Vectors are essential tools for genetic engineering that allow recombinant DNA to be introduced into and replicated in host organisms. Plasmids are the most commonly used bacterial cloning vectors. Plasmids are small, circular DNA molecules that can replicate independently of the bacterial chromosome. Important plasmid vectors include pBR322, which contains two antibiotic resistance genes for selection, and pUC19, which allows blue-white screening to identify recombinant clones through disruption of the lacZ gene. Plasmid vectors are useful for amplifying DNA inserts, producing recombinant proteins, and transferring genes for applications such as gene therapy.

1. GENETIC ENGINEERING &
APPLICATIONS -18BT56
Topic:-Vectors in Genetic Engineering
Prepared & presented by:
Ms.Salma kausar M
Assistant professor
Dept. of BT
TOCE,bangalore

2. Introduction
• Most naturally occurring vectors do not have all the
required functions for easy propagation inside host species
or accurate expression of the recombinant DNA insert.
• So vectors have been created by joining together
segments performing specific functions (called modules)
from 2 or more natural systems.
• There are several types of vectors of which some are
natural and some constructed. They can be grouped into
different classes like plasmids, bacteriophages, cosmids
and artificial chromosomes.

3. Types of Cloning Vectors
Vector Insert size Source Application
Plasmid ≤ 15 kb Bacteria Subcloning and downstream
manipulation, cDNA cloning and
expression
assays
Phage 5-20 kb Bacteriophage λ Genomic DNA cloning, cDNA
cloning and
expression library
Cosmid 35-45 kb Plasmid containing
bacteriophage λ cos
site
Genomic library
construction
BAC (bacterial
artificial
chromosome)
75-300 kb Plasmid ocntaining ori
from E.coli F- plasmid
Analysis of large genomes
YAC (yeast
artificial
chromosome)
100-1000 kb
(1 Mb)
Saccharomyces
cerevisiae centromere,
telomere and
autonomously
replicating
sequence
Analysis of large genome, YAC
transgenic mice
MAC
(mammalian
artificial
chromosome)
100 kb to > 1 Mb Mammalian
centromere, telomere
and origin of replication
Under development for use in
animal biotechnology and human
gene therapy

4. Plasmids
Definition:-
Plasmids are double-stranded and generally circular DNA sequences
that are capable of automatically replicating in a host cell. It is
physically separated from the chromosomal DNA and can replicate
independently.
Features ;
• Plasmids are found widely in many bacteria, for example in
Escherichia coli, but may also be found in a few eukaryotes, for
example in yeast such as Saccharomyces cerevisiae.
• In nature, plasmids often carry genes that may benefit the
survival of the organism, for example antibiotic resistance.
• plasmids usually are very small and contain only additional genes
that may be useful to the organism under certain situations or
particular conditions.
• Plasmids can be transmitted from one bacterium to another (even
of another species) via three main
mechanisms: transformation, transduction, and conjugation.

5. Classification of plasmids
There are five main classes of plasmids according to function:
• Fertility F-plasmids, which contain tra genes. They are capable
of conjugation and result in the expression of sex pili. Eg. F plasmid
of E. coli.
• Resistance R- plasmids, which contain genes that provide resistance
against antibiotics or poisons. Eg. RP4 of Pseudomonas sp.
• Col plasmids- which contain genes that code
for bacteriocins, proteins that can kill other bacteria. Eg. Col E1.
• Degradative plasmids - which enable the digestion of unusual
substances like toluene and salicylicacid. Eg. TOL plasmid of
Pseudomonas putida.
• Virulence plasmids - which turn the bacterium into a pathogen. Eg.
Ti and Ri plasmids.

6. Plasmids as vectors
• Plasmids are the most-commonly used bacterial cloning
vectors.
• Many different E. coli plasmids are used as vectors.
• The natural plasmids have been modified, shortened,
reconstructed and recombined both invitro and invivo to
create plasmids of enhanced utility and specific functions.
• These plasmids serve as important tools in genetics and
biotechnology labs, where they are commonly used to clone
and amplify the gene or to express particular genes.
• The bacteria containing the plasmids can generate millions
of copies of the vector within the bacteria in hours, and the
amplified vectors can be extracted from the bacteria for
further manipulation.

7. Cloning using plasmids
• Plasmid cloning vectors contain a site that allows DNA
fragments to be inserted, for example a multiple cloning
site or polylinker which has several commonly
used restriction sites to which DNA fragments may
be ligated.
• Both the plasmid and the DNA insert are digested with the
same restriction enzyme to create cohesive ends.
• Ligation is carried out using DNA ligase enzyme.
• After the gene of interest is inserted, the plasmids are
introduced into bacteria by transformation.
• A plasmid cloning vector is typically used to clone DNA
fragments of up to 15 kbp.

