Vectors in R-DNA Technology

Dr Mehul P. Dave
Associate Professor in Life Sciences (Microbiology)
Director, Centre of Extension
Indian Institute of Teacher Education, Gandhinagar
[email protected]
OUTLINES
• Vectors
• Main Features of Vectors
• Mode of action
• Classification
• Plasmid Vectors
• Bacteriophage Vectors
• Artificial Chromosomes as Vectors
• Selection of recombinant host
 Vectors
Vectors can be defined in three ways:
In epidemiology:
An organism or vehicle that transmits
the causative agent or disease-
causing organism from the reservoir
to the host.
In Molecular Biology :
A vehicle (e.g. a plasmid) used to
transfer the genetic material such as
DNA sequences from the donor
organism to the target cell of the
recipient organism.
In Biology,
A biotic agent that disperses
reproductive structures of another
organism, as a bee transmitting pollen
to the stigma of a flower.
Vectors in R
DNA Technology
DNA vectors and their properties
One of the most important elements
in gene cloning is the vector, which in
conjunction with the passenger DNA
forms the recombinant DNA which can
be propagated in suitable host cells. In
order to perform its function, a vector
must possess the following properties
• They should be capable of autonomous
replication in at least one host organism.
• They should be of small size, since this aids the
preparation vector DNA and reduces the
complexity of analyzing recombinant molecules.
• They should be capable of amplifying the cloned
sequence by occurring in multiple copies. High
copy number facilitates in maximizing expression
of cloned genes.
• There should be a unique cleavage
site for a range of restriction
endonucleases. Occurrence of
multiple cleavage sites reduces the
likelihood of functional recombinant
DNA formation.
• They should possess one or more
genetic markers enabling easy
selection of cloned molecules.
• They should permit detection by
simple genetic tests, of the presence
of passenger DNA inserted at cloning
site.
They should have appropriate
transcriptional and translational
signals located adjacent to cloning
sites for better expression of
cloned DNA sequences. They
should have host specificity when
there is biological containment for
a vector.
A variety of different cloning vectors
have been developed by using the
items mentioned above as guidelines.
They are as follows: plasmids, phages,
cosmids, phasmids, shuttle vectors,
expression vectors and single
stranded DNA
Vector Classification # 1.
On the Basis of Our Aim with Gene of
Interest:
The point is what we are targeting from
our gene of interest — its multiple copies
or its protein product.
Depending on these criteria vectors are of
following two types:
1. Cloning Vectors:
We use a cloning vector when our aim
is to just obtain numerous copies
(clones) of our gene of interest (hence
the name cloning vectors). These are
mostly used in construction of gene
libraries. A number of organisms can be
used as sources for cloning vectors.
Some are created synthetically, as in the case of
YAC and BAC while others are taken from
bacteria and bacteriophages.
The vector needs to be genetically modified in
order to accommodate the foreign DNA by
creating an insertion site where the new DNA
will fitted. Example: PUC cloning vectors,
pBR322 cloning vectors, etc.
2. Expression Vectors or Expression
construct:
We use an expression vector when our aim is
to obtain the protein product of our gene of
interest. To get the protein we need to allow
the expression of our gene of interest (hence
the name expression vector) by employing
the processes of transcription and
translation.
Apart from the three DNA sequences
discussed above (origin of replication,
selectable markers and multiple cloning
sites), the expression vectors have some
special additional sequences as well.
a. A bacterial promoter, such as the lac promoter. The
promoter precedes a restriction site where foreign DNA is
to be inserted, allowing transcription of foreign sequence
to be regulated by adding substances that induce the pro-
moter.
b. A DNA sequence that, when transcribed into RNA,
produces a prokaryotic ribosome binding site.
c. Prokaryotic transcription initiation and termination
sequences.
d. Sequences that control transcription initiation, such as
regulator genes and operators.
Expression Vector
Vector Classification # 2
On the Basis of Host Cell Used:
After construction of a recombinant DNA these can be
introduced into a host cell. So depending on the host
cell the vectors are designed and constructed. All the
parts of the vectors must be functionally compatible
with the host. For example, if we are making a vector
for a bacterial host, it must have a suitable origin of
replication which will be functional in a bacterial cell.
Depending on this basis the vectors are classified as
under:
1. Vectors for Bacteria:
These are special bacterial origin of replication and
antibiotic resistance selectable markers. Bacteria
support different kinds of vectors, e.g.; plasmid
vectors, bacteriophages vectors, cosmids, phasmids,
phagemids, etc.
2. Vectors for Yeast:
They have special origin of replication called as
autonomously replicating sequences (ARS), e.g., yeast
replicative plasmid vectors (YRp) etc.
3. Vectors for Animals:
These vectors are needed in biotechnology for the
synthesis of recombinant protein from genes that
are not expressed correctly when cloned in E. coli or
yeast, and methods for cloning in humans are being
sought by clinical molecular biologists attempting to
devise techniques for gene therapy, in which a
disease is treated by introduction of a cloned gene
into the patient, e.g., P-element, SV40 etc.
4. Vectors for Plants:
The production of genetically modified
plants has become possible due to
successful use of plant vectors. e.g., Ti-
plasmid, Ri-plasmid etc.
Vector Classification # 3.
On the Basis of Cellular Nature of Host Cell:
On this basis, the vectors are of two types:
1. Prokaryotic Vectors :
This comprises of all vectors for bacterial cells.
2. Eukaryotic Vectors :
This comprises of all the vectors for yeast,
animal and plant cells.
 PLAMID VECTORS
Plasmids are self replicating, double
stranded, circular DNA molecules that are
maintained in bacteria as independent
extra chromosomal entities. These are also
found in some yeast but not in higher
eukayotes. Plasmids are widely distributed
throughout the prokaryotes, vary in size
from less than 1 x 106 to greater than 200 x
106 Da and are generally dispensable.
 Few Definitions
Plasmid can be defined as:
An extra chromosomal self-replicating structure found in
bacterial cells that carries genes for a variety of functions not
essential for cell growth.
OR
Acircular, double-stranded unit of DNA that replicates within a cell
independently of the chromosomal DNA is called plasmid.

