning
• Protein Expression Systems and Purification Methods.
• Protein-Protein Interactions.
• Protein subcellular localisation.
• Transcriptional regulation
• Cell Cycle analysis
• Gene expression Analysis.
• Methods of detecting DNA, RNA & Proteins.
• Post Translational Modifications in Proteins.

*References being used here are only the class lectures and the prescribed textbooks, if any.
➢ Vectors:
A vector, in molecular biology, is a molecular DNA used as a transporter of foreign DNA material
into some other cell, to cause either replication or expression of that particular carried gene into the
host cell. Examples are Plasmids, cosmids, and lambda phages. Every vector, as a common charac-
teristic contain an insertion point called as origin of replication, a multi-cloning site and the se-
lectable markers.
• Vector Classification:
Cloning vectors and Expression Vectors are the two types of vectors that are being widely
used.
• Cloning vectors are the small pieces of foreign DNA fragments that are inserted for clon-
ing purposes only. These vectors can be used for expression but are very limited in applica-
tion.
• Expression vectors, also known as expression construct, are usually plasmids or viruses
that are designed specifically for protein expression in a cell.

cloning vectors expression vectors

Differences A small piece of DNA which is used A plasmid that not only introduces a
to introduce the foreign gene of in- gene of interest but also aids in the
terest into the host cell. analysis of the gene of interest via rele-
vant protein product expression.
Role it is used to obtain multiple copies of it is used to obtain or analyse the gene
the gene of interest. product, which may be RNA or protein
expresses by the inserted gene.
Types These can be plasmids, phage, BACs, Only a plasmid.
YACs or NACs .
Feature a typical cloning vector consists of a This consists of regulatory elements
origin of replication, unique re- like enhancers, promoters, termination
striction sites, reporter genes and sequences, transcription initiation
antibiotic resistance sites. sites, translation initiation sites in ad-
dition to all that in a cloning vector.
 • Available cloning & expression vectors:
• Plasmid Vectors: These are single or dual promoter vectors. There is also a Bi-cistronic
vector that express two proteins via a single promoter
Fusion Protein Expression only
prokaryotic expression
vectors • Viral vectors: These are DNA & RNA viral
GST, hIS; MBP
vectors.

PLASMID VECTORS
Eukaryotic expression
vectors
VIRAL VECTORS DNA viral vectors:
RNA viral vectors:

Baculovirus vector Adenoviral Vector

This vector is used to express a lot of therapeutic loading for This is limitedly used for protein expression but extensively
medical purposes. It is a less expensive and consists of a for the gene therapy.
strong promoter.

Onco-retrovirus vector(simple) Lenti-retro virus vector(complex)

These vectors can not infect the primary cells and are used to Lenti is greek for slow. Therefore these are slow growing vi-
transfer a gene or to express one for animal systems. ral vectors and can also effect the primary cells(non-divid-
ing).
ip vector (cloning)

ii PCI vector (mammalian vector)

iiiT/A cloning expression vector

iv Prokaryotic expression vector

Fusion expression vector: In bacterial pro-
karyotic expression vector, we do fusion
cloning. we use the bacterial expression to
express the high level expression of our
gene of interest, that is the purified form
and for purification we make a fusion pro-
tein. Fusion protiens are used according to
their affinity matrix, GST fusion protein has
high affinity to glutathione beads whereas
HIS fusion protein has high affinity to
NiCKEL.

• One significant step while using fusion proteins is to do INFRAME fusion. In inframe fusion, the translation of the
protein starts in the beginning of fusion. Upstream we will have the fusion.
v Bi-Cistronic Vector(eukaryotic vector)- different structures
Bi-Cistronic vectors have variable structures
as shown beside. Considering first one
pL-IRES-SEAP-1, in this single promoter vec-
tor, there is human Cytomegalovirus en-
hancer and promoter. Then we have lucifer-
ase gene and IRES, following it is SEAP.

Being Bi-cistronic, despite having single pro-

moter it expresses two different proteins
with single RNA as only one terminator is
there(one poly-a transcription sequence
only). Translation terminator should be in
gene only otherwise there will not be the ex-
pression of other protein.

• One important aspect of Bi-cistronic vectors is why we want to express two proteins at once?
The answer is to this is the interactions between different genes. Suppose we have two different genes A&B
and gene B may be critical for the activity of gene A, then we have to express them both together.
 Next we studied Baculovirus vector.
Baculovirus vector:
vi baculovirus is a DNA virus, 120kb big genome

Cloning in baculovirus vector is done by ho-

mologous recombination and not only by
cloning.

We need two vectors for this; a transfer vec-

tor and the baculovirus DNA. For cloning,
the gene of interest is first cloned in the bac-
ulovirus vector DNA via tranfer vector, con-
taining the baculovirus polyhydrine gene
promoter.

The double cross over event interchanges

and place the gene of interest on the place
of polyhydrine gene.

Simple Retro-viral vectors:

vii simple retrovirus vector

The above shown Simple Retrovirus have Long Terminal Repeats (LTRs), between these two LTRs at the ends,
there are 3 critically important structural genes namely GAG, POL & ENVelop genes; without these genes infectious parti-
cle will not form. Just after the LTR sequence, there is a small sequence called Primer Binding Site (PBS).

Since retro viruses are RNA viruses, retro viral vectors also have RNA as their genetic material. When the viral
vector is injected into the host, it will integrate itself into the host genome, and for that the RNA needs to be converted
to DNA for integration because the host genome is fully DNA based. For this an enzyme Reverse transcriptase is used and
the process being called as reverse transcription. The PBS is the site where the conversion of RNA to DNA is started out.
For this conversion the virus utilises host’s tRNA.

Then in between the PBS and GAG sequences, there is a PACKAGING SEQUENCE, this sequence interacts with the
virus particle and the viral infection causing genome, that is the interaction between the RNA and the infection protein.

Then we have Splice Donor & Splice Acceptor(SD & SA) sequences, after the integration into the host genome the
integrated virus genome will be expressed and the virus being a single RNA, it has to undergo splicing. The feature of this
vector is that it cleaves the GAG and POL as one single protein.
Gene Transfer Process in Simple Retrovirus Vectors: To utilise these vectors for gene transfer, we remove the POL gene,
ENV gene and a part of GAG gene and replace the SA sequence just after the TRANSGENE (which is our gene of interest).
All the proteins that were present in the vector construct are crucial for the expression and by removing and changing
them with the transgene doesn’t allow the expression of the gene so what we can do is TRANS integration; which is also a
special feature of this vector that it is not necessary to express the proteins from CIS only, that is we can express the pro-
teins from other secondary plasmid.