8. Selection of recombinant plasmids
It is very important to select for the low frequency of cells transformed by the
recombinant DNA from among the cells containing the unaltered vector
and the nontransformed cells.
• These plasmids contain a selectable marker, usually 2 antibiotic
resistance genes, such as ampicillin resistance (ampR) and tetracycline
resistance (tetR) in the vector.
• These will confer on the bacteria the ability to survive and proliferate in a
selective growth medium containing the particular antibiotics.
• The DNA insert is integrated within one of the 2 selectable markers.
• The cells after transformation are exposed to the selective media, and
only cells containing the recombinant plasmid may survive.
• In this way, the antibiotics act as a filter to select only the bacteria
containing the plasmid DNA.
• The vector may also contain other marker genes or reporter genes to
facilitate selection of plasmid with cloned insert.

9. Applications of plasmid vectors
• Amplification of DNA insert – gene of interest can be
amplified to a great extent using transcription
vectors, which can be used for transfer of the gene
to either bacterial, animal or plant cells.
• Protein production - major use of plasmids is to
make large amounts of recombinant proteins. Just
as the bacterium produces proteins to confer its
antibiotic resistance, it can also be induced to
produce large amounts of proteins from the inserted
gene. This is a cheap and easy way of mass-
producing the protein the gene codes for,
ex/ insulin.

10. Applications of plasmid vectors Contd…
• Gene therapy - Plasmid may also be used for gene
transfer into human cells as potential treatment
in gene therapy so that it may express the protein
that is lacking in the cells. Some strategies of gene
therapy require the insertion of therapeutic genes at
pre-selected chromosomal target sites within the
human genome. Plasmid vectors are one of many
approaches that could be used for this purpose.
• Disease models - Plasmids were historically used to
genetically engineer the embryonic stem cells of
rats in order to create rat genetic disease models.

11. Plasmid vectors in molecular biology
An ideal plasmid vector must have the following
functions
• Minimum amount of DNA (<10kb to avoid problems
during purification)
• Relaxed replication control
• Selectable marker gene for easy selection of
recombinant vectors
• Unique restriction site for at least one restriction
enzyme.
• Location of restriction site within the marker gene for
easy selection of recombinant vector

12. pBR322
•One of the most popular and
widely used vector is pBR322.
•It was created in 1977 was
named after the Mexican
postdoctoral researchers who
constructed it. The p stands for
"plasmid", and BR for "Bolivar"
and "Rodriguez".
•pBR322 is 4363 base pairs in
length and contains the ori of
pMB1, a close relative of ColE1.
•Due to this replication module
each cell accumulates upto 3000
copies of the plasmid easily.

13. • It has 2 selectable markers (tetracycline, tetR and ampicillin,
ampR resistance genes) which encodes two proteins which
make E. coli resistant to ampicillin and tetracycline.
• It also has unique restriction sites for several restriction
enzymes. PstI, SacI and PvuI restriction sites are located
within the ampR gene and BamHI, SacI within tetR gene.
Useful features of pBR322
• Small size easy purification and manipulation
• 2 selectable markers permit easy selection of recombinant
DNA
• High copy number of 15 per cell
• Copy number can be amplified upto 1000-3000 when protein
synthesis is blocked (by applying chloramphenicol)

14. pUC19
• The pUC series of vectors are
derivatives of pBR322 and are
much smaller.
• pUC19 is one of the most widely
used plasmid vectors.
• From pBR322 it has the
ampicillin resistance gene
(ampR) and the ColE1 origin
derived from pMB1
• Each cell produces 500-700
copies of plasmid without any
treatment for amplification of copy
number

15. • The second selectable marker is due to the E. coli gene lacZ
segment, denoted as lac Zα.
• This encodes the N-terminal fragment of β-galactosidase,
the enzyme that hydrolyses lactose.
• A polylinker sequence or multiple cloning site (MCS) is
located within the lacZ gene providing several unique
restriction sites for DNA insertion.
• pUC18, another popular cloning vector, differs from pUC19
only in the orientation of the MCS. The other vectors in the
pUC series are pUC9, pUC12 etc.
• “rop” gene is removed from this vector which leads to an
increase in copy number.

