GENETIC ENGINEERING

UNIT I
MANIPULATION OF DNA – RESTRICTION AND MODIFICATION ENZYMES
One of the most important steps in molecular biology, especially molecular genetics and analysis,
is the isolation of DNA from the human genome and make many copies of it. A restriction enzyme
is a kind of nuclease enzyme which is capable of cleaving double-stranded DNA. The enzymes
may cleave DNA at random or specific sequences which are referred to as restriction sites. The
recognition sites are palindromic in origin, that is, they are the sequences which are read the same
forward and backward. These restriction enzymes are produced naturally by bacteria. The bacterial
species use it as a form of defense mechanism against viruses.

Types
There are two different kinds of restriction enzymes:
1. Exonucleases: restriction exonucleases are primarily responsible for hydrolysis of the terminal
nucleotides from the end of DNA or RNA molecule either from 5’ to 3’ direction or 3’ to 5’
direction; for example- exonuclease I, exonuclease II, etc.
2. Endonuclease: restriction endonucleases recognize particular base sequences (restriction sites)
within DNA or RNA molecule and catalyze the cleavage of internal phosphodiester bond; for ex
EcoRI, Hind III, BamHI, etc.
The first restriction enzyme to be discovered was Hind II in the year 1970. In 1978, Daniel
Nathans, Werner Arber, and Hamilton O. Smith were awarded the Nobel Prize for Physiology or
Medicine.
Restriction Enzyme Nomenclature
The very name of the restriction enzymes consists of three parts:
1. An abbreviation of the genus and the species of the organism to 3 letters, for example- Eco for
Escherichia coli identified by the first letter, E, of the genus and the first two letters, co, of the
species.
2. It is followed by a letter, number or combination of both of them to signify the strain of the
species.
3. A Roman numeral to indicate the order in which the different restriction-modification systems
were found in the same organism or strain
Patterns of DNA Cutting by Restriction Enzymes:
Sticky ends
(i) 5′ overhangs:
The enzyme cuts asymmetrically within the recognition site such that a short single-stranded
segment extends from the 5′ ends. BamHI cuts in this manner

(ii) 3′ overhangs:
Again, we see asymmetrical cutting within the recognition site, but the result is a single-stranded
overhang from the two 3′ ends. Kpnl cuts in this manner.

A sticky-end fragment can be ligated not only to the fragment from which it was originally
cleaved, but also to any other fragment with a compatible sticky end. The sticky end is also called
a cohesive end or complementary end in some reference.
If a restriction enzyme has a non- degenerate palindromic (the sequence on one strand reads the
same in the same direction on the complementary strand e.g. GTAATG is not a palindromic DNA
sequence, but GTATAC is, GTATAC is complementary to CATATG) cleavage site, all ends that it
produces are compatible. Ends produced by different enzymes may also be compatible.
Blunts:
Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends without
overhangs. Smal is an example of an enzyme that generates blunt ends.

The 5′ or 3′ overhangs generated by enzymes that cut asymmetrically are called sticky ends or
cohesive ends, because they will readily stick or anneal with their partner by base pairing
(INCLUDE THE TABLE GIVEN IN CLASS NOTE- different RE TYPES)

Classification of Restriction Endonucleases

Based on the types of sequences identified, the nature of cuts made in the DNA, and the enzyme
structure, there are three classes:
1. Type I restriction enzymes,
2. Type II restriction enzymes, and
3. Type III restriction enzymes.
Type I Restriction Enzymes
• Type I restriction enzymes possess both restriction and modification activities. In this case, the
restriction will depend upon the methylation status of the target DNA sequence.
• Cleavage takes place nearly 1000 base pairs away from the restriction site. The structure of the
recognition site is asymmetrical. It is composed of 2 parts. One part of the recognition site is
composed of 3-4 nucleotides while the other one contains 4-5 nucleotides. The two parts are
separated by a non-specific spacer of about 6-8 nucleotides.
• For their function, the type I restriction enzymes require S- adenosylmethionine (SAM), ATP,
and Mg2+ .
• They are composed of 3 subunits, a specificity subunit which determines the recognition site, a
restriction subunit, and a modification subunit.
Type II Restriction Enzymes
• Two separate enzymes mediate restriction and modification. Henceforth, DNA can be cleaved in
the absence of modifying enzymes. Although the target sequence identified by the two enzymes is
the same, they can be separately purified from each other.
• The nucleotides are cleaved at the restriction site only. The recognition sequence is rotationally
symmetrical, called palindromic sequence. The specific palindromic site can either be continuous
(e.g., KpnI identifies the sequence 5´-GGTACC-3´) or non-continuous (e.g., BstEII recognizes the
sequence 5´-GGTNACC-3´, where N can be any nucleotide).
• These require Mg2+ as a cofactor but not ATP.
• They are required in genetic mapping and reconstruction of the DNA in vitro only because they
identify particular sites and cleave at those sites only.
• The type II restriction enzymes first establish non-specific contact with DNA and bind to them in
the form of dimmers.
• The target sequence is then detected by a combination of two processes. Either the enzyme
diffuses linearly/ slides along the DNA sequence over short distances or hops/ jumps over long
distances.
• Once the target sequence is located, various conformational changes occur in the enzyme as well
as the DNA. These conformational changes, in turn, activate catalytic center.
Type III Restriction Enzyme
• The type III enzymes recognize and methylate the same DNA sequence. However, they cleave
nearly 24-26 base pairs away.
• They are composed of two different subunits. The recognition and modification of DNA are
carried out by the first subunit- ‘M’ and the nuclease activity is rendered by the other subunit ‘R’.
• DNA cleavage is aided by ATP as well as Mg2+ whereas SAM is responsible for stimulating
cleavage.
• Only one of the DNA strand is cleaved. However, to break the double-stranded DNA, two
recognition sites in opposite directions are required.

Applications:
In various applications related to genetic engineering DNA is cleaved by using these restriction
enzymes.
• They are used in the process of insertion of genes into plasmid vectors during gene cloning and
protein expression experiments.
• Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing
single base changes in DNA known as single nucleotide polymorphisms (SNPs). This is only
possible if a mutation alters the restriction site present in the allele.
• Restriction enzymes are used for Restriction Fragment Length Polymorphism (RFLP) analysis
for identifying individuals or strains of a particular species.
DESIGN OF LINKERS AND ADAPTORS
DNA ligation
The final step in construction of a recombinant DNA molecule is the joining together of the vector
molecule and the DNA to be cloned. This process is referred to as ligation, and the enzyme that
catalyzes the reaction is called DNA ligase.
● The process of joining two pieces of linear DNA into a single piece through the use of an
enzyme DNA ligase
● It catalyzes the formation of phosphodiester bond between the 3’-hydroxyl on one piece of DNA
and 5’-phosphate on a second piece of DNA.
● Most commonly used DNA ligase is T4 DNA ligase.
● Ligation is used to join vector DNA and insert DNA
● Two ways in which DNA can be ligated into cloning vector- using DNA with sticky ends and
using DNA with blunt end.
● DNA fragment is generated by Taq DNA polymerase by a process like PCR.

DNA ligation with sticky ends

● To prepare a cloning vector for ligation with insert DNA ,it is cut with a restriction enzyme .
● The insert has sticky ends,the vector should be cut with same enzyme.the producing sticky ends
that will be complementary to the ends of the insert DNA.
● The sticky ends on the vector and the insert are complementary, when they comes into contact
during the ligation reaction they will base pair with each other using hydrogen bond
● While insert and vector are associated,T4 ligase forms a phosphodiester bond, covalently linking
two pieces of DNA.

DNA ligation with blunt ends

● Both inserted DNA and the vector have blunt ends.
● One advantage compared to Sticky end ligation in that all DNA ends are Compatible with all
other ends.
● The insert can be produced by PCR using a DNA polymerase
● Polymerase that results in 3’ deoxyadenosine overhangs is used ,ends can polished by T4DNA
polymerase to produce blunt ends.
● Blunt end inserts can also produced by cutting them out of a vector
● Preparation for the ligation step; ○ Inserts that have been produced by PCR need to be
phosphorylated. ○ Linearized , blunt end vector and blunt end insert are then combined and
incubated with T4 DNA ligase in appreciate buffer in order to form phosphodiester bonds between
insert and vector
● Relatively simple construct design and cloning procedure are advantages of blunt end ligation.
● Relatively low cloning efficiency,need for plasmid dephosphorylation to prevent circularization
are the disadvantages. Compatible sticky ends are desirable on the DNA molecules to be ligated
together in a gene cloning experiment. Often these sticky ends can be provided by digesting both
the vector and the DNA to be cloned with the same restriction endonuclease, or with different
enzymes that produce the same sticky end, but it is not always possible to do this. A common
situation is where the vector molecule has sticky ends, but the DNA fragments to be cloned are
blunt-ended. Under these circumstances one of three methods can be used to put the correct sticky
ends onto the DNA fragments.