We can do the synthesis of remaining part of GAG and POL genes in one vector
as they are cleaved into one single protein and for the ENV protein we can use another vector.

Another special feature of the simple retrovirus vectors is that the ENV genome contain a specific se-
quence that we can swap from other virus particles. To make a virus particle, the envelop gene interact
with the GAG protein. Every retrovirus infects the cell by a receptor mediator interaction; there are spe-
cific receptors on the host’s cell surface which are recognised by the ENVelop proteins on the host’s cell
surface. For example: We have two different types of retrovirus and a host cell; type A(virus) and type
B(host cell). The envelop of type A do not detect the receptor of the type B host cell; therefore we can
swap the ENV of the virus with some other retrovirus which can detect the receptor of the type B host
cell. Hence depending on the type of the cell we want to infect we can change the the envelop part.
Since this retroviral vector is of the size ranging 8kb-16kb, and we remove everything in between the
LTRs(GAG,POL,ENV sequences); we can put any number of genes not exceeding the size of the vector
otherwise the packaging will be difficult and the efficiency will be low.
There is no specificity for the integration of this virus, it is a completely random event. Wherever the vi-
rus finds the transcriptionally active region(open DNA) it will integrate itself.
The simple retroviral vectors can be utilised in dividing cells only. This is because when the cell divides,
the nuclear membrane dissolves and the DNA material is in the cytoplasm against the single plasma
membrane; no nuclear membrane now. The simple retroviral vectors do not have any mechanism to
break and cross the nuclear membrane and integrate itself into the genome but for the plasma mem-
brane it does.

Complex Retroviral Vectors: Lentiviral expression vectors

viii Lentiviral Expression Vector(complex)

Four differ-
ent plasmids
are used in
this vector
which re-
duces the ef-
ficiency of
the vector.

In the above shown Lentiviral vector, in addition to the GAG,POL & ENV genes there are some more regulatory genes
(VPU, VIF, VPR, NEF, TET, REV) which makes this vector of a complex type. Due to the presence of these extra genes only
the virus is able to infect the non-dividing cells also.

For a non-dividing cell, the nucleus is intact; so the virus has to

have the ability to transfer its viral DNA genome into the nucleus from the cytoplasm through the nuclear membrane.
VPR (in some cases TAT also) helps the whole DNA virus to get into the nucleus.

Structural differences with simple retro-virus vectors: With the GAG sequence, there is a RRE sequence also (Rev Re-
sponse Element sequence). as we know that the replication and transcription happen in nucleus but transcription
happens in cytoplasm in rough endoplasmic reticulum and mRNA is needed to be transferred to cytoplasm for that. REV
protein binds with RRE and transfer the viral RNA from nucleus to cytoplasm.

For integration of the virus into the host, synthesis of ouble stranded DNA from RNA is needed. One of the strand is syn-
thesized using the primer binding site and the other one by using central PolyPurine Tract (cPPT) as a template. This also
enables us to put any number of genes in the viral vector not exceeding the size in any direction (3’-5’ or 5’-3’).

There are 4 proteins that are synthesized in GAG,POL sequences namely;

I. Protease: This cleaves the GAG and POL proteins into functionally active or mature form.
II. Integrease: This one is for integrating the viral genome into the host genome.
III. Reverse Trabscriptase: This one is required for conversion of RNA to DNA.
IV. RNA Scratch: To dissolve the RNA strand after synthesis of RNA-DNA hybrid.

RNA scratch and Reverse Transcriptase form a hetero-dimer and then act as both in a single dimer mole-
cule. The heterodimer digests the RNA but the cPPT rsists this digestion and gets attached to RNA which
then acts as a primer for another strand.

Next is the envelope part in the structure. Here the VSV-G envelop is shown, which is a swapped envelop. This VSV-G (Ve-
sicular Stomatitis Virus- Glycoprotein) is a high efficiency envelop a it can withstand the centrifugal force applied for con-
centration management to gain high efficiency.

• PCR (Polymerase Chain Reaction):

PCR is a technique used to amplify the selected region of a piece of DNA, exponentially. The technique was devel-
oped by KARY MULLIS, a research scientist at California Biotech Company in 1983. The amplification provided by the
PC is very powerful. FOR EXAMPLE: There is E. coli bacteria in a piece of meet along with some other non-pathogenic
bacteria in it. There is huge amount of DNA in that piece of meat so, searching for the E. Coli’s GENOME will be a hec-
tic task in that large piece of DNA and PCR helps in the same task by amplifying the E.Coli’s DNA and making it easy to
identify in the bulk DNA.

PCR is an in-Vitro technique. PCR amplification is achieved by using oligonucleotide primers. These are typically short
and single stranded oligonucleotides which are complementary to the outer regions of the kmown sequences.

The Primers here are the oligonucleotides as shown abive in the diagram; and the template strands are the dena-
tured strands of the large DNA sequence resulting in the formation of the new DNA strands complementary to the
 parental DNA strands. The 5’ end is pre-defined as the 5’ end of the primer while the 3’ end is potentially ambiguous
length. in

