Vectors.pptx

Vector
Vector is a nucleic acid molecule that has the
ability to replicate in an appropriate host cell
and into which the DNA to be cloned is
integrated.
Ex. Plasmid, Bacteriophage, Plant and animal
Viruses, Cosmid, Phagemid, Phasmid, BAC,
YAC, PAC.

Properties of a good vector
1) Small size, Autonomous replication, Easy to
isolate & purify.
2) At least 2 suitable marker genes.
3) Unique target sites for many restriction
endonucleases preferably within marker genes
(Recombinant vector selection).
4) Easy to introduce recombinant vector in to host
cells and Selection of recombinants should be
easy.
5) Expression vectors should contain suitable
control elements like promoter, operator and rbs.

Vectors
Cloning Vectors: Vectors used for the
propagation of DNA inserts in a suitable host.
Expression Vector: vector designed for the
expression of a protein specified by the DNA
insert .
Expression vector should have control
elements viz., promoter, terminator, rbs.

Vectors
Natural
1) Plasmids
2) Bacteriophages
3) Plant Viruses
4) Animal Viruses
Artificial
1) Cosmids
2) Phagemids
3) Phasmids
4) BAC
5) PAC
6) YAC

Plasmids
• Joshua Lederbergh coined the term Plasmid in 1952.
• Plasmids are extra chromosomal replicons that are
stably inherited in a cell.
• Plasmids are nucleic acids (DNA/RNA) that contain
origin of replication corresponding to the host –
replicons.
• Plasmids are not a part of the genomic DNA - extra
chromosomal.
• Heterogeneous circular DNA molecules found in
Bacillus megaterium lack origin of replication and
hence they are not plasmids.

• Plasmids are seen in bacteria and in lower eukaryotes
like yeast.
• Also seen in plant mitochondria.
• Made up of both DNA & RNA. Both single stranded
and double stranded plasmids are seen.
• However the number of dsDNA plasmids > ss DNA
plsmids > ss RNA plasmids > dsRNA plasmids.
• Double stranded RNA plasmids are very rare and seen
in Sacharomyces cerevisiae.
• Majority of the plasmids are circular and double
stranded. However many linear plasmids have been
found. Ex. Borellia & Streptomyces species contain
linear double stranded plasmids.
Plasmids

Plasmids
• In a ds circular DNA plasmid if both the strands are
intact - Covalently Closed Circular (CCC) DNA. If one
of the two strands is broken - Open Circular (OC)
DNA.
• Isolated plasmid DNA exists in supercoiled state.
• Addition of Ethidium bromide introduces – ve
super coils, unwinds plasmid forms relaxed circles.
• Addition of excess ethidium bromide results in
negative supercoiling.
• However the amount of ethidium bromide that can
bind to circular DNA is limited and is exploited in
isolating plasmid DNA.

The figure shows conversion of the relaxed (a) to the
negatively supercoiled (b) form of DNA.
The strain in the supercoiled form may be taken up
by supertwisting (b)
or by local disruption of base pairing (c).

Plasmids
• Plasmid Size - < 1 kbp to >200kbp. Plasmids with
a size more than 150kbp are known as mega
plasmids. Ex. Ti plasmid, Ri plasmid.
• Plasmid copy number is the number of plasmid
copies maintained per cell.
• Stringent plasmid - copy number -1 to 3 Ex. Ti
plasmid.
• Relaxed plasmid - copy number is 5 or more ex.
pUC 18.

Plasmids
• Plasmids carry genes & their phenotypic
properties conferred on host. Ex. Antibiotic
resistance genes, heavy metal resistance genes,
hydrocarbon degrading genes, bacteriocin
producing genes.
• Plasmids with no known function is known as
called Cryptic plasmids

Plasmids
• Plasmids that contain “tra” genes are called
Conjugative plasmids because these “tra“ genes
promote conjugation. Plasmids lacking the tra
genes are known as Non Conjugative plasmids.
• Usually conjugative plasmids are maintained as
stringent plasmids and non conjugative plasmids
are maintained as relaxed plasmids.
• Some non conjugative plasmids promote
conjugation in the presence of conjugative
plasmids. Such plasmids are known as “mob”
plasmids. Ex. pBR322.

Plasmids
• The inability of 2 plasmids to exist together in the
absence of selection pressure is known as
Plasmid incompatibility.
• Plasmids that cannot co exist together (mutually
incompatible) belong to same incompatibility
group.
• Till now many such compatibility groups have
been identified Ex. In E. coli there are at least 13
such incompatibility groups.

• Plasmids have different mechanisms to be
retained by bacteria during cell division.
• Single copy plasmids have ‘Par’ site (genes and
sites) which allows equal distribution of
plasmids to daughter cells (Par functions).
– parS site, ParA and ParB proteins encoded on
plasmids.

Models for plasmid partitioning

• Plasmid Curing = loss of plasmid
• However if no partitioning mechanism is
present, random assortment would cause
some bacteria in a population to lose plasmid
entirely.
• The lower the copy number, the greater
chance for plasmid to be lost.
Plasmid Copy number
• RK2 4-7 (in E. coli)
• pBR322 24
• pUC 500 -700
• pIJ101 40-300

Plasmid Classification by function
• Fertility-F-plasmids, which contain tra-genes.
They are capable of conjugation.
• Resistance-(R)plasmids, which contain genes that
can build a resistance against antibiotics or
poisons.
• Historically known as “R” factors, before the
nature of plasmids was understood.

Plasmid Classification by function
• Col-plasmids, which contain genes that code for
(determine the production of) bacteriocins,
proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion
of unusual substances, e.g., toluene or salicylic
acid.
• Virulence plasmids, which turn the bacterium into
a pathogen

Isolation of Plasmids
• There are several techniques to isolate
plasmid DNA - Barenboim & Dolly’s method
or Alkali Lysis method, Classical Method,
Eckhart’s Method etc.
• The principle of Barenboim & Dolly’s method
or alkali lysis method is - Plasmids are more
resistant to adverse conditions than genomic
DNA and there is a narrow pH range where
the genomic DNA will be denatured but not
the plasmid DNA.

Terminology commonly used
• Transformants: The bacteria which have taken up
the foreign DNA are known as transformants
others are known as non transformants.
• Recombinants: The bacteria which have taken up
the recombinant DNA. Others are known as non
recombinants .
• Insertional inactivation: Inactivating the function
of a gene by cutting it with a restriction
endonuclease (whose target site is present within
the gene) and inserting a foreign DNA in to it.

Terminology commonly used
• Cloning vectors: A DNA molecule in which foreign
DNA can be inserted or integrated and which is
capable of replicating within host cell to produce
multiple clones of recombinant DNA. The cloned
gene may or may not be expressed. A cloning
vector should contain – Ori.
• Expression vector: A vector designed for the
expression of a protein specified by the DNA
insert. Expression vector should have control
elements viz., promoter, terminator, rbs.

Desirable features of
plasmid cloning vehicles
• Small size and low molecular weight because such
plasmids are easy to handle and more resistant to
adverse conditions & damage by mechanical shearing.
• Should have marker genes that confer readily
selectable phenotypic traits on host cells and which
permit the selection of host cells which harbor
recombinant DNA (transformants) from those which do
not (non-transformants).
• Single target sites for several restriction
endonucleases, preferably within the marker genes.
• Should have a high copy number

pSC101
• p- plasmid, SC-Stanley Cohen
• A DNA plasmid, used as first cloning vector by
Herbert Boyer and Stanley Norman Cohen in 1973.
• A gene from a frog was transferred and expressed in
E. coli using pSC101.
• Size of pSC101 is 9,263 bp.
• The copy number is around 5.
• Tetracycline resistance gene (tetR) is the marker gene.
• Unique target sites for Eco RI, XhoI outside tet
R
gene.
• Unique target sites for Bam HI, SalI, HindIII within
tet
R
gene.

Disadvantages of Plasmid pSC101
• Distinction between transformants & non
transformants and recombinants & non
recombinants is not easy.
• pSC101 has a low copy number.
• It has a big size hence the size of the DNA to be
inserted is small.
• To overcome these difficulties new plasmids with
better features have been constructed.

pBR322
P - plasmid, B- Bolivar, R- Rodriguez
• pBR322 is a popular plasmid created in 1977 using
both classical genetic techniques and recombinant
DNA methodology.
• Initial size – 4361 bp. GC bp later added – 4363 bp
• Later 2 bp (1893 & 1915) removed. Now the new
size is 4361 bp
• Ori is from plasmid pMB1
• Marker gene - ampR (ampicillin resistance) gene
from plasmid RSF2124. It’s promoter is P1
• Marker gene - tetR (tetracycline resistance gene)
from plasmid pSC101. It’s promoter is P2

pBR322
• The promoters P2 & P1 are in the same region but
on the opposite strands and initiates transcription
of tetracycline resistance gene in the opposite
direction of the beta-lactamase (bla) gene.
• pBR322 has a copy number of 24 per cell but can
be increased up to 3000 per cell by plasmid
amplification.

pBR322 Construction
• Plasmid R7268 (R1) was
isolated
↓
• Variant of this plasmid
R1drd19 was isolated
↓
• Transposon Tn3 was
transposed to pMB1 to form
pMB3.
↓
• Due to Eco R1 rearrangement
formation of pMB8 (Carries
colicin immunity)
↓
• Combination of EcoR1
fragments from pSC101 with
pMB8 (opened at its unique
EcoR1 site) to generate
pMB9.
• pMB8 codes for both
teteracycline and colicin
immunity

pBR322
• Plasmid amplification – By using antibiotics that
inhibit protein synthesis viz., chloramphenicol or
by amino acid starvation cell division is inhibited
but not plasmid replication.
• This leads to increased plasmid copy number and
the technique is known as plasmid amplification.

pBR322
• The origin of replication or ori of pBR322 is from
plasmid pMB1 (a close relative of ColE1).
• Two RNAs (RNAI and RNAII) and one protein
(called Rom or Rop) regulate replication and
hence the plasmid copy number.
• RNAII (555B long) cleaved by RNaseH acts as
primer for initiation of DNA synthesis
• RNAI and Rop protein are negative regulatory
functions: RNAI binds to RNAII stopping primer
formation; Rop protein stabilizes the complex
formed by RNAI and RNAII.