Linkers
The first of these methods involves the use of linkers. These are short pieces of double-
stranded DNA, of known nucleotide sequence, that are synthesized in the test tube. It is blunt-
ended, but contains a restriction site, BamHI in the example shown. DNA ligase can attach linkers
to the ends of larger blunt-ended DNA molecules. Although a blunt end ligation, this particular
reaction can be performed very efficiently because synthetic oligonucleotides, such as linkers, can
be made in very large amounts and added into the ligation mixture at a high concentration. More
than one linker will attach to each end of the DNA molecule, producing the chain structure.
However, digestion with BamHI cleaves the chains at the recognition sequences, producing a large
number of cleaved linkers and the original DNA fragment, now carrying BamHI sticky ends. This
modified fragment is ready for ligation into a cloning vector restricted with BamHI.

Adaptors
The second method of attaching sticky ends to a blunt-ended molecule is designed to avoid
this problem. Adaptors are short synthetic oligonucleotides. An adaptor is synthesized so that it
already has one sticky end. The idea is of course to ligate the blunt end of the adaptor to the blunt
ends of the DNA fragment, to produce a new molecule with sticky ends.
Normally the two ends of a polynucleotide strand are chemically distinct, a fact that is clear from a
careful examination of the polymeric structure of DNA. One end, referred to as the 5′ terminus,
carries a phosphate group (5′-P); the other, the 3′ terminus, has a hydroxyl group (3′-OH). In the
double helix the two strands are antiparallel, so each end of a double-stranded molecule consists of
one 5′-P terminus and one 3′-OH terminus. The sticky end adaptor molecule is modified in such a
manner that it contain OH group on both 5’ and 3’ end with the help of alkaline phosphatases.
● Ligation takes place between the 5′-P and 3′-OH ends
● Adaptor molecules are synthesized so that the The 3′-OH terminus of the sticky end is the same
as usual, but the 5′-P terminus is modified: it lacks the phosphate group, and is in fact a 5′-OH
terminus
● DNA ligase is unable to form a phosphodiester bridge between 5′-OH and 3′-OH ends. The
result is that, although base pairing is always occurring between the sticky ends of adaptor
molecules, the association is never stabilized by ligation. Adaptors can therefore be ligated to a
blunt-ended DNA molecule but not to themselves. After the adaptors have been attached, the
abnormal 5′-OH terminus is converted to the natural 5′-P form by treatment with the enzyme
polynucleotide kinase, producing a sticky-ended fragment that can be inserted into an appropriate
vector.

CLONING AND EXPRESSION VECTOR FOR PLASMID AND BACTERIOPHAGE

Cloning Vector
 Cloning vectors are vectors that are capable of replicating autonomously and thus are used
for the replication of the recombinant DNA within the host cell.
 Cloning vectors are responsible for the determination of which host cells are appropriate
for replicating a particular DNA segment.
 Cloning vectors are of further different types that are defined by different features unique
to each type of vector.
Expression Vector
  Expression vectors are vectors that enable the expression of cloned genes in order to
determine the successful cloning process.
 Usually, cloning vectors do not allow the expression of a cloned gene which is why the use
of expression vectors is required.
 The use of expression vectors facilitates the processing of introns in prokaryotes as these
are designed with restriction sites next to the regulatory region.
 The restriction sites on the vectors result in splicing of the cloned gene to permit the
expression of the gene under the regulatory system.
 The regulatory system in expression vectors consists of a promoter sequence, a termination
sequence, along a transcription termination sequence.
 The use of expression vectors is essential to determine the success of a cloning procedure
and the efficiency of selective markers on the vectors.
 Expression vectors are of varying degrees of complexity depending on whether they are to
be used in prokaryotic or eukaryotic cells.

PLASMIDS
Plasmids are small circular DNA fragments, double-stranded, self-replicating extra chromosomal
structures found in many microorganisms.
The term Plasmid was coined by Joshua Lederberg in 1952.
Plasmids are important as genetic tools, which are used to introduce, manipulate or delete certain
genes from the host cell.
Properties of Plasmids
 They are extra chromosomal DNA fragments present in the cell.
 They are double stranded structures. Exceptions are the linear plasmids in
bacteria Streptomyces spp and Borrelia spp.
 They can replicate independently.
 The absence of a plasmid in the cell does not affect cell functioning, but the presence of a
plasmid in the cell is usually beneficial.
 Plasmids are also known as sex factors, conjugants, extra chromosomal replicons, or
transfer factors.
 Copy number – the copy number refers to the number of copies of plasmid present in the
bacterial cell. Usually, small plasmids are present in high numbers and large plasmids are
present in few numbers.
 Compatibility of plasmids – this refers to the ability of two different plasmids to coexist in
the same bacterial cell.
Structure of Plasmids
1. Every plasmid has certain essential elements. These are as follows –
 Origin of replication (OR) – This refers to a specific location in the strand where the
replication process begins. In plasmids, this region is A=T rich region as it is easier to
separate the strands during replication.
 Selectable marker site – This region consists of Antibiotic resistance genes which are
useful in the identification and selection of bacteria that contain plasmids.
 Promoter region – this is the region where the transcriptional machinery is loaded.
  Primer binding site – this is the short sequence of single-strand DNA which is useful in
DNA amplification and DNA sequencing.
 Multiple cloning sites – This site contains various sequences where the restriction
enzymes can bind and cleave the double stranded structure.

Figu
re: Plasmid Structure. Created with biorender.
2. The size of the plasmid varies from 2 kb to 200 kb.
3. It is the extrachromosomal element of the cell which is not required for the growth and
development of the cell.
4. Most of the plasmids contain the TRA gene, which is the transferred gene and is essential in
transferring the plasmid from one cell to another.
Transfer of Plasmid
Plasmids are transferred by the process of Conjugation:
 The process of conjugation involves two cells: a donor cell and a recipient cell.
 The donor cells form a conjugation bridge now as pilus and attaches to the recipient cell.
 One copy of the plasmid is transferred from donor to recipient cell.

The other methods by which the plasmids can be transferred are transduction and bacterial
transformation.
Types of Plasmids
Based on the presence of the TRA gene plasmids can be classified into two types:
1. Conjugative plasmids – these plasmids contain TRA (transfer) gene and are commonly
seen in bacteria.
2. Non-conjugative plasmids – these types of plasmids lack the TRA genes.
Based on functions the plasmids can be classified into the following types:
1. F Plasmids (Fertility plasmids)
They contain the TRA genes and hence can be transferred from one cell to another.
They can replicate inside the bacterial cell.
They cause the synthesis of a pilus, which is a long protein-rich structure that helps in cell-cell
interaction.
It also contains a sequence responsible for incompatibility.
2. R plasmid (Resistance plasmids)
These plasmids contain and transmit genes for Antibiotic resistance from one cell to another.
The antibiotic resistance gene protects the bacteria from antibiotics in human medicines and
antibiotics naturally present in the soil.
These types of plasmids are usually large in size and present in low copy numbers in the cell.
3. Col Plasmids (Colicin plasmids)
These are known as bacteriocinogenic plasmids because they produce bacteriocins.
These proteins have the ability to kill the closely related bacterial cells which lack Col plasmids.
These plasmids are observed in E. coli.
4. Degradative Plasmids
These types of plasmids have the ability to digest unusual substances such as toluene, camphor,
salicylic acid, etc.
The presence of these plasmids in the organism enables the breakdown of various chemicals and
substances.
5. Virulence Plasmids
These plasmids produce virulence factors that enable the bacteria to infect other cells. Bactria
containing virulence plasmids are able to infect the plant, animal, and human cells.
Example – Ti plasmid is the virulence plasmid present in Agrobacterium tumefaciens which
causes crown gall disease in plants.
Functions and Applications of Plasmids
 The important use of plasmids is that they can be used as vectors to insert a specific gene
into other organisms due to their capacity to incorporate a gene and replicate inside the
cell.
 They are an important factor in bacteria as they carry antibiotic resistance genes.
 Degradative plasmids can be used to degrade industrial chemicals which are a threat to the
environment.
 As plasmids are easy to manipulate, they are being used in gene therapy as well.
 Because plasmids are good vectors (a vehicle/factor which is used to transfer a gene from
one organism to another) they are used in drug delivery and for hormone production in
other cells.
 Plasmids are an important source of horizontal gene transfer.
Plasmid Examples
S.N. Plasmids Organism