Plasmids are most often found in bacteria and are used in

recombinant DNA research to transfer genes between cells.
Plasmids can be grouped into two major types:
conjugative and non- conjugative. In
conjugative plasmids transfer genes (tra) and
mobilizing genes (mob) are present whereas in
non-conjugative plasmids tra genes absent. The
non-conjugative plasmids can be mobilized by
another conjugative plasmid present in the
same cell, if the mob gene is intact.
Non-conjugative differ from conjugative
plasmids by the absence of tra gene
Plasmids can also be categorized on the
basis of their being maintained as
multiple copies per cell (relaxed plasmids
or high copy number plasmids) or as
limited copies per cell (stringent plasmids
or low copy number plasmids).
The replication of stringent plasmids is
coupled to chromosome replication, hence
their low copy number. Generally conjugative
plasmids are of low molecular weight and
present in multiple copies per cell. An
exception is the conjugative plasmids RBK
which has a molecular weight of 25x106
daltons and is maintained as relaxed plasmid.
Plasmids that carry specific sets of genes
for the utilization of unusual metabolites
are called as degradative plasmids. Some
plasmids will not have any apparent
functional coding genes and are called
cryptic plasmids. Some of the plasmids do
not coexist in the same host cell in the
absence of selection pressure and are
called incompatible plasmids.
Some plasmids are capable of promoting their own
transfer to a wide range of host. These plasmids are
called as promiscuous plasmids.
Plasmids can also be grouped into narrow-host
range plasmids and wide host range plasmids
based on their nature of infectivity.
Based on the origin of plasmids, they can be
grouped into naturally occurring plasmids and
synthetic plasmids.
 A list of naturally occurring plasmids and
their properties are furnished below
Plasmid Size (kb) Conjugative Copy number Amplifiable Selectable marker
ColE1 7.0 - 10 – 15 + E1imm
RSF1030 9.3 - 20 – 40 + Apr