PCR Mixture:
o The target DNA (which is to be amplified).
o Two primers, each 20-30 nucleotides or bases long (one for forward end and other for backward end).
o Thermostable DNA Polymerases.
o Nucleotides (dNPPs)
o Mg2+ as a cofactor for DNA polymerase.
ix thermocycler for PCR

o
The whole mixture is subjected to the thermocyclers or water baths as shown above.
Primer: What is it?
A primer is a short synthetic oligonucleotide which is used in many molecular techniques and PCR is one of
them. These primers are specifically designed as the complement of the genes that we want to amplify or work
upon.
Primer Selection: significance
A primer is a oligonucleotide sequence which targets the opposite base pairing of single stranded nucleic acid
(A-T & G-C only). The primer should be fault free otherwise despite of our perfect PCR setup we wont get the
right results.
Primer Specificity: is of three aspects.
o Universality: A primer that simplifies a type of DNA in the whole sequence; for instance, all of the bac-
terial DNA is amplified in the pool of the DNA, therefore acting at different places in a line of DNA.
o Group-specific: Those amplifying all di-nitrifiers (a group of genes) for instance.
o Specific: those amplifying only a specific piece of DNA.
Primer design criteria:
Primers are not available in commercially pre-manufactured form. We have to design the primer in accordance
to what type of gene we are going to amplify and then we can produce the primer on a large scale commercial
production as per our need.
1) Primer uniqueness: the primer should be unique to what we want to amplify otherwise the genes other
than of our interest will be modified along in multiple cycles.
2) Primer length: the length is around 16-30 bases , depending upon the use.
3) Melting Temperature: Of the two primers, the melting point should not differ by much value, it should be
close and required for efficient annealing.
4) GC content range: G-C content range is critical for melting point, as G_C has 3 bonds while A-T has 2 only.
Thus giving rise to melting point and more time for annealing as well as denaturing; hence G-C=A-T.
5) 3’ clamp properties (terminal residues): DNA goes from the 5’-3’ end, and thus the binding of the primer
at the 3’ end is very critical. : if we put some half of the number of bases correctly on the primer and the
6) binding starts for the first 10 bases that are complimentary and then stops, even then it is okay. Therefore
, to ensure binding we put G or C at the 3’ end.
7) Avoid hairpins in the primers.: we have to ensure that there must not be any repeating sequences other-
wise on the other side there will be opposite to those sequences and there will be like hairpin formation
of structure.
 8) Length of Amplified regions: length should be maintained for better efficacy.
9) Avoid primer-primer interaction: there should not be any complimentary sequences on the two primers to
themselves otherwise both of them will bind to themeselvs and form a dimer rather than binding to our
targeted gene ofinterests.
10) 9. Melting temperature compatibility: temperature of melting should be nearly same forbetter annealing.
Forward and Reverse primers: two types of primers are required in PCR as it is mentioned under above PCR
mixture heading.
Types of primers:

• OLIGO-dt (16-20 T primer): This primer is at the poly-A tail of mRNA.

Every mRNA has multiple A bases at its 3’ ends for stability and transportation procedures. Suppose we want to
clone particulas mRNA which expresses a protein or a coding sequences. Then we have to use the mRNA and
not the DNA as we are having the coding along with non-coding sequences and when we express a protein,we
want only coding sequences which are present in mature mRNA form. We cannot directly express the mRNA as
we have a double stranded vector and since Poly-A tail is present in 99 % of the DNA therefore we use that as a
template for designing the primer.
• Random Hexamers: Template for cDNA.: this is used for synthesis of cDNA but not from mRNA.
• Gene Specific Primers: Usually uses reverse primers.: so, whatever we want to amplify, our primeer
should not be much costly and the must be unique and specific.
DNA polymerase- Enzyme:
This one enzyme is responsible for the copying of the sequence starting at the primer from the single DNA
strand. Commonly used polymerase is Taq DNA polymerase derived from the hyperthermophilic organism
Thermus Aquaticus.
This enzyme can withstand the 94 degree celcius temperature of denaturation, if we are having repeated cy-
cles, say 25 cycles, then we need a thermally stable enzyme. With being a thermally stable, it should of high
fidelity also. All the enzymes commits mistakes while copying DNA, sometimes mutations may even go up to
1kb. With Taq, it is more mutation prone enzyme, very likely to have a mutation while using Taq, at the same
time it is robust also and its kinetics are much faster as compared to other enzymes. More heat toleration leads
to low mismatch in copying.
WORKING OF PCR: Amplification of Target sequence
Having all the things, our target DNA, primers, and everything else, we put them all in a tube and keep the
tube in the thermocycler/ PCR machine setting up the program at the denaturation temperature around 95 de-
gree celcius.
Annealing temperature depends upon the melting temperature of Primers: 4(G+C)+2(A+T); this formula works
on the primers having less than 25 bases, otherwise we have to consider the salts and buffers that we used in
the PCR amplification as higher salts concentrations alter the melting temperature significantly. Annealing tem-
perature is 5-10 degree less, we need to check for which optimum annealing temperature is best.

Whenever we set up the PCR our template should be very low. It can be at nanogram levels or at picogram lev-
els while dealing with plasmids. For human genome amplification we deal with microgram level PCR.
 x working of PCR

beside it is shown how the amplification happens.

Blue and green are the forward and reverse primers
respectively. In the procedure we see that the re-
verse primer amplifies and it goes, so how does the
amplification stops there only? There is no reaction
or any mechanism to stop the primer amplification ,
what we rely on is the kinetics of the primer.

Every enzyme has its kinetics , for instance, the Taq

DNA polymerase can only amplify 2kb of DNA per
minute.

The primers usually don’t stop at the specified posi-

tion, they move 3-4 bases extra and after tha they
start from their specific primer sites. So if there is ex-
cessive amount of original DNA is present then we
wil get a band of different amplified regions.

Terminology and Types of PCR:

q-PCR- Real time/Quantitative PCR RT-PCR: reverse transcription PCR qRT-PCR: Quantitative reverse transcrip-
tion PCR
In this type of PCR we can specify “how much” of the gene is to be amplified.

In RT-PCR, there is conversion of RNA to DNA for viral genomes, the mechanism for reverse transcription happening natu-
rally in virions is different from what is happeining in PCR. The IN-VIVO and the IN-VITRO applications should be verified
before.

Other types of PCR are:

I. LONG PCR: this one serves the purpose of long amplifications upto 25kb. Nowadays we can extend this upto
40kb.
II. INVERSE PCR: this amplifies the unknown DNA that just adjacent to the known DNA.
III. QUANTITATIVE PCR: this amplification is done with respect to time; and compared with the standard DNA.
IV. NESTED PCR: This one involves two consecutive PCRs; for 25 or more cycles. The first PCR uses the primers that
are actually not for the gene of interest but to flank the region short containing the gene of interest. The second
PCR uses the product of the first one in conjunction with the other or same pimers to amplify the sequence of
interest.
V. HOT-START PCR: this one is used to increase the yield of the desired amplification and supress the unwanted am-
plifications simultaneously. In this technique, we add the prmers/ enzymes after 10-15 minutes of the heating the
PCR waterbath so that the complete denaturation happens before primers attaching themselves to the DNA.
Thus, supressing unwanted binding of the primers.
Can we use RNA as the target material in PCR amplification? NO! we can not use RNA as the target material for amplifi-
cation in PCR because there is no such enzyme developed or discovered to work with RNA. Taq DNA polymerase is a
DNA dependent DNA polymerase. We do not have any enzyme that is RNA dependent RNA polymerase but we do
have one which is DNA dependent RNA polymerase, which synthesizes RNA from a DNA strand.