1. pBR322 E. coli

2. pUC19 E. coli

3. ColE1 E. coli

4. RP4 Pseudomonas
 5. TOL Pseudomonas putida

6. pTiAch5 Agrobacterium tumefaciens

7. pUC8 E. coli

pBR322
pBR322 is a commonly used cloning vector in E. coli and has tremendous applications in cloning.
pBR322 full form
p = plasmid
BR = Bolivar, and Rodriguez
322 = numerical designation
 It was constructed in 1977 in the lab of Herbert Boyer at The University of California in
San Francisco.
 It is a synthetic plasmid and was the first artificial plasmid to be constructed and used as
a cloning vector.
 pBR322 is one of the most studied plasmids.
 It is 4362 base pairs long.
 It is completely sequenced, which means the whole sequence of pBR322 is known and
studied.
 Its molecular weight is 2.83 x 106 Daltons.pBR322 Vector.

Structure of pBR322
1. Origin of replication
2. Restriction enzyme sites
3. Selectable marker sites
Origin of replication
  The origin of replication in plasmid pBR322 is known as pMB1.
 The copy number of this plasmid is 15-20.
Restriction enzyme sites
 Around 40 different restriction sites are present on the genome of pBR322.
 Almost 11 different restriction sites are present in the region of tetracycline resistance
region.
 In the ampicillin resistance region 9 restriction sites of different enzymes are present.
 Some of the known restriction enzyme sites are – BamHI, HindIII, EcoRI, SaII, and many
more.
Selectable marker sites
Two selectable marker sites or antibiotic resistance genes are present on the genome of this
plasmid.
 Ampicillin resistance site – the ampicillin gene codes for β-lactamase, which can be used
for screening microorganisms when a foreign DNA is being inserted in the plasmid.
 Tetracycline resistance site – this gene degrades the antibiotic tetracycline and can be used
for screening microorganisms.
 These antibiotic resistance genes are useful in screening organisms after cloning.
Screening of recombinants containing pBR322
 Consider that the restriction enzyme BamHI is used to cut the plasmid at that specific
position.
 BamHI lies in the tetracycline resistance region of plasmid pBR322.
 Now, the recombinant molecule of pBR322, which includes the newly inserted DNA
molecule, will be sensitive to Tetracycline.
 But this recombinant molecule is resistant to Ampicillin, as the ampicillin resistance gene
is fully functional.
 Hence, when the recombinant cells are plated on the medium containing ampicillin and
incubated.
 The colonies that appear on plates containing a medium with ampicillin are transformed
colonies containing cells with the newly inserted DNA molecule.
 The replica plate technique is used to confirm the transformed colonies.
 To confirm that the colonies on the ampicillin-containing media are transformed colonies,
they are plated on the plates containing a medium inclusive of tetracycline.
 The colonies that do not grow on the medium inclusive of tetracycline are transformed
colonies as the transformed colonies are sensitive to tetracycline.
Advantages of pBR322
 Due to its manageable size, plasmid pBR322 is widely used as a cloning vector.
 The presence of two antibiotic resistance genes eases the selection process of
recombinants.
 Multiple restriction enzyme sites make the plasmid compatible in many ways.
 It has a high copy number which is highly favourable in genetic engineering.
Disadvantage
• Time consuming

BACTERIOPHAGE
Bacteriophage or Phage is a virus that infects and replicates only within the body of bacteria.
 Bacteriophages were discovered independently by Frederick W. Twort in the U.K and Félix
d’Hérelle in France.
 The term ‘bacteriophage’ has been derived from two words; ‘bacteria’ and ‘phagein’,
meaning devour. The term was coined by Félix d’Hérelle.
 These are found throughout the world in different environments and are even recognized as
one of the most abundant biological agents on earth. These are the most abundant
biological particles in water and the second most abundant component of the biomass on
land following prokaryotes.
 Bacteriophages that infect bacteria can also infect the members of the domain Archaea.
 Bacteriophages are diverse in their shape size and genome organization depending on the
type of bacteria they infect, but the basic composition remains the same.
 All bacteriophages consist of a nucleic acid genome which is enclosed inside a shell of
phage-encoded capsid proteins.
 The head structure of different phages might differ, the sizes of phages range between 24-
200 nm in length.
 The shape, size, and structure of different bacteriophages are different depending on the
type of bacteriophages.
 The studies on bacteriophages have increased over the years, as the scope of their
applications has increased.
 The ability of phages to infect and possibly kill infectious bacterial agents puts forward
their potential as a possible supplement or replacement for antibiotic agents.
 The mechanism of infection of bacteriophages remains almost the same where they first
attach to the host cell and enter their genome into the host cell to suspend the host cellular
machinery.

Structure of Bacteriophage
Even though there are different types of phages depending on the type and group of bacteria, they
infect, however, all phages share some common characteristics or properties. Some of such
characteristics or properties of bacteriophages are:
1. Like all other viruses, bacteriophages are also highly species-specific towards their host
cell. The bacteriophages only infect a single species of bacteria or even specific strains of
bacteria within a species.
2. The basic structure of all bacteriophages is the same. They consist of a core of nuclear
material surrounded by a protein capsid.
3. Bacteriophages exist in three basic structural forms; an icosahedral head with a tail, an
icosahedral head without a tail, and a filamentous form.
4. The genetic material or nuclear material of bacteriophages can be either DNA or RNA,
both of which can either be double-stranded or single-stranded.
5. Bacteriophages are obligate intracellular parasites that remain latent outside the host cell
and require host cellular machinery to conduct their metabolic activities.
6. Like bacteria, bacteriophages are also classified into different orders and families
depending on their morphology and genetic material. Some of the commonly studied
families include Inoviridae, Tectiviridae, Microviridae, and Rudiviridae.
Figure: Structure of Bacteriophage. Created with BioRender.com.
Bacteriophage Models or Types
1. λ phage
 Lambda phage or coliphage λ is a bacteriophage that infects the bacteria belonging to the
members of the bacterial species Escherichia coli (E. coli).
 The lambda phage was originally discovered by Esther Lederberg in 1951 in the US during
her studies on E. coli under ultraviolet irradiation.
 It belongs to the Siphoviridae family of the order Caudovirales which is defined by the
lack of envelope, non-contractile tail, and a linear double-stranded DNA molecule.
 Lambda viruses have been studied for various purposes to understand the lytic and
lysogenic lifestyles of various viruses and also as model viruses for viral studies.
 The virus has a temperate life cycle that enables it to either enter into the lytic phase or
reside within the host’s genome via lysogeny.
 The structure of the phage particle consists of a protein head or capsid, a non-contractile
tail, and tail fibers. The viral genome is present inside the capsid of the virus.
 The non-contractile tail of the virus indicates that the virus cannot force into the cell
membrane of the bacteria and must depend on existing pathways to invade the host cell.
 The virus consists of 12-14 different types of proteins comprised of more than 1000
protein molecules and a single DNA molecule present in the phage head.
2. T4 phage
 The T4 virus is a bacteriophage that infects the members of the bacterial species
Escherichia coli and thus, is also known as Escherichia virus T4.
 The virus is one of the seven Escherichia coliphages (name T1-T7), which were discovered
by Delbruck and coworkers in 1944 as models to study different mechanisms of the phage
community.
 The bacteriophage T4 belongs to the Caudovirales order of the Myoviridae family of
bacteriophages based on the presence of a non-enveloped head and contractile tail.
 The structure of bacteriophage T4 consists of a protein capsid, called, head which consists
of a linear double-stranded DNA molecule.
  At the end of the tail is a 925 Å long and 520 Å diameter contractile tail attached to a
special portal at the base of the head.
 There are six short tail fibers emerging from the baseplate that can recognize receptor
molecules on the host surface.
 Bacteriophage T-even viruses are among the most commonly studied and researched group
of bacteriophages that also are similar to one another in various factors.
 These are also one of the largest and most complicated groups of bacterial viruses as their
genetic makeup is made up of about 300 different genes.
Life Cycles of Bacteriophage
Viruses enter the host cell to reproduce during which the virus results in different forms of
infections to the host cell. The overall process of the entry of the virus, its replication, and exit
from the host cell comprises the lifecycle of viruses. Bacteriophages, like all other viruses, follow
a similar trajectory where the virus enters the bacterial host cell in order to replicate. There are two
types of lifecycles that differ in the mechanism of DNA replication where, in one, the viral DNA is
incorporated into the host DNA, but in the other, the DNA replicates separately from the host
DNA. These lifecycles might occur independently or alternatively in different types of
bacteriophages.
1. Lytic Cycle
 The lytic cycle is one of the two lifecycles of bacteriophages where the viral DNA remains
as a free-floating molecule and replicates separately from the bacterial DNA.
 The lytic cycle usually occurs in virulent phages as the phages result in the destruction of
the infected cell membrane during the release of the viral particles.
 The lytic cycle is a virulent infection as it results in the destruction of a cell.
.