16. Blue white screening of recombinant clones
• The lacZ gene in pUC series of vectors and several other vectors serve as
easy selectable marker for the identification of recombinant clones.
• This gene encodes for β-galactosidase enzyme that hydrolyses lactose as
well as some synthetic substrates like X-Gal (5-bromo-4-chloro-3- indolyl- β-
D-galactoside).
• Hydrolysis of X-gal by enzyme forms a blue dye, thus cells producing active
enzyme can be easily identified as those forming blue colonies.
• Vectors used in cloning include the E. coli gene lacZ segment, denoted as
lac Zα which codes for the α fragment of N-terminal fragment of β-
galactosidase enzyme.
• When pUC plasmid enters host cells, gene products of the lac Z of the
plasmid and genome together produce the active enzyme.
• Active β-galactosidase will result in formation of blue dye from X-gal, a
substrate for the enzyme. Such cells will form blue colored colonies.
• When foreign DNA is inserted into the MCS within lacZ gene, this gene gets
disrupted and active enzyme is no longer produced.
• Transformed cells produce white colored colonies on medium containing
amp, X-Gal and IPTG and are easily selected.

18. BACTERIOPHAGE VECTORS
• Bacteriophages are viruses that attack
bacteria.
• Most phages lyse the bacterial cells
they infect (lytic phages). But many
others follow either a lytic or lysogenic
cycle.
• Lysogeny is where the phage
chromosome integrates into the
bacterial chromosome and multiplies
with it ( = lysogenic phages).
• Viruses have evolved specialized
molecular mechanisms to efficiently
transport their genome inside the cells
they infect.
• Delivery of genetic material by a virus
into a bacterial cell is called
transduction and the infected cells are
said to be transduced.

19. Viral vectors are tailored to specific applications but generally share a
few key properties.
• Safety: Although viral vectors are occasionally created
from pathogenic viruses, they are modified in such a way as to
minimize the risk of handling them.
• Low toxicity: The viral vector should have a minimal effect on
the cell it infects.
• Stability: Some viruses are genetically unstable and can rapidly
rearrange their genomes. This is detrimental to predictability and
reproducibility of the work conducted using a viral vector and is
avoided in their design.
• Cell type specificity: The viral receptor can be modified to target the
virus to a specific kind of cell.
• Identification: Viral vectors are often given certain genes that help
identify which cells took up the viral genes. A common marker is
antibiotic resistance to a certain antibiotic.

20. Advantages of phages over plasmids
• Phage vectors are more efficient than plasmids for cloning of large
DNA fragments. The maximum size possible with lambda λ vector
is 24 kb while that for plasmid vector is <15kb.
• It is easier to screen a large number of phage plaques than
bacterial colonies for the identification of recombinant vectors.
Screening of phage plaques by molecular hybridization gives
clearer results.
• Plasmid vectors have to be introduced into bacterial cells, which
are then cloned and selected for the recovery of recombinant DNA.
In contrast, phage vectors are directly tested on an appropriate
bacterial lawn culture (continuous bacterial growth on an agar
plate) where each phage particle forms a plaque (a clear bacteria-
free zone in the bacterial lawn).
• Storage of viral particles is much easier than plasmid DNA.
• Shelf life of phage particles is infinite.
• Transformation of bacterial host cells is much easier using phages
rather than plasmids.

21. LAMBDA λ PHAGE VECTORS
The lambda λ genome is sized 48,502 bp, specifically infects E.
coli cells and resides inside cells by lysogeny. It contains
• An origin of replication
• Genes for head and tail proteins
• Enzymes for DNA replication, lysis and lysogeny
• Single stranded protruding cohesive ends (of 12 base pairs,
complementary)
• Lambda λ genome remains linear in the phage head but
within E. coli cells the two cohesive ends anneal to form a
circular molecule necessary for replication.
• The sealed cohesive ends are called cos sites, which are the
sites of cleavage, necessary for packaging of the mature
phage DNA into phage heads during viral assembly.

22. • The use of wild type lambda λ genome as a vector has 2 major problems, it
takes only 3kb insert and contains >1 recognition sites for every restriction
enzyme. The properties have therefore been modified to allow use of
phages as vectors.
• By mutation and recombination in vivo as well as by recombinant DNA
techniques several vectors have been produced from lambda λ genome.
These modified vectors have 2 basic features
 They can propagate as phages in E. coli cells so vector DNA can be
replicated
 They contain restriction sites which allow removal of lysogenic segment
and also provide insertion site for DNA to be cloned.
The various λ vectors are classified into 2 groups
• Insertion vectors – here a large portion of the nonessential region is deleted
and the two arms of the λ genome are ligated. There is at least one unique
restriction site within which the DNA insert is integrated. Eg. λgt10, λgt11,
λZAP II.
• Replacement vectors – insertion of DNA fragment is accompanied by the
deletion of all the major part of nonessential region of λ genome. These
vectors have 2 restriction sites useful for cloning. Eg. λEMBL4.