Control of plasmid copy number in colE1 plasmids

pBR322
• Has more than 40 unique target sites for Restriction
endonucleases
• 11 of these 40 sites lie within the tetR gene (Eco RV,
NheI, Bam H I, Sgr I, Sph I, Eco N I, Sal I, Psh A, Eag
I, Nru I, Bsp M I) and 2 sites in tetR Promoter (Hind III
and Cla I).
• There are 6 key restriction sites (Ahd I, Bsa I, Ase I,
Pst I, Pvu I & Sca I) inside the ampR gene.
• Thus, cloning in pBR 322 with the aid of any of those
19 enzymes will result in Insertional inactivation of
either ampicillin or tetracycline resistance markers.

1. Cut plasmid and insert DNA with RE at
unique site (Pst I in this example).
2. Ligate with DNA ligase.
3. Select for cells that are Tetr.
4. Detection of recombinants: Replica plate to
select Amps cells.
5. Potential problem: plasmid self-ligation.
6. Solution: pretreat plasmid with alkaline
phosphatase
Cloning with pBR322.

Selection of
Recombinant pBR322 Vectors
• If foreign DNA is inserted in to pBR 322 using PstI
then ampicillin resistance gene will become
inactivated (insertional inactivation).
• The recombinant DNA is then added to bacteria for
transformation and the transformants are selected
on a medium that contains tetracycline.
• The non transformants do not grow on tetracycline
containing medium as they lack the plasmid and
hence the tetracycline resistance gene.
• This plate in which transformants are grown is called
a master plate and is stored properly for further use.

Selection of recombinant
pBR322 Vectors
• To a new petriplate (replica plate) ampicillin is added
along with the medium.
• To a wooden block with a diameter similar to master
and replica plates, a sterile velvet cloth is tied.
• The wooden block is then gently lowered on to Master
plate.
• The bacteria of the master plate come in to contact
with the fibres of velvet cloth.
• Then the wooden block is then taken out of master
plate and gently lowered on to replica plate.
• The bacteria bound to the velvet fibres are transferred
on to the replica plate and then it is incubated under
optimal conditions.

Selection of recombinant
pBR322 Vectors
• The bacterial colonies grown on replica plate
are then compared with the master plate.
• The colonies growing in the master plate but
absent in the replica plate are sensitive to
ampicillin.
• These are recombinants and are selected from
the master plate for further use.
• This procedure is called replica plating and is
very useful in identifying the recombinants

Disadvantages of pBR322
1) The selection of recombinants in case of pBR322
requires 2 steps and replica plating. Errors may creep
in during replica plating.
2) There is a nic bom (bom = basis of mobility) region in
pBR 322 which promotes conjugation in the presence
of conjugative plasmids (Mobilization) which is not
desirable.
3) This nic bom region also interferes with the replication
efficiency of extra chromosomal DNA in monkey cells.
Such prokaryotic sequences are called poison
sequences.
4) The plasmid pBR327 was created by deleting poison
sequence from pBR322.

pUC vectors
• p-plasmid, UC -University of California
• Derivatives of pBR322, initially constructed by
Vieira and Messing in the University of California
in 1982.
• It is a circular double stranded DNA and has a
size of approximately 2.7 kbp.
• The ori and ampR are from pBR322 and lacZ’
gene which produces N-terminal fragment of β
galactosidase was taken from E. coli
chromosome.

pUC
• The copy number of pUC 18 vectors is 500-700
and can be enhanced to 1000 -3000 by
amplification.
• Note: In the construction of pUC18 the ‘rop’ gene
was deleted and a single-base was mutated in the
origin of replication of pBR322. This increased the
copy number from ~20 per cell to ~700 per cell

pUC
• pUC18 and pUC19 vectors are small, high copy
number, E.coli plasmids, 2686 bp in length.
• They are identical except that they contain
multiple cloning sites (MCS) arranged in opposite
orientations.

pUC
pUC18/19 plasmids contain:
(1) pMB1 replicon responsible for the replication of
plasmid (source – plasmid pBR322). The high
copy number of pUC plasmids is a result of the
lack of the rop gene and a single point mutation
in rep of pMB1.
(2) ‘bla’ gene, coding for beta-lactamase that
confers resistance to ampicillin (source –
plasmid pBR322). It differs from that of pBR322
by two point mutations.

pUC
pUC18/19 plasmids contain:
(3) Region of E. coli ‘lac’ operon containing CAP
protein binding site, promoter Plac, lac
repressor binding site and 5’-terminal part of
the lacZ gene encoding the N-terminal fragment
of beta-galactosidase(α lac Z).
Note: Host cells of pUC vectors have a genotype of
lacZΔM15, a mutated version of lacZ gene which
will be complemented by lacZ’ of pUC and
produce functional beta-galactosidase by
complementation.

pUC
In the presence of IPTG, bacteria synthesise both
fragments of the enzyme and form blue
colonies on media with X-gal.
Insertion of DNA into the MCS located within the
lacZ gene (codons 6-7 of lacZ are replaced by
MCS) inactivates the N-terminal fragment of
beta-galactosidase and abolishes alfa-
complementation.
Bacteria carrying recombinant plasmids therefore
give rise to white colonies.

pUC
• MCS (Multiple Cloning Site) or poly linker sequence
is a short sequence in which a series of single target
sites for several restriction endonucleases have
been designed.
• In pUC vectors a poly linker sequence or MCS is
found in the downstream of promoter and after the
first few codons of lacZ’.
• This is identical to those found in phage M13. When
DNA fragments are cloned in this region of pUC, the
lac gene is inactivated.

pUC
• The MCS of pUC series of vectors generates DNA
fragment with 2 different sticky ends to be cloned
without any additional modifications such as
addition of linker or adapter of homopolymer
tailing.
• This asymmetric cloning will aid in directional
cloning or positional cloning.

pUC
• This is an expression vector. It contains lac
promoter (Plac) - an inducible promoter, lac
operator (Olac) and lac I gene which codes for lac
repressor protein.
• Selection of recombinants is a single step process
unlike pBR322.
• These plasmids when transformed into an
appropriate E. coli strain, which is lac─ (e.g.
JM103, JMI09), and grown in the presence of
IPTG (inducer of beta galactosidase enzyme), X-
gal (substrate for the enzyme) and ampicillin to
select transformants.

pUC
• Bacteria with unaltered pUC vectors will produce
functional beta galactosidase and hence blue
colonies develop.
• Bacteria with recombinant pUC vectors lack
functional beta galactosidase (insertional
inactivation) hence produce white colonies.
• The cloning vectors belonging to pUC family are
available in pairs with reversed orders of restriction
sites relative to lac Z promoter.
• pUC8 and PUC9 make one such pair . Other similar
pairs include pUC12 and pUCl3 or pUC18 and pUC19

Selection of recombinant pUC vectors
(Blue white screening )

pGEMR series
• pGEM3Z and pGEM-4Z are standard cloning
vectors.
• They transcribe the genes invitro.
• They are very similar to pUC series of vectors.
• The size of pGEM is 2750 bp approximately.
• 2 marker genes – ampR and lacZ’ genes.
• There is a MCS present in the lacZ’ gene.

pGEMR series
• There are 2 promoters specific for SP6 and T7
phages on either side of MCS.
• These promoters are not recognized by E. coli
RNA polymerase.
• However they are very strong promoters and
produce more amounts of RNA than E. coli RNA
polymerase in a short time when supplied with
either T7 or SP6 RNA polymerase supplied
invitro.

pGEMR series
• The vectors pGEM-3Z and pGEM-4Z are identical
in all aspects except for the orientation of T7
and SP6 promoters.
• One of the advantages with this system is RNA
complementary to both the strands can by
synthesized using 2 different promoters at
different times.
• The RNA thus synthesized can be used as a
probe.

Limitations of plasmids as vectors
• Insert size - Plasmids cannot accept DNA
inserts longer than 10kbp.
• If longer DNA inserts are cloned then
transformation efficiency is lowered.
• If the DNA is introduced in to cell then the
DNA insert will not be stable.

Limitations of plasmids as vectors
• The transformation efficiency is also very low.
(1 in 10,000)
• The efficiency of transformation can be
enhanced (2 -3 in 10,000) by treatment of
competent cells with divalent cations like
Calcium, cold shock treatment etc. However
the frequency of transformation is very less &
insignificant.
• To overcome these difficulties, search for new
vectors has led to the development of Phage
vectors.

Bacteriophage Vectors
• Bacteriophages are viruses that infect
bacteria
examples -
(1) λ phage
(2) M13 phage
(3) P2
(4) T4

Plaques: the clear areas within the lawn where
lysis and re-infection have prevented the cells
from growing.

• λ phage genome is 48.5 kb in length
• Linear or circular genome (cos ends)
• Bacteriophage λ is a coliphage i.e. it infects E.
coli
• Has an icosahedron protein head (20 sides in
3 D structure) and a flexible protein tail with
tail fibre.
• Lytic phase (Replicate and release)
• Lysogenic phase (integrate into host genome)
λ phage -Features

• λ genome has 50 genes organized in to functionally
related groups.
• Left hand region includes genes of head and tail
proteins starting from Nu I to J.
• Central region has genes of (i) integration (gam) &
recombination functions (int, xis, red, gam) (ii) ‘b2’
region or the non essential region (iii) ‘att p’ site –
attachment site of λ genome in to host genome during
lysogeny.
• Many genes of this region are not essential for the lytic
life cycle of λ phage and hence can be deleted or
replaced to make space for foreign DNA incorporation.
λ phage -Features

• Right hand region – The genes to the right of
the central region include (a) regulatory genes
(N&Q), genes controlling lysogeny (cI, cII & cIII)
& lysis (cro), genes essential for replication
(O&P) and genes required for host cell lysis
(S1R, R2).
• The organization of functionally related genes
in clusters enable these genes to be
transcribed together i.e. as an operon and
controls the expression of genes as a group
rather than individually.
λ phage -Features