TROUBLESHOOTING THE PCR:

I. If little or no product is detected: in this problem, we have to check if our setup does have adequate amount of
fragments.
 II. 10-fold dilutions for template strands: for this to be the issue, we may obtain a continued carry over or different
bands of genes amplified or if there will be more un-specified fragments, the primers will bind to them also and
exhaust in the first few rounds and thus, leaving the gene of interest un- amplified properly.
III. Check for the presence of inhibitors in the template DNA: For the purification of the DNA before mixing, different
chemicals are used which may get attached with the template DNA and obstruct the primers for binding, supress-
ing the enzyme activity and ultimately inhibiting the PCR.
IV. We can add enhancers in the PCR mix like DMSO, polyethylene glycol for effective denaturation of DNA. Glycerol,
Bovine serum albumin to support the stability of the enzymes.

Applications of PCR:
• Molecular identification Sequencing Genetic Engineering
o DNA fingerprinting Bioinformatics Site-Directed Mutagenesis
o Genotyping genomic Cloning Gene-Expression Studies.
o Mutation Screening human Genome Project
o Drug Discovery
o Genetic Matching
o Detection of Pathogens

• MOLECULAR CLONING:
DNA Cloning is referred to the technique of producing copies of DNA FRAGMENTS. Again, RNA can not be
cloned before converting it DNA as was the case in retroviruses. DNA cloning canbe achieved by two different
approaches namely: 1.) cell based 2.) PCR based.

DNA copying allows any part of the DNA being copied in unlimited amounts , that canbe used for differ-
ent purposes like studying the sequences, PCR and many other basic Genetics Experiments.
Stable propagation of DNA sequences like in the case of viral DNA transferred into the bacterial cell, not
stable as it rearranges and recombines with the bacterial genome.
A single DNA molecule can be amplified allowing it to be studied, sequenced and manipulated.

Cloning methodology:
A vector and the gene of interest are cut with the same Restriction enzyme so that they will be complimentary
to each other. After the gene is inserted into the plasmid, it is ligated with the ligase enzyme and the recombi-
nant DNA molecule will be in the bacterium as extrachromosomal DNA , it will not integrate into the Bacterial
xi insertion of vector with gene of interest Genome.

Plasmid Cloning Strategy:

This include the five must to do steps:
1) Enzyme restriction digest of DNA sample; we need
an enzyme to cut the DNA that we want to clone and
the same enzyme will also cut the vector that we are
going to use.
2) Enzyme restriction digest of the DNA plasmid vec-
tor, as mentioned in above point.
3) Ligation of DNA sample products and the vector.
4) Transformation of the ligated products into bacte-
ria.
5) Growth on AGAR plates with antibiotic resistance.
Step 1: Restriction Digestion of DNA Fragment:

xii Restriction Digestion of DNA Fragment

Here, EcoR1 is the Restriction Enzyme
used for digestion of the gene. We mix
the enzyme with the fragment and it
acts at its active sites, giving out sticky
or protruding ends.

Step 2: Restriction Digestion of the plasmid vector DNA:

xiii Restriction Digestion of the plasmid vector DNA

The same Restriction enzyme EcoR1 will di-

gest the plasmid DNA. In between the LACZ
gene, there is the multi-cloning site where
the EcoR1 acts and cuts the circular DNA
into a open piece of linear DNA.

Step 3: Ligation of DNA sample and the Plasmid DNA:

xiv Ligation of DNA sample and the Plasmid DNA

so, now we mix both of the digested DNAs into one and add the ligase enzyme along with some ATPs, as this
one requires the energy to proceed further. As shown above ,we will get the combined DNA form from the 5’-
3’ end. The mixture will be kept at 16degree for overnight for the proper functioning of the ligase enzyme.
We cannot use the already freezed ATPs solution , those have to be arranged in Situation.
In the ligation step ,we may encounter two problems:

1) For both of the 5’ ends of the vector they will be having a ligation site so which orientation will ligate is can not
be determined. There are 50 % chances of each orientation and this can not be controlled.
2) Since the EcoR1 leaves the phosphate and the carboxylic group after the digestion, the vector plasmid can reu-
nite without even the need of ligase enzyme itself , hence inhibiting the ligation that we want to happen. This can
be overcome by adding a phosphatase enzyme that will eat up the phosphate group in the vector plasmid DNA
and thus restricting it to not self-ligate. Phosphatases are commercially available but the enzyme is very sticky
with the DNA thus, blocking the ligase enzyme site. So we have to make it free of Phosphatses.

xv cloning 5' and 3' protuding ends.

This figure above shows how the enzymes cut the DNA into two parts. Different enzymes do that differently.

Step 4: Transformation of ligation products:

The processby which the ligased DNA is transferred to the cell is called TRANSFORMATION. This can be achieved by two
methods: 1.) one is the CHEMICAL method by using CaCl2 and heat shocl to induce entry into the cells.
2.) other one is the method of ELECTROPORATION; which is based on short pulse of electric discharge to facili-
tate DNA uptake.

xvi transformation method with CaCl2

As in the above figure shown, the E.coli cells of particular type are used to clone. They are concentrated using a centri-
fuge and we put the concentrated bacteria with ICE and the CaCl2 solution. Then after suspension, the cells are kept at
(-)80 degree temperature for about 1-2 hours . after this we put our plasmid DNA along with the cells on the chilled ice
for ligation that takes up from 30-45 minutes. Mixing is done just by putting up a finger and shaking it. Then we take the
cell into a water bath for heat shock at 42 degree celcius but for just 45 seconds, beyond 60 seconds the cell may die. And
then first this mixture is put with LB+ on plate for about 35 minutes to60 minutes and shake for mixing, and after that the
antibiotics are added. We do this extra time wait to add antibiotics as earlier the bacteria was at negative 80 degree
celcius temperature, time is required for antibiotic gene resistance expression before adding of antibiotics. If it is not
done then we may not get any colonies of bacteria on the plate.
xvii Transformation by electroporation

ELECTROPORATON: in this, instead of resuspending in the CaCl2 solution we use STERILE WATER. This should be ex-
tremely pure not containing any salt otherwise, while giving shock, the cell may die due to spark. Rest steps are same as
in there CaCL2 method.