24. Cloning using λ vectors

25. M13 VECTORS
• M13 vectors are derived from the 6.4kb genome of E. coli
filamentous phage M13 or f1.
• This phage has a single-stranded linear DNA genome in phage
particles. It gets converted into a ds circular molecule inside host
cells.
• M13 infects only F+ and F’ cells, injects its genome inside
through the F-pili of these cells.
• M13 vectors are used to obtain ss copies of cloned DNA,
especially suited for DNA sequencing.
• Each infected cell has ~100 copies of M13 genome and about
1000 new particles are produced during each generation of an
infected cell.
• Phage M13 does not lyse the infected cells, but it forms turbid
plaques due to growth retardation of these cells. Eg M13 mp 18.
•

26. Cloning into M13 vectors
• DNA inserts are placed into the
noncoding region which also
contains the origin of
replication.
• The vector contains the E. coli
LacZ gene which is a selection
method.
• Just like in pUC vectors, the
unchanged M13 vector
produces blue plaques on the
lawn of appropriate strain of E.
coli grown on X-gal and IPTG.
• The recombinant DNA
produces colorless plaques
which can be readily identified
Advantages of M13 vectors
• Very large DNA inserts can be cloned
• Large number of ss copies of ds DNA
inserts can be obtained
• Useful in precise DNA sequencing and
synthesis of specific radiolabelled DNA
probes
• Phage infected bacterial cells remain
viable, so easy maintenance of vector
• Selection of recombinants are easy
(plaques are formed, also growth of
infected cells is slow) stable viral
particles are formed from which
recombinant DNA can be obtained.

27. COSMIDS
• Cosmids are essentially engineered plasmids that combine
unique properties of plasmids and phage vectors.
• A cosmid is a type of hybrid plasmid constructed using
recombinant DNA technology and often used in gene
cloning.
• They contain a minimum of 250bp of λ lambda DNA, which
includes the following sequences from λ phage genome –
 The cos site (the sequences giving cohesive ends)
 Sequences needed for binding of and cleavage by terminase so that
they are packaged in vitro into empty λ phage particles under
appropriate conditions.

28. • Cosmids are usually derived from
pBR322 and can easily be
maintained in E. coli cells. Cosmids
can contain 37 to 52 (normally 45)
kb of DNA, limits based on the
normal bacteriophage packaging
size.
• A typical cosmid contains a
replication origin, unique restriction
sites and selectable markers form
plasmids. Eg. pJB8.
• Unlike plasmids, they can also be
packaged in phage capsids, which
allows the foreign genes to be
transferred into or between cells
by transduction.

29. Cloning steps
• Cut cosmid by appropriate RE at a unique site
• Mix with DNA inserts prepared using same RE (usually large DNA
inserts ~40kB of eukaryotic DNA can be cloned)
• Anneal and ligate using T4 DNA ligase
• Concatamers are produced (ideal precursors for packaging into viral
particles)
• Add packaging mix
• DNA packaged into lambda heads in vivo
• Infectious particles containing recombinant DNA obtained after addition
of tail assemblies
• Transduction of host cells
• Once inside host cells the cosmid acts like a plasmid and replicates and
propagates like a plasmid. They don’t go through the developmental
sequence of phages.
• Transduced bacterial cells selected on medium with appropriate
selection agents.

30. Advantages
• Used to clone large DNA segments (upto 40kb)
• They can be packaged into lambda particles which infect host cells (many fold more
efficient than transformation with plasmid)
• Selection of recombinant DNA is simple, based on the procedures of the concerned
plasmid
• Vectors are amplified and maintained in the same easy way as the contributing plasmid.
Applications
• Widely used vectors in gene cloning to construct genomic libraries of eukaryotes
• Ideal for genome mapping.