• Cro (Control of Repressors Operator) gene –Cro
protein helps in the establishing host cell lysis.
• CI gene helps in establishing lysogeny.
• pCI is a λ repressor protein of lytic genes that
competes with pCro.
• CII – pCII is a transcription activator of λ genes that
repress lytic functions and catalyze the integration
of viral DNA in to host genome.
• CIII- pCIII stabilizes pCII by inhibiting host Hfl
protein, a cellular protease that degrades pCII.
• A, NUI – code for the enzyme terminase that binds
to cos sites and cleaves concateameric phage DNA.
λ phage -Features

• Cro (Control of Repressors Operator) gene –
Cro helps in the establishing host cell lysis.
• CI helps in establishing lysogeny.
• pCro is protein obtained from Cro gene (an
early gene product) transcribed from PR
promoter.
• pCro protein is a repressor of PCI (promoter of
CI) and early genes.
• CI - pCI is a λ repressor protein of lytic genes
that competes with pCro. CI helps in
establishing lysogeny.
λ phage -Features

• CII – pCII is a transcription activator of λ genes that
repress lytic functions and catalyze the integration of
viral DNA in to host genome. It is a delayed early
gene product and is transcribed by PL promoter. The
pCII concentration is high during poor nutritional
conditions and higher multiplicity of infection.
• CIII- pCIII stabilizes pCII by inhibiting host Hfl protein,
a cellular protease that degrades pCII. This is a
delayed gene product transcribed from PL
promoters. The concentration of pCII will be high
during poor nutritional conditions and higher
multiplicity of infection.
λ phage -Features

• A, NUI – code for the enzyme terminase that
binds to cos sites and cleaves concateameric
phage DNA.
• λ bacteriophage has a 15 bp long sequence (att
p) in its genome which is homologous with a
sequence seen in E. coli genome.
• Prophage – During lysogeny the phage genome
gets integrated in to host genome using this site.
Such an integrated phage form is called
prophage.
λ phage -Features

-PL ( promoter) for transcription for the left side of
l with N and cIII
-PR (promoter) for right, including cro, cII and the
genes encoding the structural proteins.
-OL and OR is short non-coding region of genome,
they control the promoters.
-cI (repressor) protein of 236 a.a. which binds to
OR and OL, preventing transcription of cro and N,
but allowing transcription of OL, and the other
genes in the left hand end.
-cII and cIII encode activator proteins which bind to
the genome.
λ phage -Features

-Cro (66 aa) protein which binds to OR and OL,
blocking binding of the repressor to this site to
prevent lysogeny.
-N codes an antiterminator protein and allows
transcription from PL and PR. It also allows RNA
polymerase to transcribe a number of phage
genes, including those responsible for DNA
recombination and integration of the prophage,
as well as cII and cIII.
-Q is an antiterminator similar to N, but only
permits extended transcription from PR
-Two Termination sites- One between N and CIII
and other between cro and CII.
λ phage -Features

• Immediate early phage genes in phage lambda
are equivalent to the early class of other phages.
They are transcribed immediately upon infection
by the host RNA polymerase.
• Delayed early genes in phage lambda are
equivalent to the middle genes of other phages.
They cannot be transcribed until regulator
protein(s) coded by the immediate early genes
have been synthesized.
λ phage -Features

• Middle genes are phage genes that are regulated
by the proteins coded by early genes. Some
proteins coded by middle genes catalyze
replication of the phage DNA; others regulate the
expression of a later set of genes.
• Late genes are transcribed when phage DNA is
being replicated. They code for components of
the phage particle.
• Two sets of transcripts are independent; as a
consequence, early gene expression can cease
after the new sigma factor or polymerase has
been produced.
λ phage -Features

Genes are clustered by function in the
lambda genome
Recombination Control region Replication Lysis
Virus head
&tail
origin
oR
Pint oL
PL PRM PR PRE PR‘
tR3
tL1 tR1 tR2 t6S
att
int
xis
red
gam
cIII N cI cro cII O P Q S R A…J
promoter
operator
terminator
Late control
cos
Not to scale!

λ Phage
Lysogenic
life cycle
Part1

λ Phage
Lysogenic
life cycle
Part2

DNA
Protein
coat
cos cos
Nonessential region
Long (left)
arm
Short (right)
arm
Exogenous DNA
(~20-23 kb)
λ phage

The phage λ cos ends
Circular form
Linear form

Which proteins determine the cycle?
• Lysogenic cycle: cI proteins predominate
• Lytic cycle: cro proteins predominate

Lambda Phage killing Host cell

Temperate and lytic phage have a different
plaque morphology
Lytic phage: clear plaques
Temperate phage
generate turbid
plaques
lysogenized cells
lysed cells
uninfected cells
lysed cells
Mutants of phage that
have lost the capacity to
lysogenize form clear
plaques

Induction and immunity of lysogens
l
A l lysogen
l
Spontaneously,
1/1000 lysogens will
induce, i.e. the l
prophage will
excise, replicate and
lyse the cell.
UV treatment leads
to induction of
virtually all lysogens
in a culture.
Lysogens are immune to
further infection with similar
(lambdoid) phage
+
l

Induction and immunity of lysogens
l

λ phage - Vectors
• λ Insertion vectors
• λ Replacement vectors

λ Insertion vectors
• Insertion vectors are the simple lambda cloning
vectors. This vector itself can be grown on bacterial
lawn (therefore must contain at least 75% of the
wild-type genome length).
• To accommodate large DNA stretches, a portion of
non essential region is deleted. However the size of
the genome after non essential region removal
should be at least 38 kbp or more.
• Foreign DNA fragment is inserted into a unique
restriction site in the vector genome.
• Packaging requirements thus limit insert fragment
size to 0 - 10 KB - due to the limitations on viral
genome size. Ex. λ gt10, λgt11 etc.

λgt10
• λgt10 is a 43 kb double stranded DNA for cloning
fragments that are only 7 kb in length.
• The insertion of DNA into λgt10 inactivates cI+
(repressor) gene, generating a cl- bacteriophage.
• Non recombinant λgt10 is cl+ and forms cloudy
plaques on appropriate E. coli host, while
recombinant λgt10 are cl- and form clear plaques
permitting screening of recombinant plaque.
• Note: cI gene is responsible for lysogeny.

The phage λ insertion vectors

• Further, in an E. coli strain carrying hflA 150
mutation (high frequency lysogeny mutation) only
cl-
phage will form plaques, because cl+ will form
lysogens (integrate with bacterial genome) and will
not undergo lysis to form any plaques.
• Recombinant λgt10 plaques can thus be easily
selected.

Fusion Proteins
• Fusion proteins or chimeric proteins are proteins
created through the joining of two or more genes
which originally coded for separate proteins.
• Translation of this fusion gene results in a single
polypeptide with functional properties derived from
each of the original proteins.

Fusion Proteins
• Fusion genes may occur naturally in the body by
transfer of DNA between chromosomes.
• The bcr-abl gene found in some types of leukemia is
a fusion gene that makes the BCR-ABL fusion
protein.
• Recombinant fusion proteins are created artificially
by recombinant DNA technology for use in
biological research or therapeutics.
• Many whole gene fusions are fully functional.
Some, however, experience interactions between
the two proteins that can modify their functions.

λgt11
• λgt11 is an expression vector, where inserted DNA is
integrated within the lacZ gene present in the
vector and expressed as β galactosidase fusion
protein.
• λgt11 is a 43.7 kb double stranded phage for cloning
DNA segments, which are less than 6 kb in length
(usually for cDNA).
• It has a marker gene (lacZ) with a unique target site
for EcoRI
• Foreign DNA can be expressed as β galactosidase
fusion proteins.

• Recombinant λgt11 can be screened using either
nucleic acid or antibody probes (λgt10 cannot be
screened using antibodies).
• The recombinant λgt11 becomes lacZ─ and form
clear plaques.
• The non recombinant λgt11 remains lacZ+ and form
blue plaques permitting screening in the presence
of IPTG (inducer) and Xgal (substrate).

λ replacement vector
• Replace the nonessential region of the phage
genome with exogenous DNA (~ 20 kb)
• High transformation efficiency (1000-time
higher than plasmid)

λ Replacement
Vector
2. Packing with a
mixture of the phage
coat proteins and phage
DNA-processing enzymes
1. Ligation
3. Infection and
formation of plaques

• λ Replacement vector has a loading capacity of 10-
23 kb and consists of a full length lambda-molecule
with two identical restriction sites flanking a non-
essential region called stuffer fragment which is
deleted and replaced by foreign DNA.
• Again a selection system is required to differentiate
between wild type and recombinant phage.
• The selection of recombinants is done by placing
relevant genes onto the stuffer fragment, the loss of
which gives rise to a detectable phenotypic signal.
• Phage libraries consist of course of plaques; their
main advantage is that the clone density can reach
108-109 pfu per μg of ligated DNA.

EMBL-3 Vector
• EMBL-3 is a vector designed to replace Lambda
vectors derived from ʎ1059.
• It has a multiple cloning region containing sites for
Bam H1, Eco R1 and Sal 1 which flank a 14Kb stuffer
fragment.
• DNA fragments from 9 - 23 Kb in size may be
cloned.
• The vectors offer Spi (Sensitive to Ps Interference)
phenotype selection of recombinants.
• Recombinant λ DNA may be purified from phage
particles from plaques or from liquid culture.

Spi (Sensitive to Ps Interference) phenotype
• Wild-type lambda phage do not efficiently infect E
coli lysogenic for an unrelated bacteriophage P2.
• Thus, wild-type phage are Sensitive to Ps
Interference (spi+).
• They are are red+gam+

Spi (Sensitive to Ps Interference) phenotype
selection of recombinants.
• Phages that are red+gam+ are sensitive to the P2
lysogenic repressor molecule (spi+).
• Spi selection means ‘sensitive to P2 inhibition’.
• If the genes are removed (red-gam-) then it can
grow on a P2 lysogen. ( However the size of plaques
is small)
Phenotype
Growth on P2 lysogens?
(bacterial strain)
spi+ (red+gam+) poor
spi- (red-gam-) good

EMBL-4 Vector
• EMBL-4 is a vector designed to replace Lambda
vectors derived from λ1059.
• It has a multiple cloning region containing sites for
Bam H1, Eco R1 and Sal 1 which flank a 14Kb stuffer
fragment.
• EMBL-3 and EMBL-4 differ in the orientation of the
restriction sites within the multiple cloning regions
or poly linker sequence.
• DNA fragments from 9-23 Kb in size may be cloned.
The vectors offer Spi phenotype selection of
recombinants.