Electroporation is more efficient than chemical based methods. Irrespective of the size of the plasmid, it may be even
some 10kb long , no issues in this method. We just require a electroporator machine for this.

Step 5: Growth on Agar Plate:

xviii antibiotic X-GAL plate identification

Blue colonies rep-

resent Ampicillin-
resistant bacteria
that contain pVec-
tor and express a
functional alpha
fragment from an
intact LacZ alpha
coding sequence.

White colonies
represent Ampicil-
lin-resistant bacte-
ria that contain
pInsert and do not
produce LacZ al-
pha fragment
The presence of blue colonies represents the re-ligation of the plasmid vector, as this is exhibited by the intact LACZ gene
only , that will happen after re-ligation. If the vector and the insert is ligated, then we see the white colonies along with
very few blue ones. White colonies appear due to absence of lacz alpha fragment resulted by no re-ligation.

BLUE-WHITE screening:

After the all five steps above , for a few bacteria we may get the insert with vector and for some we may not. For this we
do the blue-white screening test.

Only the E.Coli with resistant plasmids will grow on the antibiotic plate. Therefore those whose
LacZ gene is intact ( without the insert , self re-ligation) can grow on X-GAL and give blue colonies as mentioned above
also. This verifies that there is no insertion at all. On the other note , if the LacZ is negative, that is not expressed with the
X-GAL, there is insertion and that gives out white colonies with Recombination. All this happens due to the alpha-com-
plementation.

α -Complementation:

The Lacz gene encoding first 146 amino acids, which is called the Alpha- fragment is on the plasmid. The remaining of the
Lacz gene is found on the host chromosome. If this lacz with alpha fragment is intact, it will interactwith the host chromo-
some and produce the enzyme called beta-galactosidase enzyme.

Enzymes used in molecular Biology:

Alkaline phosphatase: This removes phosphate groups from 5' ends of DNA (prevents unwanted re-ligation of cut DNA).

DNA Ligase: This one Joins compatible ends of DNA fragments (blunt/blunt or complementary cohesive ends) via Using
ATP as the driving energy source.

DNA Polymerase I: This Synthesises DNA complementary to a DNA template in the 5'-to-3'direction; starting from an oli-
gonucleotide primer with a 3' OH end.

Exonuclease III: Digests nucleotides progressively from a DNA strand in the 3' -to-5' direction.

Polynucleotide Kinase: Adds a phosphate group to the 5' end of double- or single-stranded DNA or RNA; using ATP as the
enrgy source.

RNase A: Nuclease which digests RNA, not DNA.

Taq DNA Polymerase: Heat-stable DNA polymerase isolated from a thermostable microbe (Thermus aquaticus).

Restriction enzymes:
The restriction enzymes used in molecular biology labs cut within their recognition sites and generate one of three dif-
ferent types of ends namely; 5’ overhangs and 3’ overhangs. Overhangs , sticky ends and protruding ends are all same.
5’ overhangs: When the enzyme cuts the DNA Asymmtrically, leaving behind a short strand of a few bases at one strand
only, from the 5’ end. BAM h1 cuts in thus manner.

3’ overhangs: Again the same asymmetrical cutting , but the xtending segments are from the two 3’ ends in this 3’ over-
hangs. Usually kpn1 cuts in this manner.

Blunt Ends:
Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends without overhangs. SMA1 is
an example of an enzyme that generates blunt ends.
Converting a 5’ overhang to blunt end:
T4 DNA polymerase can be used to fill in 5’ protruding ends with dNTPs. This technique is used in joining the incompatible
ends of the DNA fragments. Once the ends have been blunted, the ligation can proceed.this leaves the fragments too
much sticky therefore we have to wash that with phenol.

Converting the 3’ overhang into blunt end:

T4 DNA polymerase has a 3’-5’ exonuclease activity. In the presence of excess dNTPs, will convert a 3’ protruding end to a
blunt end too. After this Ligation can now proceed.

Directional Cloning:
As we have discussed the problem of orientation in cloning, one absolutely wants the cloning to be done in the preferred
orientation. While using a single restriction enzyme it is not fairly possible, but using two different enzymes makes this
work out. We construct the foreign DNA with the same two different enzymes, this will solve the problem of 50% orienta-
tion as we encountered in the above discussions. Now since the sites were same due to the same but two different en-
zyme consumption. Foreign DNA will only be inserted in one direction. The usage of two different enzymes gives two sin-
gle stranded ends cut which lowers the chance of self ligation.

Alkaline Phosphatase:
Alkaline phosphatase enzyme removes 5' phosphate groups from DNA and RNA. It will also remove phosphates from
nucleotides and proteins. These enzymes are most active at alkaline pH.

As showm in the figure nearby, after the removal of phosphate groups, the self-ligation for one strand is not happening
and the other strand stays nicked. The one strand that is ligated will break off by the bacterial mechanismand the self-
ligation is supressed.

There are two primary uses for alkaline phosphatase in DNA manipulations:

• Removing 5' phosphates from plasmid and bacteriophage vectors that have been cut with a restriction enzyme.
In subsequent ligation reactions, this treatment prevents self-ligation of the vector and thereby greatly facilitates
ligation of other DNA fragments into the vector (e.g. subcloning).
• Removing 5' phosphates from fragments of DNA prior to labelling with radioactive phosphate. Polynucleotide
kinase is much more effective in phosphorylating DNA if the 5' phosphate has previously been removed.
Removal of alkaline phosphatase before the cloning is very essential for good efficiency, by washing.
xix alkaline phosphatase mechanism

Generating new Restriction Enzyme Site at a blunt end:

We can create anew restriction enzyme site at the blunt ends by using oligonucleotide linkers. These linkers place the
sticky ends in place of the blunt ends.