31. PHAGEMIDS
• A phagemid is a vector that combines
the features of a filamentous phage and
a plasmid.
• It contains an f1 origin of
replication from an f1 phage and can be
used as a type of cloning vector in
combination with filamentous
phage M13.
Features
• Phage f1 origin of replication
• A portion of lacZ gene driven by lac
promoter
• A multiple cloning site (MCS) within
lacZ gene
• ColE1 origin of replication
• ampR gene for ampicillin resistance

32. Cloning steps
Applications
 Phagemids were originally used to generate single stranded DNA templates for
sequencing purposes.
 It can also be applied into RNA transcription, restriction mapping, sequencing of
single and double stranded nucleic acids, generation of deletions etc.
 Using phagemids, peptides and proteins can be expressed as fusions to phage
coat proteins and displayed on the viral surface and so this technique is useful to
study protein-protein interactions and other ligand/receptor combinations.

33. pBluescript (pBS) is a commercially
available phagemid containing
several useful sequences for use
in cloning with bacteriophage. Size
is 2958 bp and is a cloning vector
derived from pUC19 and M13
phage.
pBLUESCRIPT SK (+/-)
Features;
 M13 origin of replication
 A portion of lacZ gene driven by lac promoter
 A multiple cloning site (MCS) located within the lacZ gene, with 21 unique
restriction enzyme recognition sites
 Phage T7 and T3 promoter sequences flanking the MCS sequence (promoters
that can be used to synthesize RNA in vitro)
 Col E1 origin of replication
 ampR resistance gene
Applications
This multipurpose vector can serve as :Cloning vector,Expression vector,Riboprobe
vector &Sequencing vector.

34. ARTIFICIAL CHROMOSOMES
Artificial chromosome vectors are linear or circular vectors that
are stably maintained in usually 1 or 2 copy per cell. There are
several types of such vectors.
• BAC – bacterial artificial chromosome
• P1 derived artificial chromosome (PAC)
• YAC – yeast artificial chromosome
• MAC – mammalian artificial chromosome
• HAC – human artificial chromosome
YAC are used for cloning in yeast, while MAC and HAC are
used in mammalian and human cells.

35. BACTERIAL ARTIFICIAL CHROMOSOMES
• A bacterial artificial chromosome (BAC) is a vector based on a
functional fertility plasmid (or F-plasmid), used for transforming
and cloning in bacteria.
• The bacterial artificial chromosome's usual insert size is 150-350
kb.
• Vector pBeloBAC11 is c convenient vector of 7.4 kb, allows
selection of recombinants cloned by LacZ complementation.
• This vector is maintained in E. coli cells at single copy per cell. It
contains
 oriS – origin of replication from E. coli F plasmid
 repE – encodes a Rep protein required for plasmid replication and
regulation of copy number
 parA, parB and parC loci - for partitioning F plasmid DNA to
daughter cells during division and ensures stable maintenance of
the BAC

36. • CMR – chloramphenicol
resisitance
• Cos N – lambda phage cos site
• Lox P – site on lambda phage P1
genome where extensive
recombination occurs
• lacZ gene – beta gal gene
• T7, bacteriophage T7 RNA
polymerase driven promoter
• SP6, bacteriophage SP6 RNA
polymerase driven promoter
Applications of BACs
1. Sequencing
2. Contribution to models of
disease - Inherited disease
3. Contribution to models of
disease - Infectious disease
4. Gene mapping

37. YEAST ARTIFICIAL CHROMOSOME
• Yeast artificial chromosomes (YACs) are genetically
engineered chromosomes derived from the DNA of the
yeast, Saccharomyces cerevisiae, which is then ligated into a
bacterial plasmid.
• pYAC3 is essentially a pBR322 plasmid into which the yeast
sequences have been integrated.
• Several yeast vectors based on pYAC3 have been
constructed. The vector is propagated in E. coli while cloning
is done in yeast.
• The primary components of a YAC are the ARS, centromere,
and telomeres from S. cerevisiae.
• Additionally, selectable marker genes, such as antibiotic
resistance and a visible marker, are utilized to select
transformed yeast cells.

38. Construction Basic functional elements
• an ARS sequence
• CEN4 sequence
• Telomeric sequence
• One or two selectable markers, eg.
TRP1 and URA3
• SUP4 a selectable marker into
which the DNA insert is integrated
• Plasmid sequences for selection
and propagation in bacteria

39. Applications
• Yeast expression vectors, such as
YACs, YIps, and YEps, have an
advantage over BACs in that they can
be used to express eukaryotic
proteins that require posttranslational
modification.
• By being able to insert large
fragments of DNA, YACs can be
utilized to clone and assemble the
entire genomes of an organism. With
the insertion of a YAC into yeast cells,
they can be propagated as linear
artificial chromosomes, cloning the
inserted regions of DNA in the
process
• Two processes can be used to obtain
a sequenced genome, or region of
interest: physical mapping and
chromosome walking.

40. Thank you