Genetic markers for the selection
and screening of λ vectors
(a)β-galactosidase: Some of the vectors contain a
segment of E. coli lacZ gene coding for α
peptide, capable of complementing the
defective lacZ gene (deletion in the region of α
peptide) in the host giving blue plaques.
Whenever there is an insertion of foreign DNA
these vectors give clear plaques, due to lack of
complementation.

Genetic markers for the selection
and screening of λ vectors
(b) Lysogeny: Some of the vectors contain a cI gene
with an EcoRI site.
• The hosts used carry a mutation hflA150 (high
frequency lysogenization).
• When wild type vector with intact cI infects
such, mutant hosts, the lytic growth is greatly
reduced.
• But when foreign DNA is inserted, cI gene is
disrupted (insertional inactivation) leading to
lytic growth and plaque formation.

Genetic markers for the selection and
screening of λ vectors
• (C) While using lambda vectors you want to
maximize the number of resulting phage particles
that contain foreign DNA or minimize the number of
wild type particles.
• One approach is through spi selection. This (spi)
refers to sensitivity to P2 interference.

Phenotype
Growth on P2 lysogens
(bacterial strain)
spi+ (red+gam+) poor
spi- (red-gam-) good
EMBL 3/4 vectors have placed the red and gam genes in the
stuffer fragment.
Thus only those particles from which the stuffer has been
replaced can grow well in a P2 lysogen bacterial cell but
plaque size is small.

(C) Sensitivity to P2 interference (Spi+/Spi-): In some
of the replacement vectors, genes red and gam
(genes involved in recombination) are present in
the stuffer region, rendering them Spi+ (sensitive
to P2 prophage interference, i.e. restricted growth
on P2 lysogens).
• When stuffer region is removed, red and gam are
lost rendering the recombinant vector red- gam-
and consequently Spi- (growing well in P2 lysogens
as long as they carry chi site and rec+).
• The vectors carrying red and gam in the stuffer
region include λ2001, λDASH and EMBL series.

(d) Chi (Crossover hot-spot instigator) site is a 8-mer
DNA sequence (5'-GCTGGTGG) present on the
Escherichia coli chromosome as well as lambda
genome.
• It increases plaque size of bacteriophage lambda.
• Chi site is responsible for both the attenuation of
RecBCD exonuclease activity and the promotion of
RecABCD-mediated homologous recombination.

(d) Chi site: Crossover hot-spot instigator (Chi) sequences
(5′-GCTGGTGG-3′) are orientation-dependent, strand-
specific sequences implicated in RecA-mediated DNA
recombination.
• Chi site is a suppressor of small plaque phenotype in
red- gam- .
• Lambda phage form normal plaques if they are red+
and gam+, but form small sized plaques when they
are red- and gam-.
• However if they contain chi site they form normal
sized plaques even if they are red- and gam-.

Genes or foreign sequences may be
incorporated essentially permanently into
the genome of E. coli by integration of a l
vector containing the sequence of interest.
λ Lysogens In Cloning
Techniques

Induction And Immunity Of Lysogens
l
A l lysogen
l
Spontaneously,
1/1000 lysogens will
induce, i.e. the l
prophage will
excise, replicate and
lyse the cell.
UV treatment leads
to induction of
virtually all lysogens
in a culture.
Lysogens are immune to
further infection with similar
(lambdoid) phage
+
l

• M13 is a filamentous, temperate
bacteriophage that infects only F+ E. coli cells
and turbid plaques are formed due to
decreased cell growth.
• The phage has 6.4 kb long circular ssDNA as
genome and an outer protein coat surrounds
it.
• There are 10 genes present in M13.
M13 Phage

Gene Function
1 NS membrane protein; required for assembly
2
Site- & strand-specific endonuclease/
topoisomerase; required for replication of RF
X (10)
N-terminal fragment of gene 2; required for
replication of RF
3
Minor Coat Protein that binds to F pilus of host
cell (receptor)
M13 phage

Gene Function
4 Membrane protein; required for assembly
5
Major structural protein during replication;
controls expression of g2p; binds to DNA and
converts RF replication to progeny (+)stand
synthesis; replaced by g8p during assembly
6
Present at same end of particle to g3p; involved
in attachment & morphogenesis
7/9
Present at opposite end of particle to g3p/g6p;
involved in assembly
8 Major coat protein
M13 phage

• The coat's dimensions are flexible & size range
varies from less than 100b to 13kb.
• The protein coat is made up of 2,800 copies of p8
(major coat) protein and 5 copies each of p7 and
p9 proteins on one side and p3 protein on the
other.
• All these 3 proteins (p7,p3 & p9) are called minor
coat proteins.
• The number of p8 copies is adjusted to
accommodate the size of the ss genome as it
packages.
M13 phage

• A deletion of a phage protein (p3) produces M13
phage that are 10-20 times the normal length
with several copies of the phage genome seen
shedding from the E. coli host.
• Protein pII nicks the double stranded form of the
genome to initiate replication of the + strand.
• The protein p2 is essential for M13 replication.
The phage genome can not replicate without p2.
M13 phage

M13 Life cycle
• M13 phage p3 tip uses F pilus to infect E. coli.
• Viral (+) strand DNA enters cytoplasm
• Complementary (-) strand is synthesized by bacterial
enzymes
• DNA Gyrase, a type II topoisomerase, acts on double-
stranded DNA and catalyzes formation of negative
super coils in double-stranded DNA
• Final product is parental replicative form (RF) DNA
• A phage protein, pII, nicks the (+) strand in the RF
• 3'-hydroxyl acts as a primer in the creation of new viral
strand
• pII circularizes displaced viral (+) strand DNA
• Pool of progeny double-stranded RF molecules
produced.

M13 Life cycle
• Negative strand of RF is template of transcription
• mRNAs are translated into the phage proteins
• Phage proteins in the cytoplasm are pII, pX, and pV,
and they are part of the replication process of DNA.
• The other phage proteins are synthesized and
inserted into the cytoplasm or outer membranes.
• RF DNA synthesis continues and amount of pV
reaches critical concentration.

M13 Life cycle
• pV dimers bind newly synthesized single-stranded
DNA and prevent its conversion to RF DNA
therefore DNA replication switches to synthesis of
single-stranded (+) viral DNA
• pV-DNA structures from about 800 nm long and
8 nm in diamter
• pV-DNA complex is substrate in phage assembly
reaction

M13 Phage Cloning Vectors
• First vectors used – M13mp18 & M13mp19 (Fig.
3.3)
• M13 phage with lacZ ' containing multiple
cloning site
• Same gene and cloning site as pUC18 & pUC19

M13 Phage Vectors
Advantages – blue/white screening system
– genes cloned in pUC18 or pUC19
– can be subcloned to same sites in M13mp
equivalent
– different directions for multiple cloning sites
–both strands of cloned DNA
– converted to single-stranded form
– in different vectors
Disadvantages – limits to size of cloned DNA (2 kb)
– low yield of DNA
– cannot amplify phage genome numbers much
– phage proteins toxic in high concentrations

M13 phage vectors - Applications
1) Replication form (RF, dsDNA) of M13 phage can
be purified and manipulated like a plasmid.
2) Phage particles (ssDNA): DNA can be isolated in a
single-stranded form
3) DNA sequencing
4) Site-directed mutagenesis
5) Cloning (RF, like plasmid)  transfection
(recombinant DNA)  growth (plating on a cell
lawn)  plaques formation (slow growth)
6) The insert size of foreign DNA is small (<1000b).

Phage display
1. Phage display is used for the high-throughput
screening of protein interactions. High-throughput
screening allows a researcher to quickly conduct
millions of chemical, genetic, or pharmacological
tests.
2. In the case of M13 filamentous phage display, the
DNA encoding the protein or peptide of interest is
ligated into the pIII or pVIII gene, encoding either
the minor or major coat protein, respectively.
3. Multiple cloning sites are sometimes used to
ensure that the fragments are inserted in all three
possible reading frames so that the cDNA fragment
is translated in the proper frame.

Phage display
4. The phage gene and insert DNA hybrid is then
inserted (a process known as "transduction") into
Escherichia coli (E. coli) bacterial cells such as TG1,
SS320, ER2738, or XL1-Blue E. coli.
5. If a "phagemid" vector is used (a simplified display
construct vector) phage particles will not be
released from the E. coli cells until they are infected
with helper phage, which enables packaging of the
phage DNA and assembly of the mature virions with
the relevant protein fragment as part of their outer
coat on either the minor (pIII) or major (pVIII) coat
protein.

Phage display
6. By immobilizing a relevant DNA or protein
target(s) to the surface of a microtiter plate
well, a phage that displays a protein that
binds to one of those targets on its surface
will remain while others are removed by
washing.
7. Those that remain can be eluted, used to
produce more phage (by bacterial infection
with helper phage) and so produce a phage
mixture that is enriched with relevant (i.e.
binding) phage.

Phage display
8. The repeated cycling of these steps is referred
to as 'panning', in reference to the
enrichment of a sample of gold by removing
undesirable materials.
Phage eluted in the final step can be used to
infect a suitable bacterial host, from which
the phagemids can be collected and the
relevant DNA sequence excised and
sequenced to identify the relevant,
interacting proteins or protein fragments.

Helper Phage
• Plasmids carrying the intergenic region of
filamentous phage (oriF1) can package as
ssDNA in viral particles in the presence of a
phage.
• When wild-type phages are used, interference
of the plasmid with the phage replication
leads to reduction in the phage copy number
and drastic decrease in virion production.
• Helper phages are designed to overcome
interference, maximize virion production and
keep packaging of their own ssDNA at a low
level.

Helper Phage
• Helper phages provide all the necessary gene
products for particle formation when using
phagemid vectors.
• Helper phages are mutated wild‐type phage
containing the whole genome, with a
defective origin of replication or packaging
signal, and hence, are inefficient in
self‐packaging. e.g. M13K07

Hybrid vectors
Cosmid vectors,
Phagemid,
Phasmid,
Bacterial Artificial Chromosome,
PAC,
Fosmid

Cosmid vectors
1. Utilizing the properties of the phage λ cos
sites in a plasmid vector.
2. A combination of the plasmid vector and the
COS site which allows the target DNA to be
inserted into the λ head.
3. The insert can be 37-52 kb.