Identification of the positive clones:

• One of the first steps is to identify clones carrying the recombinant plasmid, with the desired DNA insert.
• This can be done by 'picking' clones - choosing individual bacterial colonies in order to isolate the plasmid DNA
from each of them.
• Single bacterial colonies are grown in culture broth containing the selection antibiotic in order to maintain the
plasmid. If there is ampicillin restriction in plasmid, we have to add ampicillin in the plate.
• The plasmid DNA is extracted by the standard minipreparation technique and then analysed by restriction digest.
• After digesting the DNA, different sized fragments are separated by agarose gel electrophoresis and the sizes de-
termined by comparison with known DNA molecular weight marked.
Biological Role of restriction enzymes:
• Most bacteria use Restriction Enzymes as a defence against bacteriophages. Restriction enzymes prevent the rep-
lication of the phage by cleaving its DNA at specific sites.The bacterial DNA is protected by Methylases which add
methyl groups to adenine or cytosine bases within the recognition site thereby modifying the site and protecting
the DNA.
• They do the workof cutting DNA by hydrolyzing the phosphodiester bond.
• R.E. are a useful tool for analysing Recombinant DNA
▪ checking the size of the insert
▪ checking the orientation of the insert
▪ determining pattern of restriction sites within insert

Isoschizomers and Neochischizomers:

• Restriction enzymes that have the same recognition sequence as well as the same cleavage site are Isoschi-
zomers.
• Restriction enzymes that have the same recognition sequence but cleave the DNA at a different site within that
sequence are Neochizomers.

PCR cloning:
There are three clonking methods for PCR products:
1) Blunt end cloning
2) Sticky end cloning
3) T-A cloning
Factors affecting the PCR cloning:
• Nature of the Insert: All PCR fragments will not clone with the same efficiency into the same vector.
• Insert Size: The size of the fragment being cloned is a primary contributor to the overall cloning effi-
ciency. Large fragments of DNA (≥ 5 kb) are amenable to cloning in high-copy number vectors, yet at a
much lower efficiency.
• Vector-to-Insert Ratio: Optimization of molar concentration ratios of the vector to insert is critical to
ensure efficient cloning. insert ratios: 1:1, 1:3; based on the size and insert.

• PROTEIN EXPRESSION:
After the cloning is done, now we want to express our proteins which are encoded in the insert or in the genome of the
plasmid.understanding the Protein expression helps in various discoveries and research goals.
To understand the working and essential functioning of the proteins , for therapeutic purposes, for target spe-
cific drug discovery, For vaccine manufacturing and for producing biotechnological enzymes. Protein expression is also
effected by the proteinprotein interaction. Insulin production is also possible only by using recombinant protein expres-
sion.

Recombinant proteins and their expression:

Recombinant proteins can be made by first cloning or synthesizing the gene and putting it through the
expression construct or vector and then transfecting the cells but all this process may not be successful if
we don’t know hat type of protein we are going to express, is the protein be expressible in the cells that
we chose to transfect,will the protein be functional in the pH conditions of our system, temperature
changes and many other factors.

Host selection for the expression of the protein depends if the protein is prokary-
otic or eukaryotic , is the protein is cytoplasmic or organelle specific. Amount of protein needed to be ex-
pressed depends onour downstream application, for functional studies nanograms levels will do, for
 antibody production we go for micrograms to milligrams levels, and for commercially used proteins we
may go for kilogram levels.
For expression of human proteins, mammalian cells serves the purpose best but are expensive to use and are
slow on process.insect cells like that of baculovirus gives the many advantages of post translational modifica-
tions and etc, and are also cheaper and faster to give results. The cheapest among all is E.Coli,but it does not
work for many of the proteins.

Getting the gene by using PCR is very ineffective. Amplification is error prone most of the time; even in the
case of high fidelity , if there will be a mutation , our work goes off to zero progress and we have to restart our
work. Also the size of genes expressing proteins are relatively big so that also attracts the error possibility.

Protein expression in PET vector system:

We have to clone our gene inframe .expression. Three bases are responsible for one codon. The + and – are
just used to denote the orientation of the multi cloning site. Recognisation sequence plays the important role
in expression , should be in correct frame.
Vectors, tags and solubility while expressing protein:
❑ The highest level of expression is not always the best. High expression can also give the most insoluble
material. Sometimes reducing the temperature and the expression level can give more overall soluble
material.
❑ Tags and protease sites can effect protein solubility/ stability. Most tags will make protein more solu-
ble, but it may not stay soluble if the tag is pulled off..
❑ Proteins are effected by Some proteolytic sites. (Incorporating a TEV cleavage site reduces the solubility
of proteins).

Protein purification (gene fusion strategy):

Most target protein lack a suitable affinity ligand usable for capture on a solid matrix. A way to circumvent this
obstacle is to genetically fuse the gene encoding the target protein with a geneencoding a purification tag.
When the chimeric protein is expressed, the tagallows for specific capture of the fusion
protein. This will allow the purification of virtually any protein without any prior knowledge of its biochemical
properties.

His tag allows easy purificationof the protein. Imidazole is used as a matrix separator here, use of imidazole
depends on the tag used. For example is GST tag is used , we would have taken glutathione for this.
Using TAGS in fusion proteins allows to produce a good yield of protein and prevent proteolysis with
incresead solubility. On the contrary, using TAGS sometimes resulted in the toxicity and cleaving the tag is an
expensive task as it requires some specialised enzymes like enterokinase.
Commonly used tag affinity systems in recombi- Cleavage sites
nant protein expression
Maltose binding protein fusion Factor Xa site
Glutathione-s-transferas Thrombine or factor Xa site
Thirodexin fusion protein Enterokinase site.