CloDF13 10.0 - 10 + DF13 i mm

pSC101 9.7 - 1 -2 - Tcr

R6K 42 + 10 – 40 - AprSmr

F 103 + 1–2 - -

R1 108 + 1–2 - AprCmrSn

RK2 56.4 + 3–5 - AprKmrTcr

In general plasmid cloning vectors are designated by a lowercase ‘p’ which

stands for plasmid, and some abbreviations that may be descriptive.
 What is mode of action of a
plasmid vector?
Plasmids are pieces of DNA that can be
manipulated through the use of restriction
enzymes. Since these plasmids are already in
the bacteria, the restriction enzymes are able to
act as "molecular scissors" and cut up the part
of the plasmid that is desired to be changed.

Then, the gene of interest is inserted as a new

part of the plasmid, allowing the bacteria to
now demonstrate this gene.

39
What is mode of action of
plasmid vector?
In the case of plasmids utilized
as transcription vectors,
incubating bacteria with
plasmids generates hundreds or
thousands of copies of the
vector within the bacteria in
hours, and the vectors can be
extracted from the bacteria, and
the multiple cloning sites can be
cut by restriction enzymes to
excise the hundredfold or
thousand fold amplified insert.
These plasmid transcription vectors
characteristically lack crucial
sequences that code for
polyadenylation sequences and
translation termination sequences in
translated mRNAs, making protein
expression from transcription
vectors impossible.
What are the types of plasmid
vectors?
Plasmid vectors may be:
Conjugative/transmissible vectors:

They mediate DNA transfer through conjugation and

therefore spread rapidly among the bacterial cells of
a population; e.g., F plasmid, many R and some col
plasmids.
Non-conjugative vectors:
They do not mediate DNA through conjugation, e.g.,
many Rand col plasmids.
pBR322 plasmid
Plasmid pBR322 is the one of the best
studied and most often used ‘”general
purpose” plasmids.
The BR of the pBR322 recognizes the work
of the researchers F. Bolivar and R.
Rodriguez
pBR322 is 4361 base pair long and
completely sequenced.
I. pBR322:
This was the first widely used, purpose built plasmid
vector. pBR322 has a relatively small size of 4,363 bp.
This is important because transformation efficiency is
inversely proportional to size and above 10 kbp is very
low.
Thus, there is ‘room’ in pBR322 for an insert of at least
six kbp. Also this vector has a reasonably high copy
number (~15 copies per cell), which can be increased
200-fold by treatment with a protein-synthesis
inhibitor—chloramphenicol amplification.
The nomenclature of pBR 322:
The nomenclature of ‘pBR 322’ can be understood
with following explanation:
1. ‘p’ indicates as a plasmid
2. ‘BR’ identifies Bo-liver and Rodriguez, the two
researchers who developed it
3. ‘322’ distinguishes those plasmids from others
(like pBR 325, pBR 327, etc.) developed in the same
laboratory.
The Construction of pBR322:

1. Origin of Replication:
It carries a fragment of plasmid pMB1 that acts
as an origin for DNA replication and thus
ensures multiplication of the vector.
pBR322 carries two antibiotic resistance genes. One
confers resistance to ampicillin (Ampr) and the other
confers resistance to tetracycline (Tetr) There are eleven
known enzymes which cleave pBR 322 at unique sites.
For three of the enzymes, Hind III, Bam HI and Sal I, the
target site lies within the Tetr genes and for another two,
Pst I and Pru I, they lie in Ampr genes.