Cosmids
1. Cosmid was first described by Collins and
Hohn in 1978.
2. Cosmid is a hybrid plasmid that contains cos
sequence of a Lambda phage.
3. Cosmids' (cos sites + plasmid = cosmid)
4. Cosmids are often used as a cloning vectors

• Features of both plasmid and phage cloning
vectors.
• Do not occur naturally.
• Circular in shape.
• Origin (ori) sequence for E. coli.
• Selectable marker, e.g. ampR.
• Restriction sites.
Cosmid Cloning Vectors

• Phage l cos site (1 or 2) permits packaging into
l phages and introduction to E. coli cells.
• Can accommodate 30-45 kbp.
• Unlike plasmids, Cosmids can also be packaged
in phage capsids, which allows the foreign
genes to be transferred into or between cells
by transduction.
• Cosmids always form colonies and not plaques
Cosmid Cloning Vectors

Cos Sequences
• Cos sequences are ~280 base pairs long and
essential for packaging.
• They contain a cosN site where DNA is nicked at
each strand, 12bp apart, by terminase which
linearizes the circular cosmid with two "cohesive" or
"sticky ends" of 12b.
• The DNA must be linear to fit into a phage head.
• The cosB site holds the terminase while it is nicking
and separating the strands.
• The cosQ site of next cosmid (as rolling circle
replication often results in linear concatemers) is
held by the terminase after the previous cosmid has
been packaged, to prevent degradation by cellular
DNases

Digestion
Ligation
C) Packaging and infect
Formation of a cosmid clone

Cloning in to Cosmid Vectors
• Cosmids are extracted from bacteria and
mixed with restriction endonucleases.
• Cleaved cosmids are mixed with foreign DNA
that has been cleaved with the same
endonuclease or an isocaudamer.
• Recombinant cosmids are packaged into
lambda caspids and is injected into the
bacterial cell where the r-cosmid arranges into
a circle and replicates as a plasmid.
• It can be maintained and recovered just as
plasmids.

Cloning in a cosmid
vector
cos
B
SmaI
B B
S S
cos cos
cos
B
B

Target sites of BamHI & Sau3A
BamHI Bacillus
amyloliquefaciens
5'G↓GATCC
3'CCTAG↑G
5'---G GATCC---3'
3'---CCTAG G---5'
Sau3A Staphylococcus
aureus
5'↓GATC
3‘ CTAG↑
5'--- GATC---3'
3'---CTAG ---5'
SmaI Serratia
marcescens
CCC↓GGG
GGG↑CCC

Phagemid
• Definition: A plasmid vector that
contains origin of replication from a
phage like M13, f1 or fd in addition to
that of a plasmid Ex. p Bluescript SK (+/-)
• Hybrid vector

Hybrid plasmid-M13 vectors
• Small plasmid vectors (pBluescript) being
developed to incorporate M13 functionality &
are known as Phagemids
• Contain both the plasmid and M13 origin of
replication
• Normally propagate as true plasmids
• Can be induced to form single-stranded phage
particles by infection of the host cell with a
helper phage

Helper Phage
• Plasmids carrying the intergenic region of
filamentous phage (oriF1) can package as ssDNA in
viral particles in the presence of a phage.
• When wild-type phages are used, interference of
the plasmid with the phage replication leads to
reduction in the phage copy number and drastic
decrease in virion production.
• Helper phages are designed to overcome
interference, maximize virion production and keep
packaging of their own ssDNA at a low level.

p Bluescript
• Size-2958 bp, a derivative of pUC19 & has
1) Phage f1(M13) origin of replication.
2) A portion of lacZ driven by lac promoter
3) MCS within lac Z
4) T7 &T3 promoter sequences flanking MCS
5) A colE1 origin of replication
6) Ampr gene

p Bluescript
1) Cloning vehicle
2) Expression vehicle
3) Riboprobe vector
4) Sequencing vector

Incorporation
of the VH/K
gene protein
into the
phage coat
proteins.
Gene VIII is a
phage coat
protein gene

Phasmid
• Phasmids are λ insertion vectors with a
shortened linear λ genome containing DNA
replication, lytic functions & cohesive ends of
λ Phage.
• The central non essential portion is replaced
by a linearized plasmid with an in tact origin
of replication.

Phasmid
• A Phasmid usually contains several copies of
the linear plasmid which make the size of the
vector at least 38 kb required for packaging in
to λ Phage heads.
• The plasmid has λ att B site using which the
plasmid is integrated in to λ genome (lifting of
the plasmid) by homologous recombination.
Ex. λZAP

Phasmid
• Phasmids are particularly useful in the
generation and analysis of mutations exhibiting
non-selectable or lethal phenotypes, such as
those affecting the replication of plasmids.
• Phasmids may also be used as phage
replacement vectors and for directing the high
level expression of protein from cloned
sequences by replication in the phage mode.

Bacterial Artificial Chromosome (BAC)
• BAC vectors have origin of replication (oriS)
from F plasmid, repE protein which initiates
replication, parA, parB and parC regions
required for partitioning of plasmid during cell
division and a marker gene.
• BACs can hold up to 300 kbp.

Bacterial Artificial Chromosome(BAC)
• The F factor of E. coli is capable of handling
large segments of DNA.
• Recombinant BACs are introduced into E. coli
by electroportation ( a brief high-voltage
current).
• Once in the cell, the rBAC replicates like an F
factor.
Example: pBAC108L, pBeloBAC11

Bacterial Artificial Chromosome (BAC)
pBeloBAC11

pBeloBAC11
•Has a set of regulatory genes, OriS, and repE
which control F-factor replication, and parA
and parB which limit the number of copies to
one or two, a chloramphenicol resistance
gene, and a cloning segment

pBeloBAC11
Advantages
• 300 kb of DNA can be accommodated
• Copy number 1-2 per cell
• No deletions or rearrangements in the inserted
fragments unlike in YAC.
• More user-friendly than YAC
Disadvantages
• Laborious construction
• Invitro manipulations to be performed in agarose
plugs to prevent DNA shearing due to large size.

Fosmids
• A BAC vector (F Ori, par etc.,) that contains λ
phage Cos sites to facilitate packaging in
heads of λ phage.
• Ex. pFos1
• Can accommodate up to 40kb DNA
• Useful for library construction similar to
cosmids.
• Low copy number hence more stable than
cosmids

• P1 is a temperate bacteriophage infects
Escherichia coli and a some other bacteria.
• Exhibits both Lytic & Lysogenic life cycles
• During lysogenic cycle the phage genome
exists as a plasmid in the bacterium unlike
other phages (e.g. the lambda phage) that
integrate into the host DNA.
• P1 has an icosahedral "head" containing the
DNA attached to a contractile tail with six tail
fibers.
P1-derived Artificial Chromosome

• The genome of the P1 phage is 93Kbp in
bacteria and is longer (110Kbp) than the
genome in the viral particle .
• It is created by cutting an appropriately sized
fragment from a concatemeric DNA chain
having multiple copies of the genome.
• Due to this the ends of the DNA molecule are
identical. This is referred to as being
"terminally redundant".
• This is important for the DNA to be
circularized in the host.

• The P1 plasmid has a separate origin of replication
(oriL) that is activated during the lytic cycle.
• Replication begins by a regular bidirectional theta
replication at oriL but later in the lytic phase, it
switches to a rolling circle method of replication
using the host recombination machinery.
• The end of the concatemer is cut a specific site
called the pac site or packaging site and is followed
by the packing of the DNA into the heads till they
are full.

• The rest of the concatemer that does not fit
into one head is separated and the machinery
begins packing this into a new head.
• The location of the cut is not sequence
specific. Each head holds around 110kbp of
DNA so there is a little more than one
complete copy of the genome (~90kbp) in
each head, with the ends of the strand in each
head being identical.

• After infecting a new cell this "terminal
redundancy" is used by the host
recombination machinery to cyclize the
genome if it lacks two copies of the lox locus.
• If two lox sites are present (one in each
terminally redundant end) the cyclization is
carried out by the cre recombinase.

• The P1 phage can be used to transduce the
phenotype of a target bacterium.
• As it replicates during its lytic cycle it captures
fragments of the host chromosome.
• If the resulting viral particles are used to infect
a different host the captured DNA fragments
can be integrated into the new host's
genome.
• This method of in vivo genetic engineering
was widely used for many years and is still
used today, though to a lesser extent.

• P1 can also be used to create the P1-derived
artificial chromosome cloning vector which
can carry relatively large fragments of DNA.
• Also, P1 encodes a site-specific recombinase,
Cre, that is widely used to carry out cell-
specific or time-specific DNA recombination
by flanking the target DNA with loxP sites.
• The phage P1 vector system has a capacity to
clone DNA as large as 100 kb, about twice
capacity of cosmid and less than that of yeast
artificial chromosome (YAC)

P1 clones features
• Clones are maintained in E. coli as low-copy-
number plasmids.
• A high copy number can be induced acting on
the P1 lytic replicon.
• P1 clones can be used to construct genomic
libraries ( mouse, human, fission yeast and
Drosophila .

P1-derived artificial chromosome - PAC
• The P1-derived artificial chromosome are DNA
constructs that are derived from the DNA of P1
bacteriophage.
• They can carry large amounts (about 100- 300
kilobases) of other sequences for a variety of
bioengineering purposes.
• It is one type of vector used to clone DNA
fragments (100- to 300-kb insert size; average,
150 kb) in Escherichia coli cells.
• Similar λ phage they also require invitro
packaging.