There are problems faced whem expressing proteins. The major problem is the insolubility of the protein. To
tackle this one out, we need to re fold the protein and give heat shocks to produce chaperons that may help in
re folding of the protiens. Sometimes the expression goes off useless at the end and we have to change our fu-
sion protein and restart the work.
Other common problems in E.Coli expression system are the toxicity, incorrect folding, unstability and the for-
mation of inclusion bodies with low solubility. To overcome these we have to choose our tag ,standard E.Coli
strains , fusion proteins and leading sequences.
Protein expression in mammalian cells via gene therapy:
When there is a need of wild type protein or deficiency of the same, due to which there is a defectin function-
ing of a particular protein, then via gene therapy a functional copy of that gene is transferred into the cell to
overcome the deficiency or the defectcaused by the same. There are many diseases like OTC deficiency, sickle
cell anaemia and many more. Applications of gene therapy extends to the treatment in cancer , by correcting
the tumor suppressor gene .
GENE THERAPY- METHOD OF GENE DELIVERY:
By what means the gene will be delivered , to which tissue , depends on the vehicle we use to caary the gene
into the organism. We can use the viral vectors like adenoviruses, retroviruses and lentiviruses, as discussed
before, lentiviruses can be used for dividing cells also. Then we can use plasmids, that are non viral vector
based transmission at some particular site or target organ, but not for the whole body.

Viral vectors have good response in gene therapy: as virus cells are very efficient in transferring the viral DNA
into the host cell. Viruses can also be deployed for specific target cells, depending on the viral attachment pro-
tein (like glycoproteins). Non essential genes(virus pathogenicity) can be replaced by the gene of interest (exog-
enous genes).
Viral vectors are generated for genetherapy using replication competent or defective ways, provided they do
not induce any pathogenicity , which is very important. We go for replication defective as for transferring a
gene , only infection is needed and not the replication of the virus; therefore these do not encode for structural
proteins and can not go beyond first cycle of infection, not replication. Elements needed are the promoters,
origin of replication, packaging signal and gene of interest. Gene specific promoter enables us to make the virus
active at the particular site we want , as all the cells carries the gene but the genes are active at a particular tis-
sue only , by gene regulation. VSV-G is used for high efficiency as itcan withstand the centrifugal force also. We
have got a transfer vector, a packaging vector and the helper vectors like VSV-G. we try to split as much as pos-
sible because if we have a single plasmid containing multiple genes there will be chance of recombination and
the virus particle may complement with the other genes and turns out more pathogenic.

Adenoviral vectors for gene therapy: if we use adenoviral mediated gene therapy ,it will have respiratory in-
fections in humans. Therefore it is completely forbidden to be used in gene therapy as few decades ago there
was a death disaster due to high dosage for adenoviral vector, stating high immunogenicity for the adeno viral
vector.
Retroviral vectors can only infect the dividing cells as it does not have any specific mechanism to get into the
nucleus and integrate, rather it does integration when the DNA is suspended in cytoplasm during the cell divi-
sion.
Pseudotyped virus is a retroviral vector, in which all the structural genes are removed as we don’t want the
virus to be expressed but our protein,so we introduce the protein only gene via another vector. Hence the vec-
tor is called pseudotyped. The promoter sequence of the virus is used to express the protein gene. The inte-
grated form of the virus is called provirus. As per our downstream target tissue, we have to decide which vec-
tor we need to use.
retroviral vectors gives an advantage ofpseudotyped vector virus and permanent expression. On the
contrary , these can only infect the dividing cells and may inhibit tumor suppressor genes.

lentiviral vector can infect the non-dividing cells also.HIV, can be used with its accessory genes, providing a
envelop from a non retrovirus vector(VSV).These viruses replicates faster in non dividing cells than the dividing
ones , the reason being unknown. For integration, interaction between the integrase and the reverse transcrip-
tase is required.
 ➢ Transfection and Protein Localisation:
When we don’t know the function of a protein, we go for the location where it is located. Protein localization
refers to the accumulation of the protein at a site , for example nucleolus has some proteins which are not uti-
lised in ribosomal RNA synthesis, yet they are present in the nucleolus. Localisation decides most of the pro-
tein’s functions , with some exceptions.
For Exploring the protein function, we have two approaches : first is by immunofluorescence by using antibod-
ies and the other is to expresss the protein with tag fusion. Limitation to the immunofluorescence method is
when the protein is present inmultiple sites in the cells and antibody cannot be used in live cells as it is difficult
to pass the plasma membrane, that is why we use the GFP. By using antibody we can not specify if the protein
is blocking any stage of the cell cycle, this can be done only by the GFP. For what the protein is doing in the cell,
we observe the changes caused by altering the protein levels, this can be achieved by RNA interference, over-
expressing the protein and expressing the mutant versions of the proteins. Mutants can be in the form of
phosphorylated or methylated.
Transfection: when we express the protein by fusing with GFP, we overexpress a protein or we are introducing
a mutant gene , all this is done by TRANSFECTION. Transfection is the introduction of DNA into mammalian
cells. The gene is transcribed and translated into a protein, which we refer to expression.
Methods for direct introduction (without mixing any other cehmical) of DNA in transfection:
• By calcium chloride CaCL2 mediated tranfection; cheap but less efficient than the lipid based one.
• By lipids mediated transfection, efficiency is still much lower than electroporation.
• By the method electroporation, electric fields generated disrupts the plama membrane temporarily so
that a foreign gene can be introduced.
• Biolistics, which is a gunfire , of the DNA coated particles into the cell.
• Microinjection.
Methods for virally mediated transfection:
We use recombinant viruses to deliver DNA ,like retro and adenoviruses.
Types of transfection: Transient and stable. Transient is for short term expression, for 24 hours post transfec-
tion whereas the stable one is done by using selectable markers , these markers are different than the antibi-
otic markers.

GFP fusion construct:

If we are dealing with a multifunctional protein, our potein resides in different compartments during the cell
cycle, we use GFP protein fusion construct. Wecan do the fusion on either sides of the terminus, C or N.

➢ Protein-protein Interaction:
Proteins are the backbone of the biological phenomenons, as the proteins also determine the phenotype of
the organism, by interacting with our proteins , not alone they can perform all the functions.proteins may also
interact with other clas of molecules than protein itself, like RNAs and DNAs.
Protein interaction generally means physical contact between proteins and their interacting partners.Protein
associate physically to create macromolecular structures of various complexities and heterogeneities. Protein
pair can form dimers, multi-protein complexes or long chains. Other than physical interaction , proteins may
be genetically interacting as co-expressed , may be in same localization or may be found in the same metabolic
Pathway.
 Protein interactions :
◼ Genetic approach
 Yeast 2-hybrid: based on the transcription factor domain. Some transcriptional
xx yeast 2hybrid preferred
activators have of
attachments
separable DNA binding and transcriptional activation binding. bait and target.