The lytic lifecycle of bacteriophage is completed in the following steps;

a. Attachment and Penetration
 The first step in the lifecycle of a bacteriophage is attachment, where the ligands on
specific molecules on the surface of the viral particles bind to the receptor molecules on
the plasma membrane of the host cell.
 The receptors depend on the type of viruses as most orthomyxoviruses use receptors like
terminal sialic acid on an oligosaccharide side chain of a cellular glycoprotein.
 The ligand, however, is an aperture at the distal end of each monomer of the trimeric viral
hemagglutinin glycoprotein.
  Even though there is a high degree of specificity between the receptors and the ligands, a
number of viruses might use the same receptors.
 Besides, some bacteriophages might use other membrane glycoproteins as their receptors.
 Once attached, the virus injects its nuclear material into the cytoplasm of the bacterial cell.
 The viral genome (either DNA or RNA) remains in the cytoplasm, and in some cases
becomes circular and resemble the bacterial plasmid.
b. Biosynthesis and Transcription
 Once in the cytoplasm, the viral genome hijacks the host cellular mechanism and utilizes it
to produce more viruses.
 In the case of DNA viruses, the DNA undergoes transcription to produce messenger RNA
that then directs the ribosome of the host cell.
 In the case of the lytic cycle, the mRNA encodes for various polypeptides, the first of
which destroy the host’s DNA.
 In the case of RNA viruses, an enzyme called reverse transcriptase is involved which
transcribes the viral RNA into DNA.
 The DNA is then transcribed back to mRNA, which then directs the destruction of host
DNA.
 The viral DNA then takes control of the host cell and produces different proteins required
for the assembly of new viruses.
 The viral DNA also undergoes replication to produce more genetic material for new viral
particles.
 The process of biosynthesis and DNA replication is mediated by different genes and
enzymes.
c. Assembly and Lysis
 As biosynthesis and replication continue, a large number of viral proteins and genomes are
formed.
 Once enough viral particles are formed and matured, these particles under assembly during
which the genetic material of the virus is incorporated into the viral protein, capsid.
 The newly assembled bacteriophages release the enzyme, lysin, into the cytoplasm. The
enzyme causes the lysis of the bacterial cell wall, resulting in the release of newly formed
phage particles.
 Thus, at the end of the lytic lifecycle, the infected bacterial cell and cell membrane are
destroyed.
2. Lysogenic Cycle
 Lysogenic is one of the two lifecycles of bacteriophages defined by the incorporation of
the bacteriophage genome into the host genome.
 During the lysogenic lifecycle, the host bacteria continue to live and reproduce normally
after the replication of bacteriophages.
 The genetic material of bacteriophage incorporated in the bacterial DNA during the
lysogenic lifecycle is called a prophage which can be transmitted to daughter cells during
the bacterial cell division.
 The lysogenic cycle is a temperate and non-virulent infection as the bacteriophage doesn’t
kill the host cell.
The process of lysogenic lifecycle occurs in the following steps;
a. Attachment and Penetration
 The first step of the lysogenic lifecycle is identical to the first step of the lytic lifecycle.
 The bacteriophage ligands attach to the receptors on the surface of the bacterial cell wall.
 The attachment is highly specific as it is determined by the interaction between the ligands
and the receptors present on the surface of the bacterial cell wall.
 After attachment, the viral genome is injected into the cytoplasm of the host cell.
 The infective viral DNA or prophage is then incorporated into the host chromosome, which
converts the infective prophage into a non-infective prophage.
b. Replication
 The viral DNA then uses the host machinery to replicate as it continues to replicate with
the host chromosomes during cell division.
 In some cases, the prophage might be ejected from the host chromosome, and the viral
DNA might enter the lytic cycle.
 Unlike the lytic cycle, the bacterial cellular mechanism is not hijacked by the viral
particles, and no biosynthesis of viral proteins takes place.
 The prophage, however, can be transferred to the daughter cells during the bacterial cell
division.
 The process of replication continues until there are some stressors which can either be
physical stressors like UV radiation, low nutrient condition or chemical, which might
result in the transition of the lysogenic cycle into the lytic cycle.
 Once converted into the lytic cycle, the viral DNA undergoes transcription to produce viral
proteins. The proteins and viral genome are then assembled to form complete viral
particles which then are released from the host cell by lysis.

Application
Bacteriophages have been considered to be potential antibacterial therapeutics for the treatment of
various infectious bacterial diseases in humans and animals. In the beginning, the clinical
application of bacteriophages was limited to the treatment of acute intestinal infections and skin
infections. Later, however, the application of bacteriophages in surgical practices for the treatment
of prurient infectious complications was initiated. The following are some of the application of
bacteriophages in different areas;
Treatment of bacterial infections
 With the increasing cases of bacterial resistance against numerous antibiotics, the potential
use of bacteriophage a possible treatment has been explored.
 As the bacteriophage infects only bacteria and is harmless to humans, the administration of
such bacteriophages into humans helps in the destruction of such infectious bacteria.
 Besides, the application of bacteriophages on burn wounds has shown to reduce the
chances of infection and sepsis by a large number.
In food hygiene and safety
 Bacteriophages are used to control and eliminate bacterial contaminants from food surfaces
and food-borne spoilage.
 Bacteriophages are highly specific, which makes them attractive for sanitization of ready-
to-eat foods like milk, vegetables, and meat products.
 Many bacteriophages have been commercialized for their use as spray sanitizers to
disinfect cattle hides prior to slaughter in order to reduce contamination in the meat.
 Some bacteriophages are also useful as surface and environment decontaminants as they
can disinfect stainless stain as efficiently as a quaternary ammonium compound.
In agriculture
 Some bacteriophages that are specific to plant bacteria have also found their application in
agriculture.
 These phages are used for the treatment and prevention of bacterial diseases in plants. The
use of bacteriophages in the place of antibiotics prevent the clumping of antibiotics on the
plant surface, which then might be harmful to the health of the consumers.