Eukaryote Vectors
• Yeast vectors (Yeasts)
• Agrobacterium tumefaciens (Ti plasmid)
(Plants)
• Baculovirus (Insects)
• Mammalian viral vectors (Mammalian)
• Transfection of eukaryotic cells
• Shuttle vectors

Yeast Episomal Plasmids (YEps)
1) Vectors for the cloning and expression of genes in
Saccharomyces cerevisiae.
2) Based on 2 micron (2m) plasmid which is 6 kb in
length. Has One origin, Two genes involved in
replication, A site-specific recombination protein
FLP.
3) Normally replicate in yeast similar to plasmids in E.
coli, and may integrate into the yeast genome.
4) High copy number (50-100/cell)
5) Replication in yeast by the replication elements of
the circular 2 µ plasmid
6) URA3 selection marker in yeast e.g. YEp24

Yeast Replicating Plasmid (YRp)
• E. coli elements, ARS = ‘Autonomous Replicating
Sequences’for replication in yeast. Do not
integrate in to yeast genome.
• Intermediary copy number
• More transformants
• URA3, TRP1 selection markers
• e.g. YRP17
• Disadvantages: lower stability ; tendency to
segregate with the parent cell – in spite of
selection pressure

Yeast Replicating Plasmid (YRp)

Yeast Integrating plasmids (YIp)
• A bacterial plasmid which can insert it self into
yeast chromosome.
• Genes integrated are more stable.
• Low transformation efficiency
• Copy number is 1

Yeast centromere plasmids plasmids
(Ycp)
• Have a region around Centromere
• Have chromosomal ori
• Stably maintained
• Single copy

pYAC or Yeast artificial
chromosome vector
• Hybrid vector has ARS, Centromeres and
telomere sequences
• Ex. pYAC3, which is essentially a pBR 322 into
which a number of yeast genes have been
inserted.
• URA3 and TRP 1 selectable markers for Yip5 and
YRp7 - respectively.
• Can accommodate up to 1400kbp of DNA.
• Centromere ensures proper segregation of
chromosomes

1. Saccharomyces cerevisiae selectable markers do
not confer resistance to toxic substances
2. Growth of yeast on selective media lacking
specific nutrients can serve for selection.
Auxotrophic yeast mutants are made as host
strains for plasmids containing the genes
complementary to the growth defect .
Selection in S. cerevisiae

For example: TRP1 mutants can’t make tryptophan,
and can only grow on media supplemented with
tryptophan.
The presence of a plasmid containing gene
encoding tryptophan enables the cell to grow on
media without tryptophan.
Selection in S. cerevisiae

Why Genetically Engineer Plants?
• To improve the agricultural or horticultural
value of plants
• To serve as living bioreactors for the
production of economically important
proteins or metabolites
• To provide a renewable source of energy
(biofuels)
• To provide a powerful means for studying the
biological action of genes and gene products

Plant transformation with the Ti plasmid
of Agrobacterium tumefaciens
• A. tumefaciens is a Gram-negative, non-spore
forming, motile, rod-shaped bacterium,
closely related to Rhizobium.
• It is found on and around root surfaces
(rhizosphere) –
• It survives by nutrients that leak from the root
tissues.
• It infects only through wound sites.

Crown galls caused by
A. tumefaciens

Plant transformation with the Ti plasmid
of Agrobacterium tumefaciens
• A. tumefaciens naturally transforms plant
cells, resulting in crown gall (cancer) tumors
• Tumor formation is the result of the transfer,
integration and expression of genes on a
specific segment of A. tumefaciens plasmid
DNA called the T-DNA (transferred DNA)
• The T-DNA resides on a large plasmid called
the Ti (tumor inducing) plasmid found in A.
tumefaciens

Crown Gall Tumors
• Tumor: Collection of cells growing in an
undifferentiated, uncontrolled manner.
• Crown gall tumors occur usually at wound sites
• Agrobacteria
A. tumefaciens- causes crown galls on
many dicots
A. rubi- causes small galls on a few dicots
A. rhizogenes- hairy root disease
A. radiobacter- avirulent

Crown Gall Tumors &
Agrobacterium tumefaciens
• Tumor formation is the result of the transfer,
integration and expression of genes on a
specific segment of A. tumefaciens plasmid
DNA called the T-DNA (transferred DNA)
• The T-DNA resides on a large plasmid called
the Ti (tumor inducing) plasmid found in A.
tumefaciens

Ti plasmid
• Tumor-causing ability (virulence) of
Agrobacterium correlates with the presence of
a large extrachromosomal element in the
bacterium - the Ti plasmid
• Virulent Agrobacterium tumefaciens have this
plasmid
• Crown Gall Tumorigenesis is due to the
"activation" of unregulated phyto hormone
synthesis in the transformed cells

Tumor characteristics
• Synthesize a unique amino acid, called
“opine”
• Octopine and Nopaline - derived from arginine
• Agropine - derived from glutamate
• Opine depends on the strain of A. tumefaciens
• Opines are catabolized by the bacteria, which
can use only the specific opine that it causes
the plant to produce.
• Has obvious advantages for the bacteria, what
about the plant?

Ti Plasmid
1. Large (~200-kb)
2. Conjugative
3. ~10% of plasmid transferred to plant cell after
infection
4. Transferred DNA (called T-DNA) integrates
semi-randomly into nuclear DNA
5. Ti plasmid also encodes:
– enzymes involved in opine metabolism
– proteins involved in mobilizing T-DNA (Vir
genes)

auxA auxB cyt ocs
LB RB
LB, RB – left and right borders (direct repeat)
auxA + auxB – enzymes that produce auxin
cyt – enzyme that produces cytokinin
Ocs – octopine synthase, produces octopine
T-DNA
These genes have typical eukaryotic expression signals!

Ti plasmid
structure
and
function.

DNA Transport Between Kingdoms
1. (Virulent) strains of A.
tumefaciens contain a
200-kb tumor inducing
(Ti) plasmid
2. Bacteria transfer a
portion of the plasmid
DNA into the plant
host (T-DNA).

The wound-induced plant phenolics induce the vir
genes on the Ti plasmid.

The infection process
1.Wounded plant cell releases phenolics and
nutrients.
2.Phenolics and nutrients cause chemotaxic
response of A. tumefaciens
3.Attachment of the bacteria to the plant cell.
4.Certain phenolics (e.g., Acetosyringone,
Hydroxyacetosyringone) induce vir gene
transcription and allow for T-DNA transfer and
integration into plant chromosomal DNA.

The infection process
5. Transcription and translation of the T-DNA in
the plant cell to produce opines (food) and
tumors (housing) for the bacteria.
6. The opine permease/catabolism genes on the
Ti plasmid allow A. tumefaciens to use opines
as C, H, O, and N sources..

Agrobacterium tumefaciens
Ti plasmid
Ti plasmid
200kb
T-DNA
plant chromosome
Integrated T-DNA
Gene induce crown gall

Ti-plasmid based vectors
Binary systems Co-integrated vectors
Needs 2 vectors:
Needs 3 vectors
Disarmed Ti plasmid
with gene of interest
(no vir genes)
Helper vector
for infection
(with vir genes)
Disarmed Ti plasmid
used for infection
Intermediate vector with T-
region and gene of interest
(transferred by conjugation)
Form
co-integrated
plasmid
after
homologous
recombination
on T-DNA Helper vector for transfer of
intermediate plasmid into A. tumifaciens

Co-integrated vectors
(hybrid Ti-plasmids)
Right now rarely used

Co-integrated Vectors (Hybrid Ti-plasmids)
DISADVANTAGES:
1) Long homologies required between the Ti
plasmid and the E. coli plasmids (pBR322
based Intermediate vectors) making them
difficult to engineer and use.
2) Relatively inefficient gene transfer compared
to the binary vector

Binary Vectors Strategy
1. Move T-DNA onto a separate, small plasmid.
2. Remove aux and cyt genes.
3. Insert selectable marker (kanamycin
resistance) gene in T-DNA.
4. Vir genes are retained on a separate plasmid.
5. Put foreign gene between T-DNA borders.
6. Co-transform Agrobacterium with both
plasmids.
7. Infect plant with the transformed bacteria.

Ti plasmid vector systems
are often working as binary vectors
Virulence
region
T DNA region removed
ori for A. tum
Gene of interest
Plant selectable marker
Bacterial selectable
marker
ori for A. tumefaciens
ori for E.coli
HELPER
plasmid
Disarmed Ti
plasmid

Ti plasmid vector systems
are often working as binary vectors

Ti Plasmid Binary Vectors
DISADVANTAGE: Depending on the orientation,
plasmids with two different origins of replication
may be unstable in E. coli
ADVANTAGE: small vectors are used, which
increases transfer efficiency from E. coli to
Agrobacterium.
No intermolecular recombination is needed

Plant
Genetic
Engineering
Using
T-DNA
Vector

Why Virus-mediated Gene Transfer?
• Viral genome do not integrate into plant
genome.
• Viral vectors have high copy number per cell
and they are not subjected to the “position
effect”.
• The gene product is very rapidly accumulated.
• Viral genome sequences are excellent source
of promoters, enhancers and other
components useful for designing gene vectors.
• Virus exhibits systemic infection in plants.
• Generally have wide host range.

Most Notable Plant Virus Vectors
1.DNA Virus-
• CaMV based vectors.
• Gemini virus based vectors.
2. RNA Virus
• TMV based vectors.
• Brome Mosaic Virus (BMV)

CaMV
1) Genome is 8kb long circular dsDNA with 3
discontinuities (2 in 1 and 1 in other).
2) 5’ terminus of discontinuities is covalently
bound to oligoribonucleotides.
3) Genome packaged as nucleosome.
4) It produces spherical particles.
5) Replication involves reverse transcription similar
to Retroviruses.
6) Has 8 tightly packed genes- only genes II and VII
are non-essential hence very little DNA can be
replaced.

CaMV
Gene I: plasmadesmata
movement
Gene IV: translation
transactivation
Gene V: reverse
transcriptase
Gene III/IV: assembly
Gene II/VI: inclusion
bodies

CaMV
6. CaMV genomes exceeding natural size (8,024bp)
even by a few 100bp are not packaged in to
virions - A major limitation.
7. Use of a helper virus to accommodate long
stretches of DNA are usually unstable because of
recombination between helper and vector
viruses.
8. The bacterial gene dihydrofolate reductase
(dhfr) was expressed in plants replacing gene II
of CamV.
9. CaMV though has strong promoters is of limited
use as a vector.