◼ Biochemical approach
 Co-immunoprecipitation activation
 Fusion protein affinity chromatography
domain
◼ Cell-biology
 FRET - fluorescence resonance energy transfer
◼ Computational
 Rosetta Stone

target
 Co-regulation
 Phylogenetic analysis

bai
Immunoprecipitation: t
Immunoprecipitation is a precipitation technique that allows the protein
Isolation from the biological smaples. The general steps in the process are : DNA binding
1) Incubation of sample with antibody against protein of interest. protein
2) Separation of the antibody- protein complex from the remaining sample.
3) Analysis.
There are different types of immunoprecipitation:
Individual protein immunoprecipitation: (IP) this involves the usage of an antibody that is specific of for a par-
ticular protein, to isolate that particular from the mixture of the other proteins. HIGH specificity in antibody
binding will result in successful IP.
Protein complex immunoprecipitation(CO-IP) precipitation of intact protein complex is called CO-immuno-
precipitation. Working mechanism is similar to the individual protein immunoprecipitation, only difference is
that the protein is targeted from the complex.
Chromatin immunoprecipitation: (CH-IP) this method is used to determine the location of the DNA binding
sites in the genome for a particular proteins. Knowing the sites , we can specify the protein interactions hap-
pening in the cell.
RNP immunoprecipitation targets ribonucleoproteins (RNPs): in this the live cells are lysed, and then the tar-
get proteins and the associated RNA are immunoprecipitated using antibody.

Applications of the immunoprecipitation techmiques:

We can identify unknown proteins in a protein complex,study its protein protein interactions, synthesize the
proteins, isolate them for studies. we can also verify protein expression in various tissues.

DNA Footprinting:
In this technique , theDNA Nuclease like Dnase1 is used to degrade the DNA molecule. Nucleases cannot de-
grade DNA if it is bounded by the proteins, hence that region remains protected from the nucleases. The pro-
tect region is called the FOOTPRINT.

DNA footprinting is used to for the qualitative study of DNA-Protein binding and localizing binding sequences.
Cell cycle Analysis:
The cell cycle is a ubiquitous and complex process that takes place in a cell giving two new daughter
cells as an end stage. Accurate regulation of this process is necessary to ensure the genetic integrity of a
cell. Cyclins and cyclin-dependent kinases are central to this cycle. Cell cycle progression is also con-
trolled by negative regulatory proteins. Loss of cell cycle control is a hallmark of cancer. Besides cancer,
hundreds of xenobiotics are known to influence cell cycle-related events causing cell injury and cell
death.

In eukaryotes , the process is divided in two main stages namely, interphase and mitotic {M} phase (include
both mitosis and cytokinesis). During interphase, the cell grows accumulating nutrients for mitosis , replicates
its DNA and perform other several necessary functions which are critical for living, for example a nerve cell
transforming signals through out the nervous tissues, lung cells doing respiration etc.
The eukaryotic cell cycle is briefly divided into stages , and checkpoints where the cell checks and promote the
cellular machinery to the next task if the previous ones are completed and qualifies for the next ones.
Phases in cell cycle:
There are four different phases in a eukaryotic cell cycle namely : G1 phase, S phase (synthesis), G2 phase (col-
lectively known as interphase) and M phase (mitosis and cytokinesis).
M phase is itself composed of two tightly coupled processes: mitosis, in which the cell's nucleus
divides, and cytokinesis, in which the cell's cytoplasm divides forming two daughter cells. Activation of
each phase is dependent on the proper progression and completion of the previous one. Cells that have
temporarily or reversibly stopped dividing are said to have entered a state of quiescence
called G0 phase.
State Phase function
Resting G0 In this phase the cell leaves the cell cycle and dividing and do its reg-
ular job.
Interphase G1 The cell increases its size in G1 and prepares itself for DNA replica-
tion. The cell also checks at G1/S checkpoint by some mechanism
whether it to proceed for synthesis of DNA or not.
Interphase S DNA replication happens in this phase.
Interphase G2 During the gap between DNA synthesis and mitosis, the cell will
continue to grow. The G2 checkpoint control mechanism ensures
that everything is ready to enter the M (mitosis) phase and divide.
M phase M Cell growth is tsopped in this phase and the cellular energy is uti-
(cell divi- lised for the precise cell division into two daughter cells. First the
sion) nucleus divides and the actual cell divided alon with the cytoplasm
and the organelles in cytokinesis.

Analysis of the cell cycle:

• Looking at cells under a microscope reveals the phase of cell cycle that is in progress.
• Alternatively, the cells can be stained with DNA binding fluorescent dyes or antibodies that bind to spe-
cific components of the cell such as the spindle microtubule.
• Cells in S phase are revealed by supplying the cells with the thymidine analogue, bromodeoxyuridine
(BrdU). Following with the nuclei are revealed with anti-BrdU antibodies.
• A more versatile way to determine the phase of the cell cycle is by using flow cytometry to measure the
DNA content that doubles in the S phase.
 ➢ Gene Regulation and Expression:
Each cell contains copy of genome. They express only a subset of genes depending upon the functions of
different cell in different tissues.
This ability of same genome set to perform different functions is due to gene expression regulation.
Gene expression is controlled by chromatin modifications, transcription control, splicing, transport and
translational control.

Transcriptional regulation:
• Gene expression regulation is directed by short set of conserved sequences in a promoter region. These
sequences are recognized by Transcription factors(TFs) (DNA binding protein) assisting RNA polymer-
ase.
• Factors required for initiation of RNA synthesis are Basal TFs. They form complex with RNA poly II and
determines the initiation site.
• And those factors which binds to specific consensus sequences located upstream of TSS are Regulatory
TFs .
• TFs upon binding to promoter either activate or repress gene expression
• These promoter, a cis acting element contains several short DNA sub-sequences called binding sites.
• The specific combination of Binding sites with the promoter determines the condition of the gene ex-
pression

Transcriptome:
• TFs upon binding to promoter either activate or repress gene expression. These promoter, a cis acting
element contains several short DNA sub-sequences called binding sites.The specific combination of
Binding sites with the promoter determines the condition of the gene expression.