VECTORS FOR INSECT, YEAST AND MAMMALIAN SYSTEM

VECTORS FOR INSECT
The baculovirus vector system is widely used for the expression of recombinant proteins in
cultured insect cells. It is one of the most versatile and powerful systems for eukaryotic expression
of recombinant proteins. This system is particularly advantageous for large-scale preparation of
proteins that require expression in eukaryotic host cells. Many eukaryotic proteins undergo
posttranslational modifications that can only take place in eukaryotic cells (e.g. glycosylation), or
they need an eukaryotic cellular milieu for proper folding (e.g. membrane proteins). In these cases,
prokaryotic expression systems are often inadequate and the baculovirus expression system could
be a good alternative.
Baculovirus is a double-stranded DNA virus that commonly infects insects, particularly
members of the order Lepidoptera (moths, butterflies and skippers). The cloning vector in our
baculovirus expression system, pBV, is optimized for use with the baculovirus shuttle vector
(known as bacmid) derived from the baculovirus strain AcMNPV (Autographa californica
multicapsid nucleopolyhedrovirus), which has a 134 kb genome in its wildtype form.
The gene of interest is first cloned into the pBV vector under the control of a strong promoter. This
entire expression cassette, along with a gentamicin resistance gene, is flanked by the Tn7
transposon terminal elements, Tn7L and Tn7R. This vector is then transformed into E. coli
carrying the bacmid shuttle vector and a helper plasmid. The bacmid is essentially a very large
plasmid containing the baculovirus genome modified to carry a lacZ gene and an attTn7 docking
site inserted in the lacZ coding region. The helper plasmid expresses the Tn7 transposase. The
transposase would then mediate the transposition of the region flanked by Tn7R and Tn7L on the
pBV vector, which contains the expression cassette for the gene of interest and gentamicin
resistance, into the attTn7 docking site of the bacmid. Colonies containing recombinant bacmids
can be identified by gentamicin selection and blue/white screening (non-recombinant colonies are
blue due to lacZ expression whereas recombinant colonies are white due to disruption of lacZ by
transposon insertion). Purified bacmid DNA can then be used to transfect insect cells to generate
live baculovirus, which can be used to produce the recombinant protein of interest.
The most commonly used cell line for expressing recombinant proteins from baculovirus vectors
is Sf9. This clonal line was derived from ovarian tissue of Spodoptera frugiperda (fall armyworm).
This cell line is adaptable to a variety of culture and media conditions, including suspension or
monolater culture and serum-free media. Larvae and other Lepidoptera cell lines have also been
used extensively, and there are some reports of baculovirus being an effective vector for
mammalian cells.
Key components

AcMNPV polyhedrin promoter. It drives high-level expression of the gene encoding your
recombinant protein.
ORF: The open reading frame of your gene of interest is placed here.
SV40 early pA: Simian virus 40 early polyadenylation signal. It facilitates transcriptional
termination of the upstream ORF.
Tn7L: Tn7 transposon left terminal element. It is recognized by Tn7 transposase. DNA flanked by
Tn7R and Tn7L can be transposed by Tn7 transposase into attTn7 docking sites.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin
selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E.
coli.
Tn7R: Tn7 transposon right terminal element. It is recognized by Tn7 transposase. DNA flanked
by Tn7R and Tn7L can be transposed by Tn7 transposase into attTn7 docking sites.
Gentamicin: Gentamicin resistance gene. It allows for drug selection of E. coli carrying
recombinant bacmids.
Highlights
Our baculovirus recombinant protein expression vector system enables efficient production of
recombinant proteins in insect cells. This system allows for expression of proteins with
posttranslational processing characteristic of eukaryotic cells, and with good adaptability to large-
scale applications.
Advantages
Eukaryotic system: Insect cells carry out posttranslational processing of proteins similar to that
of mammalian cells. Our system is thus particularly suitable for expressing mammalian and other
eukaryotic proteins whose function requires proper post-translational processing not present in
prokaryotic expression systems, such as covalent modifications or membrane targeting.
Strong expression and good solubility: In most cases, the protein of interest is highly expressed,
soluble, and can be easily recovered from infected cells.
Ease of scale-up: In our system, baculovirus obtained from initial transfection of insect cells can
be used to infect more cells to further amplify viral titer. Protein production with our system can
therefore be reproducibly scaled up.
Suspension culture: Sf9 and other Lepidoptera cell lines grow well in suspension cultures,
allowing for the production of recombinant proteins in large-scale bioreactors.
Safety: Baculovirus cannot replicate outside of insect cells and are nonpathogenic to mammals
and plants. Thus our expression system can be used in insect cell lines under minimal biosafety
conditions.
Disadvantages
Technical complexity: Protein production using the baculovirus expression system requires
multiple steps, including cloning the gene of interest into pBV, generating recombinant bacmid
from pBV, and transfecting bacmid into insect cells. These procedures are technical demanding
and time consuming relative to recombinant protein expression in bacterial systems. These
demands can be alleviated by choosing our recombinant bacmid generation and baculovirus
packaging services when ordering your vector.

VECTOR FOR YEAST

The yeast Saccharomyces cerevisiae is one of the most important organisms in
biotechnology. As well as its role in brewing and breadmaking, yeast has been used as a host
organism for the production of important pharmaceuticals from cloned genes . Development of
cloning vectors for yeast was initially stimulated by the discovery of a plasmid that is present in
most strains of S. cerevisiae. The 2 µm plasmid, as it is called, is one of only a very limited
number of plasmids found in eukaryotic cells.
Yeast 2µm plasmid  Excellent basis for a cloning vector.  Covalently closed circular DNA
molecule  6 kb in size  Copy number of between 70 and 200.  Located in nucleus of yeast
cell  REP1 and REP2 are involved in replication of the plasmid  FLP codes for a protein that
can convert the A form of the plasmid to the B form, in which the gene order has been rearranged
by intramolecular recombination  D encodes recombination specific factors.
Yeast integrative plasmids (YIps)  Bacterial plasmids carrying a yeast gene  This is PBR322
plasmid with URA3 gene inserted  URA3 gene codes for orotidine-5′-phosphate decarboxylase
and is used as a selectable marker  Does not contain 2 µm plasmid and cannot replicate as
plasmid  Have all features of yeast cloning vectors except origin of replication
These plasmids therefore replicate only by integration into yeast chromosome  One copy per cell
 Produce very stable recombinants  Eg:YIp5

Yeast Episomal Plasmid  First constructed by Beggs (1978) by recombining an E. coli cloning
vector with the naturally occurring yeast 2 µm plasmid  Vectors derived from the 2µm plasmid
are called yeast episomal plasmids (YEps)  Eg:YEp13
Features of YEp13  It can replicate as an independent plasmid  It is a shuttle vector(It can
propagate in two different host species).  Contain 2 µm origin of replication and the selectable
LEU2 gene  Includes the entire pBR322 sequence.  Replicate and be selected for in both yeast
and E. coli  Has a copy number of 50–100 per haploid cell  Easy to recover the recombinant
DNA molecule from a transformed yeast colony
The word “episomal” indicates that a YEp can replicate as an independent plasmid, but also
implies that integration into one of the yeast chromosomes can occur. Integration occurs because
the gene carried on the vector as a selectable marker is very similar to the mutant version of the
gene present in the yeast chromosomal DNA. With YEp13, for example, homologous
recombination can occur between the plasmid LEU2 gene and the yeast mutant LEU2 gene,
resulting in insertion of the entire plasmid into one of the yeast chromosomes . The plasmid may
remain integrated, or a later recombination event may result in it being excised again.
Yeast Replicative plasmids (YRps)  Constructed by Struhl et al. (1979)  They isolated
chromosomal fragments of DNA which carry sequences that enable E. coli vectors to replicate in
yeast cells.  Such sequences are known as ars (autonomously replicating sequences)  ars acts
as an origin of replication  Able to multiply as independent plasmids  It is made up of pBR322
plus the yeast gene TRP1 Autonomous replicating system  This gene, which is involved in
tryptophan biosynthesis, is located adjacent to a chromosomal origin of replication.  The yeast
DNA fragment present in YRp7 contains both TRP1 and the origin.  Eg:YRp7

Yeast Centromeric Plasmids(YCps):  Autonomously replicating  Considered low copy vectors

and incorporate part of an ARS along with part of a centromere sequence (CEN).  Found as a
single copy.  CEN vectors are stable without integration.  Transform yeast at high efficiency 
Eg: YCp 50

Yeast Artificial Chromosome (YAC)  Yeast artificial chromosomes (YACs) are genetically
engineered chromosomes derived from the DNA of the yeast.  First described by Murray &
Szostak in 1983  It is a human-engineered DNA molecule used to clone DNA sequences in yeast
cells.  They are the products of a recombinant DNA cloning methodology to isolate and
propagate very large segments of DNA in a yeast host.  By inserting large fragments of DNA,
the inserted sequences can be cloned and physically mapped using a process called chromosome
walking.  The amount of DNA that can be cloned into a YAC is, on average, from 200 to 500 kb.
 However, as much as 1 Mb can be cloned into a YAC.  pYAC3 being a typical example
Structure of YAC cloning vector  Ori →PBR322 replication origin  Yeast selectable markers
These are genes which are compliment to specific auxotrophy (Organism that does not synthesize,
so we need to provide that biochemical into the media) and thus requires host cell(Yeast) to
contain a recessive non reverting mutation. Eg:LEU2,TRP1,URA3 &HIS3  ARS
→Autonomous replicating sequence elements It act as an origin of replication and initiates and
propagates the sequence elements of chromosome in yeast  CEN(Centromere) is required to
ensure segregation of sister chromatids in mitosis and homologous chromosome at first meiotic
division  TEL(telomere):Seals the ends of the chromosome and ensure its survival by protecting
from nuclease attack  SUP4:It is a selectable marker into which new DNA is cloned at Sma1
site.