CaMV
• Mechanical and aphid mediated transmission
• Virion DNA alone or cloned CaMV DNA is
infectious when simply rubbed on leaves
• Up to 106 copies per cell.
• 3-4 weeks for systemic infection through
plant.

transcription
nucleus
35S RNA
19S RNA
translation
Reverse
transcription
uncoating
Gene IV
Gene V
Gene III/IV
assembly
Inclusion body
(gene VI)
Gene I
CaMV
activity in
plant cell

Challenges with CaMV vector
• Small insertions (10-30 bp) in various sites
abolished infectivity.
• Only gene II could tolerate insertion of
significant size and could be entirely removed
• But the largest insert tolerated so far is 256-
531 bp.
• Complicated polycistronic design (ATG of
cloned DNA must not interfere with the
termination of gene I).
• CaMV genome was inserted into Ti vector to
integrate into genome (Agroinfection).
• Due to these limitations, CaMV vectors have
not be widely used.

Gemini viruses
• Gemini Viruses are plant viruses with ss
circular DNA genomes.
• Genes diverge (coded) in both directions
from a virion strand origin of replication
(i.e. geminivirus genomes are ambisense).
• A single-stranded genome that contains
both positive-sense and negative-sense is
said to be ambisense.

Gemini viruses
• Geminiviruses are dependent on host cell
factors for replication- DNA polymerases,
repair polymerases, transcription factors.
• The genomic ssDNA is replicated in the
nucleus of the host cell by a rolling-circle
mechanism utilizing double-stranded DNA
(dsDNA) intermediates similar to the
ssDNA-containing bacteriophages.
• Infect both Monocots and Dicots.

Gemini viruses: smallest viral genome ~2.7 kb
WDV, TGMV and MSV: ssDNA genome replicates as dsDNA
intermediate

Gemini viruses
• dsDNA form of virus genome is infectious
and coat protein gene is not required for
systemic infection.
• Most have insect mediated transmission.
• No mechanical transmission which may be
overcome by cloning viral genome into Ti
plasmid and carrying out “Agroinfection”
• WDV genome allows insertion of up to 3
kb foreign sequence

Gemini viruses
• Maize Streak Virus (MSV) belongs to
Gemini virus & has a circular, ~2.7-Kb
monopartite single-stranded (ss) DNA
genome.
• It encodes only four proteins.
• MSV produces infection only when it is
transmitted by leaf hopper Cicadulina
mbila, but other leafhopper species, such
as C. storeyi, C. arachidisand C. dabrowski,
are also able to transmit the virus.

Gemini viruses
• Agroinfection of MSV as well as WDV was
successful.
• Note: In case of many viruses and viroids
when T-DNA contains one complete and
one incomplete copy of viral genome
arranged in tandem, single copies of viral
genome escape & initiate infection.

RNA Viruses
• Genome of majority of plant viruses is RNA(+).
• Vectors based on RNA viruses include
1. Tobacco Mosaic Virus and
2. Brome Mosaic Virus.

TMV
1) ssRNA virus of 6395 bases with 4 ORFs
2) 126 / 183 kDa: replicase complex translated
from a single frame (183 kDa is generated by
read through of a leaky amber termination
3) codon of 130 kDa gene
4) 30 kDa: movement protein (M P)
5) 17.5 kDa: capsid protein

TMV based vectors
126 / 183 kDa 30 kDa CP 3’
Insert (size limit)
TB2
Foreign gene is inserted into 3’ end of MP in such a way
that native CP promoter drives the expression of foreign
gene, and a related virus (ORSV: Odontoglossom ring
spot virus) CP promoter drives the expression of native
CP ORF.

Procedure
1. Use of cDNA copy of viral genome for cloning
in E. coli & for manipulation of transgene in
to viral genome.
2. Invitro transcription of the recombinant viral
genome cDNA to produce infectious RNA
copies to be used for plant infection.
Ex. Gene ‘cat’ (chloramphenicol acetyl
transferase) was expressed in tobacco leaves
but there is no systemic infection.

Brome Mosaic Virus (BMV)
• Infects several species of Graminae including
Barley.
• Has 3 genomic segments- 1,2,3, each
packaged in to separate particles.
• Coat Protein gene is located on RNA segment3
and is the only target site for DNA insertion.
However this prevents formation of virus
particles.

Brome Mosaic Virus (BMV)
• A high Cat activity was noticed when Cat gene
was inserted in to the CP gene of RNA 3 (using
its cDNA) and its RNA transcripts were used
along with RNA1 and RNA2 to infect barley
protoplasts.
• A transgene is placed in the downstream to
the regulatory sequences of cp gene of BMV
result in high yields of protein.

Animal Vectors
• Most animal vectors replicate and express in
animal cells.
• However passive transducing SV40 vectors
cannot replicate.
• Retroviruses and transposon based vectors
integrate in to host genome.

Animal Vectors
1. Insect Vectors -
• P elements
• Bacculovirus
2. Mammalian Vectors –
• SV 40 virus.
• Bovine Papilloma Virus Vector
• Retrovirus
• Adenovirus
• Adenovirus-Associated Viral Vector

P elements
• Cloning in Drosophila makes use of a
transposon called the P elements, since no
plasmids are known in Drosophila.
• P elements are 2.9kb in length, contain 3
genes flanked by short inverted report
sequences.
• Transposase, carries out transposition by
recognizing the inverted terminal repeats of
the inserted transposon.

P elements
• P elements jump within or between
chromosomes.
• P elements also move between a plasmid
carrying a “p” element and one of the fly’s
chromosomes which contains the insertion
site for the DNA that will be cloned.
• Insertion of the new DNA into this P
elements results in disruption of its
transposase gene, so this element is inactive.

P elements
• P element of plasmid contains an intact
transposase gene lacking the terminal
inverted repeats.
• Once the gene to be cloned has been
inserted into a vector, the plasmid DNA is
microinjected into fruit fly embryos.

P elements
• The transposase from the P element lacking
the terminal inverted repeats directs transfer
of the engineered P element into one of the
fruit fly chromosomes.
• If this happens within a germline nucleus
then the adult fly that develops from the
embryos will carry copies of the cloned gene
in all its cells.

Baculovirus
• Baculovirus – used for transfecting insects.
Not infectious for vertebrates & plants.
• Baculovirus - two groups
• Nucleopolyhedroviruses (NPV)
• Granuloviruses
• 2 NPV viruses – Ac NPV (Autographa
californica NPV) infecting Spodoptera
frugiperda and Bm NPV (Bombyx mori NPV)
infecting cells & larvae of silk worm (Bombyx
mori) were exploited for transfecting insects.

Baculovirus
• Rod shaped Virus
• Genome - covalently closed circular ds DNA
(134 kbp).
• Can accommodate large foreign DNA
fragments
• Insect expression system is an important
eukaryote expression system.

Baculovirus Vectors
• Baculovirus produces nuclear inclusion bodies
which consist of virus particles embedded in a
protein matrix.
• This protein matrix is polyhedrin and accounts
for 70% of total coded protein. (Extremely
active promoter).
• The strong promoter expressing polyhedrin
protein (not essential for replication) can be
used to over-express foreign genes engineered
& large quantities of proteins can be produced
in infected insect cells.

Baculovirus Vectors
• Genetic manipulation of the viral DNA is not
possible as it has a very large DNA with many
restriction sites for a single enzyme.
• Hence, the gene of interest is cloned into the
small recombination transfer vector and co
transfected into insect cell lines along with the
wild type of virus in the cell.
• Homologous recombination takes place
between the polyhedrin gene and our gene of
interest.

Baculovirus Vectors
• Thus, our gene of interest will be transferred
from the vector plasmid into the wild type of
virus, polyhedrin gene will be transferred from
the virus on the plasmid.
• This is something like displacement reaction.
This displacement of gene will not effect the
replication of virus, as polyhedrin gene is not
required for replication.

Baculovirus Vectors
• The recombination virus replicates in the cells
and generates characteristic plaques (without
inclusion bodies).
• Normally the virus is cultured in the insect cell
line of Spodoptera frugiperda.
• The foreign gene is expressed during the
infection and very high yields of protein can be
achieved by the time the cell lyses.

Mammalian Viral Vectors
• SV 40 virus.
• Bovine Papilloma Virus Vector
• Retrovirus
• Adenovirus
• Adenovirus Associated Virus Vectors

SV40 Virus
• SV 40 is a spherical virus.
• Genome - 5.2 kbp long circular DNA.
• Codes for 5 proteins - small T & large T (early
transcription), VP1, VP2 & VP3 (late
transcription).
• SV 40 virus is grown in monkey kidney cell
lines.

Life Cycle
• The virus travels to the nucleus and gets uncoated.
• Then both the T -genes located near the origin are
translated in the clockwise direction.
• The large T protein is important for virus DNA replication
and starts after the translation of large T -protein.
• Replication starts at the origin and is bi-directional. It
terminates when two replication forks meet.
• About 105 molecules of duplex DNA are synthesized per
cell. Along with DNA replication, VP1, VP2 and VP3
proteins are synthesized.
• Then packing of DNA occurs to form new virions, which
are released by the lysis of cell.
• The entire process can also be initiated by transfection
with naked SV 40 DNA.

SV 40 Vectors
• SV 40 vectors are constructed similar to phage
vectors.
• Viral genome Portions are replaced by foreign
DNA segments.
• There are three types of SV 40 vehicles each of
which have a distinct advantage or disadvantage
among themselves.
1) Animal Vectors - SV40 Transduction Vectors
2) Animal Vectors - SV40 Plasmid Vectors
3) Animal Vectors - SV40 Passive Transforming
Vectors

SV40 Transduction Vectors
• Replicate & package into virion particles.
• Contain a 300 bp segment - ori & provides the
transcriptional regulatory signals for mRNA
synthesis.
• The non essential genes for replication - VP1,
VP2 & VP3 were deleted to accommodate
foreign DNA.
• A helper virus or genes cloned in to host
genome provide the above deleted products in
trans.

SV40 Transduction Vectors
• Can accommodate inserts of 3.9 to 4.5 kb.
• Normally the recombinant SV 40 vectors are
transformed into the COS cell line, a kidney cell
line of the African green monkey kidney into
which T -protein gene was incorporated.
• So when the vector is transfected into these
cells, virion particles are yielded with the help of
helper virus.
• They multiply both in E. coli and monkey cell
lines (shuttle vectors) but are not packed as
virions.

SV40 Plasmid Vectors
• Normally the recombinant is multiplied in E. coli
cells to high copy number and then transferred into
the cell line.
• These cells are stable in bacterial cell and are
efficiently transferred from parent cells to daughter
cells.
• However, the plasmid vectors are unstable in most
animal cells and cannot be maintained indefinite.