VECTOR FOR MAMMALIAN

Mammalian expression vectors are used to introduce a specific fragment of DNA into
mammalian systems for RNA or protein expression. These systems include human and mouse cell
lines, and even in vivo in live animals. Mammalian expression vectors are often considered to be
more beneficial for the expression of mammalian genes due to the similarity in cellular machinery.
Adenoviruses, retroviruses, plasmids and lentiviruses are common expression vectors, each with
varying characteristics on the nature of protein expression. When choosing an expression vector, it
is also important to consider the target species, the size of the insert, and which detection tag is
most compatible. Learn more about a given vector by visiting the supplier page.

Mammalian cell transfection is a technique commonly used to express exogenous DNA or

RNA in a host cell line. There are many different ways to transfect mammalian cells, depending on
the cell line characteristics, desired effect, and downstream applications.
Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral
methods. Using various chemical or physical methods, this gene transfer technology enables the
study of gene function and protein expression in a cellular environment. The development of
reporter gene systems and selection methods for stable maintenance and expression of transfected
DNA have greatly expanded the applications for transfection. Assay-based reporter technology,
together with the availability of a wide array of transfection reagents, provides the foundation to
study mammalian promoter and enhancer sequences, trans-acting proteins such as transcription
factors, mRNA processing, protein: protein interactions, translation and recombination events.
Selecting the cell type is one decision that is vital for a successful transfection experiment.
Due to the variability in cell responses that may occur with different reagents or methods,
choosing the appropriate cell type when designing an experiment can help maximize transfection
success.
Expanding and maintaining cells for use in transfection experiments can prove quite
challenging due to the large amount of effort and time needed.
After cells are transfected, how will you determine success:
Cells are typically harvested 24–72 hours after transfection for studies designed to analyze
transient expression of transfected genes. The optimal time interval depends on the cell type,
research goals and specific expression characteristics of the transferred gene. Analysis of gene
products may require isolating RNA or protein for enzymatic activity assays or immunoassays.
The method used for harvesting cells will depend on the end product assayed. For example,
expression of the firefly luciferase gene in the pGL4.10 [luc2] is generally assayed 24–48 hours
after transfection, whereas the pGL4.12 [luc2CP] Vector with its protein degradation sequences
can be assayed in a shorter time frame (e.g., 3–12 hours), depending on the research goals and the
time it takes for the reporter gene to reach steady-state expression.
When performing a transient transfection, you can choose between a standard or reverse
transfection protocol. In a standard transfection protocol, the cells are plated on day 1, transfected
on day 2 and assayed on day 3 or 4. In a reverse transfection protocol, cells are added directly to a
plate containing the transfection reagent/DNA mix and assayed on day 2 or 3. Because the cells
are added directly to the DNA, this process reduces the experimental time by one day and allows
for high-throughput transfection of DNA in a plate or microarray format.
PROKARYOTIC AND EUKARYOTIC HOST
With the beginning of recombinant DNA technology, cloning and expression of numerous
mammalian genes in different systems have been explored to produce many therapeutics and
vaccines for human and animals in the form of recombinant proteins. But selection of the suitable
expression system depends on productivity, bioactivity, purpose and physicochemical
characteristics of the protein of interest.
Choice of host organism:
A good host should have the following properties:
• Easy to grow and transform.
• Do not hinder replication of recombinant vector.
• Do not have restriction and methylase activities.
• Deficient in recombination function so that the introduced recombinant vector is not
altered.
• Easily retrievable from the transformed host.
Various hosts are used in rDNA technology depending on the goal: For example bacteria, yeast,
plant cells, animal cells, whole plants and animals.

Prokaryotic systems such as E. coli are commonly used due to various advantages like, They
have-
• Well studied expression system,
• Compact genome,
• Versatile,
• Easy to transform,
• Widely available, and
• Rapid growth of recombinant organisms with minimal equipment.
Only disadvantage is that they lack post-translational modification (PTMs) machinery required for
eukaryotic proteins.
Escherichia coli
Escherichia coli is a Gram negative, facultative anaerobic, rod-shaped bacterium.
o E. coli is found in the environment, foods, and intestines of human and animals.
o E. coli is a large and diverse group of bacteria.
o The harmless strains produce vitamin-K and prevent colonization of the intestine
by pathogenic bacteria.
o E. coli is a typical prokaryotic expression system and one of the most attractive
heterologous protein producers.
o To date improved E. coli is the extensively used cellular host for foreign protein expression
because of its rapid growth rate which is as short as 20-30 minutes.
o Have the capacity for continuous fermentation and relatively low cost.
o The expression of proteins in this system is the; Easiest, High-level expression, Quickest
(Fast expression), Cheapest (Low cost), Simple culture conditions.
Bacillus subtilis is Gram-positive rod- shaped, spores forming bacterium found in the soil
o Also, the normal flora of the body and can found gastrointestinal tract of ruminants and
humans.
o Catalase positive and obligate aerobe
o B. subtilis, also known as hay bacillus or grass bacillus
It is an alternative to the E. coli expression system. It can secrete degradative enzymes or
antibiotics, produce spores and can become competent for genetic transformation Its
biotechnology companies secreted enzyme producing bacteria which produce a large scale of
industrial products. It is estimated that Bacillus species, including B. subtilis, produce 60% of
commercially available enzymes.
Expression System
Eukaryotic systems are difficult to handle in contrast to bacterial hosts. They are favoured
for expression of recombinant proteins which require post translational modification and only if
they can grow easily in continuous culture.
2. Choice of vector:
Vector is an autonomously replicating (inside a host cell) DNA molecule designed from a
plasmid or phage DNA to carry a foreign DNA inside the host cell. Transformation vectors are of
two types:
• Cloning vector is used increasing the number of copies of a cloned DNA fragment.
• Expression vector is used for expression of foreign gene into a protein.
• If a vector is designed to perform equally in two different hosts, it is called a shuttle
vector.
Properties of an ideal vector: A good vector should have the following characteristics:
• Autonomously replicating i.e. should have ori (origin of replication) region.
• Contain at least one selectable marker e. g. gene for antibiotic resistance
• May contain a scorable marker (β-galactosidase, green fluorescent protein etc.)
• Presence of unique restriction enzyme site.
• Have multiple cloning sites.
• Preferably small in size and easy to handle.
• Relaxed control of replication to obtain multiple copies.
• Presence of appropriate regulatory elements for expression of foreign gene.
• High copy number
Eukaryotic system for the expression of protein include:
o Yeast (EXPLAIN THESE POINTS SHORTLY REFER RESPECTIVE TOPIC)
o Mammalian cells (EXPLAIN THESE POINTS SHORTLY REFER RESPECTIVE TOPIC)
o Baculovirus cells (Insects) (EXPLAIN THESE POINTS SHORTLY REFER
RESPECTIVE TOPIC)
All these systems are great eukaryotic systems for the expression of recombinant proteins.
The selection of a suitable vector system depends mainly on the size limit of insert DNA and the
type of host intended for cloning or expression of foreign DNA.
The mechanism of regulation of gene expression in prokaryotes is greatly different from
eukaryotes. Regulation of gene expression in prokaryotes is mainly for environmental adaptation.
Regulation of gene expression in eukaryotes is for cell growth, differentiation and development.
There are four recombinant protein expression systems: bacteria (E.coli), yeast, insect and
mammalian cells.