SV40 Passive Transforming Vectors
• These vectors neither replicate nor produce
virions, but simply integrate the DNA
segments into the cellular DNA.
• These transformed cells replicate the new
DNA as an integral part of their own genomes.
• These plasmids are also shuttle vectors and
include selective markers like herpesvirus,
thymidine kinase or neo genes.
• Apart from the selective markers, they include
transcriptional regulator signals and
polyadenylation sites

Cloning strategy - SV40 viral vector

Bovine Papilloma Virus Vector
• Bovine papilloma virus (BPV) causes warts
(uncontrolled epithelial proliferation) and
papillomas in a range of mammals including
cattle.
• BPV normally infects terminally differentiated
squamous epithelial cells.
• They do not integrate into host genome and
are maintained as episomes in the host
nucleus.

• BPV has circular ds DNA (79 kbp) surrounded
by a capsid protein.
• 69% of this genome is important for viral
function, whereas 31 % (~24kbp) of the
genome can be replaced by the insert.
• The recombinant BPV is constructed by
ligating the insert and BPV vector (69%) onto
the pBR 322 plasmid, thus generating the
shuttle vector containing plasmid “ori” site
and virus replication sequences.

• These shuttle vectors are multiplied in E. coli
cells first and then they are transformed into
mouse cell line.
• It has been observed that if these plasmid
sequences are removed prior to transfection,
the vector exists at high copy number i.e., 200
copies per cell.
• When transfected with pBR 322 sequences, it
exists at low copy number, i.e., less than 10
copies cell.

• The major advantage of BPV is the generation of
permanent cell line.
• As the infected cells are not killed, a stable
plasmid number is found even when the insert is
of large size.
• The selection of transformants is very easy as
they form a pile of cells on the transferred
monolayer of cells called "Focus".
• The transformed cells are then selected by the
presence of marker gene which is mostly the
neomycin phospho transferase gene coding for
resistance against G418 (aminoglycoside).

Retrovirus Vectors
• Probably the most studied group of viruses in
molecular biology
• Unique morphology and replication
• Enveloped with two copies of positive single-
stranded RNA.
• Encodes only 3 genes

Retrovirus Vectors
• Gag proteins - Group Specific Antigens- form
nucleocapsid
• Pol (Reverse Transcriptase, Integrase, and
RNase H)--bound to diploid RNA
• Env glycoprotein coded by env gene, which
along with lipids obtained from the host
plasma membrane during budding process
form the outer envelope
• Host is range determined by envelope proteins

Retrovirus Vectors
• Genomic RNA is converted to ds DNA by
Reverse Transcriptase
• Duplication of terminal segments creates LTR's
(U3-R-U5; LTR's = Long Terminal Repeats)
• This double-stranded DNA form is first circular
and is integrated into genomic DNA by
integrase, coded by the Pol gene.
• This form of the viral genome is called the
‘provirus’.

Integration
Of Retrovirus
In To Host
Cell Genome

MMLV (Moloney Murine Leukemia Virus)
1. Ability to integrate into the host genome in
stable fashion (provirus)
2. It has been used in a number of FDA-approved
clinical trials (e.g. SCID-X1, insertion near
LMO2).
3. Target cells should be actively dividing for
transduction (e.g. neuron?).
4. Concern for insertional mutagenesis (insertion
at random position).

Lentiviral Vector
• A subclass of retroviruses.
• Ability to integrate into the genome of non-
dividing as well as dividing cells.
• Concern for insertional mutagenesis (insertion
at random position).

Adenovirus properties
• Nonenveloped icosahedra 65-80nm
• Linear dsDNA 30-38 kbp contains 5’TP
• Encode 25-30 proteins, 15 are structural
• Both strands transcribed in nucleus
• Ordered, timed expression of viral genes
• Virus assembly in nucleus
• Cause respiratory, eye, and intestinal infections
(Ex. Conjunctivitis)
• Some induce tumors in rodents but not in
humans.

Adenovirus - Structure
1 = penton capsomeres, 2 = hexon capsomeres,
3= viral genome (linear dsDNA)

Adenovirus
type 2
Central Section
Adenoviruses

Adenovirus structure and genome organization

Adenoviral Vector
• Adenoviral DNA do not integrate into the
genome remain as episome and is not replicated
during cell division.
• Used for gene therapy and vaccine.
• Naturally, adenoviruses cause respiratory,
gastrointestinal and eye infections in human.
• Pre-existing immunity in human (May be the
reason for failure in gene delivery or fatal side
effect).
• No possibility of insertional mutagenesis.

ITR – Inverted Terminal Repeat
(origin of viral replication)
E – Early Response Genes
• Initiation and activation of viral replication
• Suppression of host cell gene expression and
protein synthesis
• Activation of late response genes (L)
L – Late response (viral structural components)
The Adenoviral Genome

Recombinant Adenovirus (DE1/E3)
• To ensure replication deficiency of the virus, the E1
region is deleted allowing it to safely be used as a
gene delivery tool.
• To accommodate larger recombinant genes (up to 8
Kb), 1st generation adenoviruses are both E1 and
E3 deleted (E1/E3), since the E3 region is not
essential for in vitro viral growth.
• The adenovirus vector is able to deliver genes with
100% efficiency to a wide selection of cell types
including dividing or non-dividing cells, or primary
cells or cell lines.
• This ability far surpasses the gene delivery
efficiencies of lipid-based transfection approaches
or other viral-based gene delivery systems.

Advantages Of Using Recombinant
Adenovirus To Introduce Genetic
Material Into Host Cells
• Recombinant adenovirus represents a
homologous system for human genes.
• Adenoviral vectors use a human virus as vector
and human cells as host.
• Therefore, human proteins have identical post-
translational modifications as native proteins.

Advantages Of Using Recombinant
Adenovirus To Introduce Genetic
Material Into Host Cells
• Adenoviral vectors have the ability to infect most
mammalian cell types (both replicative and non-
replicative)
• Accommodates reasonably large transgenes (up
to 8 kb)
• Allow high expression of the recombinant
protein.

Advantages using Recombinant
Adenovirus to introduce genetic
material into host cells
• May be grown at high titer (1010 VP/mL, which
can be concentrated up to 1013 VP/mL)
• Are well tolerated, with post-infection viability of
the host cells being almost 100%
• Remains epichromosomal, i.e. do not integrate
into the host chromosome so do not inactivate
genes or activate oncogenes.
• All these have made recombinant adenovirus the
vector of choice for functional genomics research,
protein-over-expression and pre-clinical studies

Risks Associated with Adenoviruses
• Adenovirus is transmitted by inhalation, contact
with mucus membranes (eyes, nose and mouth),
fecal-oral transmission and waterborne
transmission.
• Adenovirus infections most commonly cause
illness of the respiratory system with symptoms
ranging from the common cold to pneumonia,
croup, and bronchitis.
• Depending on the infecting serotype, adenovirus
infection may also cause other illnesses such as
gastroenteritis, conjunctivitis and rash.

Adenovirus-Associated Viral (AAV)
Vector
• Adeno-associated virus (AAV) is not related to
adenovirus, but first discovered as a contaminant in
an adenoviral isolate.
• AAV is a ss DNA virus, a member of the parvovirus
family.
• It is naturally replication defective, requires another
virus (usually adenovirus or herpesvirus) to complete
its infection cycle.
• AAV genome is small (about 5 kb).
• Has rep (replicase) and cap (capsid) genes in central
region flanked by 145-b inverted terminal repeats

Adenovirus-Associated Viral (AAV)
Vector
• AAV is not known to cause disease.
• AAV causes a very mild immune response.
• AAV can infect both dividing & non-dividing
cells.
• AAV incorporate its genome into the host
genome.
• Predictable insertion: insertion at a specific
site (AAVS1) in the human 19th chromosome.
• Disadvantage of AAV vectors -the limited
capacity for foreign DNA

Shuttle vectors
Vectors contain sequences required for
replication and selection in both E. coli and the
desired host cells, so that the construction and
many other manipulation of the recombinant
plasmids can be completed in E. coli.
Most of the eukaryotic vectors are constructed
as shuttle vectors

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Gene Transfer
Genes may be transiently or permanently
introduced into cultured eukaryotic cells
without the use of vector in strict sense.
• Transient expression
• Integration

1. Transfection: The take-up of DNA into
eukaryotic cells
2. More problematic than bacterial transformation
3. Much lower efficiency in the progress
4. Transfection methods
5. Electroporation
6. Microinjection
7. Liposome

Insects And Insect Cell Lines
• Baculovirus infects lepidopteran (butterflies
& moths) insects and insect cell lines
• Commonly used cell lines are sf9 & sf21
derived from the pupal ovarian tissue of the
fall army worm Spodoptera frugiperda and
high five derived from the ovarian cells of
the cabbage looper.

Baculovirus Expression
System
• Heterologous genes placed under the
transcriptional control of the strong
polyhedrin promoter of the Autographa
californica polyhedrosis virus (AcNPV)
• Based on site specific transposition of an
expression cassette (pfast Bac with gene of
interest) into a baculovirus shuttle vector
(bacmid)

Recombinant Baculovirus
Production
• Clone the gene of interest in pfast Bac donor plasmid
• Expression cassette in pfast Bac is flanked by left and
right arms of Tn7 and also an SV40 polyadenylation
signal to form a miniTn7
• Cloned pfast Bac is transformed in E.coli host strain
(DH10Bac) which contains a baculovirus shuttle
vector bacmid having a mini-attTn7 target site
• Helper plasmid which allows to transpose the gene
of interest from pfast to bacmid (shuttle vector)
• Transposition occurs between the mini-att Tn7 target
site to generate a recombinant bacmid
• This recombinant bacmid can now be used to
transfect insect cell lines.

Recombinant Baculovirus Production

Recombinant Baculovirus Selection
• PCR amplification using M-13 Forward and Reverse primers
• If no transposition, then a region a bacmid alone will amplify to
gave product of 300bp
• In condition of transposition then the amplified size will be
2300bp+size of insert
• Recombinant bacmid is now ready to transfect to insect cell lines

Vectors.pptx

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