INTRODUCTION OF RECOMBINANT DNA INTO HOST CELLS AND SELECTION

METHODS
The delivery of DNA into the host is required for generation of genetically modified organism.
DNA delivery to host is a 3-stage process, DNA sticking to the host cell, internalization and
release into the host cell.
Griffith Experiment Transformation - it is the natural process, through which bacterial
population transfer the genetic material to acquire phenotypic features. The event of
transformation was first time demonstrated by Frederick Griffith in 1928. Griffith has used two
different Streptococcus pneumonia strains, virulent (S, causes disease and death of mice) and
avirulent (R, incapable of causing disease or death of mice). In a simple experiment he injected 4
different combination of bacterial mixture, (1) live S, (2) heat killed S, (3) live R, (4) mixture of
live R and heat killed S in to the mice. The observation indicates that live S has killed the mice
where as mice were healthy with heat killed S or live R. Surprisingly, mice injected with mixture
of live R with heat killed S were found dead, and bacteria isolated from these dead mice were
virulent. Based on these observations, Griffith hypothesized the existence of a transforming agent
(Protein, DNA) being transferred from heat killed virulent strain to the avirulent strain and
proposed the concept of transformation. Later, Oswald has proved that the transforming factor is
DNA rather than protein.
Mechanism of Transformation- Transformation is the process by which cell free DNA is taken up
by another bacteria.The DNA from donor bacteria binds to the competent recipient cell and DNA
enters into the cell. The DNA enters into the recipient cell through a uncharacterized mechanism.
The DNA is integrated into the chromosomal DNA through a homologous recombination.
Naturally transformation is common between closely related species only.

Transduction/Transfection includes three methods of transfer chemical, physical and Biological.

In this process by which foreign DNA is introduced into a cell by a virus or viral vector
(biological method). Transduction does not require physical contact between the cell donating the
DNA and the cell receiving the DNA, it involves physical or chemical method to incorporate the
rDNA into host. Transduction is a common tool used by molecular biologists to stably introduce a
foreign gene into a host cell's genome (both bacterial and mammalian cells).

CHEMICAL METHOD
Calcium phosphate
In this method, DNA is mixed with calcium chloride in phosphate buffer and incubated for
20mins. Afterwards, transfection mixture is added to the plate in dropwise fashion. DNA-calcium
phosphate complex forms a precipitate and deposit on the cells as a uniform layer. The particulate
matter is taken up by endocytosis into the internal storage of the cell. The DNA is then escapes
from the precipitate and reach to nucleus through a unknown mechanism. This method suited to
the cell growing in monolayer or in suspension but not for cells growing in clumps. But the
technique is inconsistent and the successful transfection depends on DNA-phosphate complex
particle size and which is very difficult to control.
Uses
• This method is mainly used in the production of recombinant viral vectors.
• It remains a choice for plasmid DNA transfer in many cell cultures and packaging cell lines. As
the precipitate so formed must coat the cells, this method is suitable only for cells growing in
monolayer and not for suspension cultures.

Advantages
• Simple and inexpensive
• Applicability to generate stably transfected cell lines
• Highly efficient (cell type dependent) and can be applied to a wide range of cell types.
• Can be used for stable or transient transfection
• Toxic especially to primary cells
• Slight change in pH, buffer salt concentration and temperature can compromise the efficacy
• Relatively poor transfection efficiency compared to other chemical transfection methods like
lipofection.
• Limited by the composition and size of the precipitate.
• Random integration into host cell.
Optimal factors (amount of DNA in the precipitate, the length of time for precipitation reaction
and exposure of cells to the precipitate) need to be determined for efficient transfection of the
cells. This technique is simple, expensive and has minimal cytotoxic effect but the low level of
transgene expression provoked development of several other methods of transfection

Liposome and lipoplex method-Another approach of DNA transfection in animal cell is to pack
the DNA in a lipid vesicle or liposome. In this approach, DNA containing vesicle will be fused
with the cell membrane and deliver the DNA to the target cell. Preparation of liposome and
encapsulating DNA was a crucial step to achieve good transfection efficiency. Liposome prepared
with the cationic or neutral lipid facilitates DNA binding to form complex (lipoplex) and allow
uptake of these complexes by endocytosis. The lipoplex method was applicable to a wide variety
of cells, and found to transfect large size DNA as well. Another advantage of liposome/lipoplexes
is that with the addition of ligand in the lipid bilayer, it can be used to target specific organ in the
animal or a site within an organ.

PHYSICAL METHOD
Electroporation
Electroporation is a mechanical method used for the introduction of polar molecules into a
host cell through the cell membrane. This method was first demonstrated by Wong and Neumann
in 1982 to study gene transfer in mouse cells. It is now a widely used method for the introduction
of transgene either stably or transiently into bacterial, fungal, plant and animal cells. It involves
use of a large electric pulse that temporarily disturbs the phospholipid bilayer, allowing the
passage of molecules such as DNA.
 This method is based on the use of the short electrical pulses of high field strength.
Electroporation causes the uptake of DNA into protoplasts by temporary permeabilization of the
plasma membrane to macromolecules. Protoplasts and foreign DNA are placed in a buffer
between two electrodes and a high intensity electric current is passed. Electric field damages
membranes and creates pores in membranes. DNA diffuses through these pores immediately after
electric field is applied, until the pore are resealed. The technique is optimized by using
appropriate electric field strength (defined as the applied voltage divided by the distance between
two electrodes).
The optimum field strength is dependent on the following:
1. The pulse length of electric current
2. Composition and temperature of the buffer solution
3. Concentration of foreign DNA in the suspension
4. Protoplasts density, and
5. Size of the protoplasts.

Particle Gun or Biolistic Method

This is latest technology to transfer DNA into intact tissues. The method for regeneration
should be previously standardized and proper tissues be selected for bombardment. (a) Instrument:
The instrument is commercially available. Prototype was designed by Klein and co-workers. It
uses the explosive force of gun powder (0.22 caliber gun cartridge) to accelerate a polypropylene
cylindrical macroprojectile. Thin piece of polypropylene macroprojectile is loaded with
microprojectiles coated with DNA. Gun powder explosion forces this macroprojectile to move
with high speed toward another end of barrel, where it is blocked by a polycarbonate disc having
an aperture. Macroprojectile is stopped but microprojectiles move fast through the aperture
towards tissue placed in the same direction. For each transfer, 50 mg tungsten is accelerated upto
2000 ft per second in a partial vacuum. With this speed, particles reach upto lower layers of cells
in target tissues (Fig). The other devices are similar in basic design concept but use different
methods to accelerate particles like use of compressed air or gas. Compressed air (130 kg/cm2
pressure) has been used to accelerate microprojectiles at velocities (approximately 440 m/ sec)
necessary to achieve DNA delivery to plant cells (Fig). An electric discharge particle acceleration
device differs in basic design from the above described devices. In this device, a high voltage
discharge (14 KV current) delivered to a small water droplet which quickly vaporizes and releases
energy to propel DNA coated gold spheres into target cells (Fig.). In a similar way to above
devices, a DNA carrier is attracted (accelerated) due to potential differences, stopped in-between
by a screen, DNA coated particles cross the screen and fly towards target tissue and deliver the
DNA into cells.

Microinjection
Delivery of nucleic acids to protoplasts or intact cells via microinjection is a labour
intensive procedure that requires special capillary needles, pumps, micromanipulators, inverted
microscope and other equipment. However, injection into the nucleus or cytoplasm is possible and
cells can be cultured individually to produce callus or plants. In this way selection of
transformants by drug resistance or marker genes may be avoided. This method involves skill of
the worker to insert needle into the cytoplasm or in the nucleus. The basic technique is similar to
that used for animal cell microinjection. In order to microinject protoplasts or other plant cells, the
cells need to be immobilized

The cells are immobilized by:

1. The use of a holding pipette which holds the cells by vacuum.

2. Attachment of cells to poly-L-lysine coated cover slips. 3. Embedding the cells in

agarose, agar or sodium alginate.
Glass micropipette are prepared to have openings of about 0.3 uM in diameter and are
inserted into plant cell cytoplasm and nuclei with the aid of a micro manipulators device. A
syringe like device is used for the controlled delivery of volume (10-11 - 10-4 ul) into the plant
cell. Most plant cells are injected while keeping inside microdroplets (2-50 ul) of medium using a
chamber which is sterile, vibration free and permits temperature and humidity regulation. A
maximum of 100-200 cells per hour can be microinjected by this method. The recovery of
transformants is dependent upon the regeneration ability of the microinjected cells. Different
methods have been used to grow injured (microinjected) single cells or protoplasts. Hanging
droplets, covered under thin layer of agar or agarose, and micro culture have been used (Fig).
Attempts have been made to inject linear, or super coiled DNA, in cytoplasm or in nucleus.
Nuclear injections are found better for transformations.
Advantages
• No requirement of a marker gene.
• Introduction of the target gene directly into a single cell.
• Easy identification of transformed cells upon injection of dye along with the DNA. 9
• No requirement of selection of the transformed cells using antibiotic resistance or
herbicide resistance markers.
• It can be used for creating transgenic organisms, particularly mammals.
Disadvantages:
• Only one cell receives DNA per injection.
• Handling of protoplast for microinjection requires skilled persons.
• Sophisticated equipment.
• Requirement of regeneration process from microinjected cells.
BIOLOGICAL METHOD
Agrobacterium Method of Transfer
Agrobacterium is a phytopathogen that infects plants through wound sites, causing crown
gall disease, and is one of the most popular plant transformation tools used in agriculture to date.
 Agrobacterium tumefaciens is the soil pathogen that utilizes its bacterial type IV secretion
system for the transfer of its transferred (T)-DNA into the host cells.
 The genus Agrobacterium consists of different species depending on their disease
symptomology and host range. Some of the species
of Agrobacterium include A. radiobacter, A. vitis, A. rhizogenes, A. rubi and A.
tumefaciens.
 The organisms of this genus are most notably known as plant transformation tools used in
a wide range of host cells.
 The host range of the bacteria is determined by different bacterial as well as plant factors.
Bacterial factors include virulence genes and T-DNA oncogenes, whereas the plant factors
include genes required for transformation and tumor formation.
 The natural diversity of the bacteria is determined based on the presence of the primary
pathogenic determinant, the Ti/Ri plasmid.
 Different strains of Agrobacterium can be isolated from all around the world in a wide
range of host plants. Some of the common host plants include roses, poplar, weeping fig,
chrysanthemum, and other fruit trees.
 Agrobacterium is a Gram-negative rod-shaped bacterium ranging in size from 1.5 to 3 µm
in length and 0.6 to 1.0 µm in width. The bacterium is motile with one or as many as six
flagella.
 These do not form spores and are strictly aerobic organisms residing in soil with clinical as
well as biotechnological applications.
Structure of Ti plasmid

T-DNA region – this region carries genes responsible for inducing tumors in plants. Other
foreign genes of interest can be inserted in this region.
For inducing a tumor, only the T- DNA is transferred from one cell to another.
Vir region – this region consists of virulence genes. It is responsible for the excision,
integration, and transfer of T-DNA into the plant chromosome.
Opine catabolism region – this region catabolizes opines, which are specialized amino acids.
It is responsible for catabolizing opines produced by the T-DNA region. Opines often act as a
source of Nitrogen in the bacteria.
  Other regions include the origin of replication, T-DNA border sequence, plant hormone
synthesis region, and conjugation region.
Plant hormone synthesis region – this region is responsible for the synthesis of essential
plant hormones like auxins and cytokinin.
Ti plasmid Conjugation region – process involving Ti plasmid includes the transfer of whole
Ti plasmid from one cell to another.
The T-DNA border sequence is 24bp with direct repeats. It marks the border of the T-DNA and
is important for the transfer of plant chromosomes.
Principle of Agrobacterium-mediated Gene Transfer
 The basis of Agrobacterium-mediated transformation is the ability of the organism to
transfer its T-DNA into the host cells efficiently.
 The biology of the process consists of two components; the T-DNA consists of 25 bp
repeats that end at the T-region and the virulence (vir) region composed of seven major
loci.
 The mechanism of Agrobacterium-mediated transformation is based on the transfer of a
piece of plasmid by the bacteria into the plant cells during infection.
 The plasmid then integrates into the nuclear genome in order to express its own genes and
affect the hormonal balance in the host cell.
 Besides, the bacteria also produce a number of enzymes that are involved in the synthesis
of opines that is then used by the bacteria as nutrients.
 Some of the essential components of the bacteria involved in infection are T-DNA present
on the plasmid called Ti (tumor-inducing) plasmid along with other functional components
like virulence (vir), conjugation (con), and origin of replication (ori).
 The infection begins with the entry of the bacteria through wounded sites. The binding of
bacteria to the plant cells is enhanced by the release of phenolic acetosyringone (AS) by
the injured plant cells.
 The AS activates the VirA proteins on the bacteria, which activates VirG via
phosphorylation of its aspartate residue.
 The activated form of VirG then binds to other vir genes, inducing their expression. VirD
activated by this process stimulates the T-strand generation (a single-stranded copy of the
T-DNA).
 The VirD2 covalently binds to the 5’ end of the T-strand as the 5’ end is the leading end
during the transfer. Other factors like VirE2 and VirB proteins also bind to the T-strand,
forming a T-complex.
 The complex is then passed into the nucleus by the nuclear target signals released by the
Vir proteins. The T-DNA strand is integrated into the plant genome randomly as either a
single copy or multiple copies.
 The integration usually occurs in the transcription active or repetitive regions of the
genome by the process of recombination.
 Even though much is known about the molecular biology of T-DNA transfer
in Agrobacterium cells, not much is known about the plant-encoded factors involved in the
process.

Applications of Agrobacterium-mediated Gene Transfer

 The following are some of the important applications of Agrobacterium-mediated
transformation;
1. The Agrobacterium-mediated transformation has been used as a method of genetic
modification of plants for the production of various substances like proteins, antibodies,
and even vaccines.
2. Different plants have also been modified to produce life-saving pharmaceutical products
like anticoagulants, human epidermal growth factors, and interferons.
3. Transgenic plants prepared with Agrobacterium serve as biomonitors to detect the presence
of toxic compounds in the environment as well as to detoxify the contaminated soil and
water.
4. Agrobacterium-mediated transformation has also remarkably increased crop yields by
modifying the shelf-life and biosynthesis of the plants.
5. Plants can be modified to enhance tolerance against biotic and abiotic factors, nutrient
capture with increased pest resistance.
6. Agrobacterium-mediated transformation has been used to produce insect resistance crops
by the incorporation of various toxic genes like the Bt toxin genes.
7. The increase in pest resistance results in a reduction in the use of harmful agrochemicals
and herbicides.

Bacteriophage (REFER BACTERIOPHAGE CONTENT GIVEN ABOVE)

SELECTION METHODS FOR RECOMBINANTS

Screening for Recombinants
After the introduction of r-DNA into a suitable host cell, it is essential to identify those
cells which have received the r-DNA molecule. This process is called screening. The vector or
foreign DNA present in recombinant cells expresses the characters, while the non-recombinants do
not express the characters or traits. For this some of the methods are used and one such method is
Blue-White Selection method.
1. Insertional Inactivation - Blue-White Colony Selection Method
It is a powerful method used for screening of recombinant plasmid. In this method, a reporter
gene lacZ is inserted in the vector. The lacZ encodes the enzyme β-galactosidase and contains
several recognition sites for restriction enzyme.
β-galactosidase breaks a synthetic substrates called X-gal (5-bromo-4-chloro-indolyl-β -D-
galacto-pyranoside) into an insoluble blue coloured product. If a foreign gene is inserted into lacZ,
this gene will be inactivated. Therefore, no- blue colour will develop (white) because β-
galactosidase is not synthesized due to inactivation of lacZ.
Therefore, the host cell containing r-DNA form white coloured colonies on the medium contain X-
gal, whereas the other cells containing non-recombinant DNA will develop the blue coloured
colonies. On the basis of colony colour, the recombinants can be selected.

Antibiotic resistant markers

An antibiotic resistance marker is a gene that produces a protein that provides cells with
resistance to an antibiotic. Bacteria with transformed DNA can be identified by growing on a
medium containing an antibiotic. Recombinants will grow on these medium as they contain genes
encoding resistance to antibiotics such as ampicillin, chloro amphenicol, tetracycline or
kanamycin, etc., while others may not be able to grow in these media, hence it is considered useful
selectable marker.
Replica plating technique
A technique in which the pattern of colonies growing on a culture plate is copied. A sterile
filter plate is pressed against the culture plate and then lifted. Then the filter is pressed against a
second sterile culture plate. This results in the new plate being infected with cell in the same
relative positions as the colonies in the original plate. Usually, the medium used in the second
plate will differ from that used in the first. It may include an antibiotic or without a growth factor.
In this way, transformed cells can be selected.
Colony Hybridization (REFER UNIT II NOTES ON THIS TOPIC).