Thus cloning in pBR 322 with the aid of these enzymes

results in insertional inactivation where the inserted DNA
disrupts the function of the gene containing the cloning
site.
• Where the cloning site is within in an
antibiotic resistance gene, such insertional
inactivation results in transformants sensitive
to the appropriate antibiotic. Thus, insertional
inactivation helps in the selection of
recombinants.
Insertional Inactivation
Blue White Screening
Blue White Screening
Colony Hybridization
Uses of pBR322:
It is widely used as a cloning vector. In addition to
this, it has been widely used as a model system for
study of prokaryotic transcription and translation.
Advantages of pBR322:
1. Small size (~ 4.4 kb) enables easy purification and
manipulation.
2. Two selectable markers (amp and tet) allow easy
selection of recombinant DNA.
3. It can be amplified up to 1000-3000 copies per cell
when protein synthesis is blocked by the application
of chloramphenicol.
Disadvantages of pBR322:
1. It has very high mobility i. e; it can move to
another cell in the presence of a conjugative
plasmid like F-factor. The nic-bom (bom=basis
of mobility) region of pBR322 is responsible
for this feature. Due to this, the vector may
get lost in a population of mixed host cells.
2. There is a limitation in the size of the gene
of interest that it can accommodate.
3. Not a very high copy number is present as is
expected from a good vector.
4. Although insertional inactivation of an
antibiotic resistance gene provides an effective
means of recombinant identification, the
method is made inconvenient by the need to
carry out two screenings, one with the antibiotic
that selects for trans-formants, followed by the
second screen, after replica plating, with the an-
tibiotic which distinguishes recombinants.
This makes the screening process time-
consuming and laborious.
pUC Vectors:
pUC are obtained by modifying the pBR322
vector. pUC vectors are smaller than pBR322
of being only ~2.7 kb. But comparatively they
have a high copy number. A mutation within
the origin of replication produces 500 to 600
copies of the plasmid per cell without
amplification.
The Nomenclature of pUC Vectors:
The nomenclature of ‘pUC’ can be understood
with the following explanation:
1. ‘p’ indicates the plasmid.
2. ‘UC’ stands for university of California where
it was first developed by J. Messing et al.
We also see many numbers after this like pUC8,
pUC18, pUC19 and so on. They are just the
series of pUC and have been named just to
separate from each other.
The construction of pUC vectors:
1. Origin of Replication: It is derived from the
origin of replication of pBR322.The ColE1origin
of replication of pBR322 has been modified by
carrying out a chance mutation so that each
transformed E. coli cell has 500-600 copies of
the plasmid.
2. Selectable Marker: It has an ampicillin
resistant gene. The transformed host cells can
grow on media having ampicillin whereas non-
transformed cells die.
3. lac Z’ gene having MCS.
4. The lac Z’ is incorporated into this vector codes for the
enzyme beta-galactosidase which acts on a chromogenic
substrate called X-gal (present in bacterial culture media).
The expression of lac Z’ gene is induced by another
compound present in the same media called Iso-propyl-
thiogalactoside (IPTG).
When the enzyme substrate reaction takes place, then the
X- gal is converted from white to a blue compound. Now
the lac Z’ gene itself has MCS. Hence, when the gene of
interest has been introduced into the lac Z’ gene, then it
fails to code for beta-galactosidase and thus in this case
the substrate (X-gal) is never converted to any other
colour. This type of screening is called blue-white screen-
ing.
Advantages of pUC Vectors:
The pUC vectors offer following major advantages
over pBR322 vectors:
(a) High copy number of 500-600 copies per cell.
(b) Easy and single step selection.
(c) The unique restriction sites used for cloning are
clustered within the MCS. This allows cloning of a
DNA fragment having two different sticky ends.
Disadvantages of pUC:
It cannot accommodate a gene of interest larger
than 15kb.
 What are the types of plasmid
vectors?
1)Ti plasmid:
A Ti or tumor inducing plasmid is a circular plasmid is often, a part
of the genetic equipment of Agrobacterium tumefaciens and
Agrobacterium rhizogenes. Used to transduce its genetic material
to plants.
•Classification of Ti plasmid:
•Based on the type of opine produced by their genes.
The different opines specified by pTi are octopine, nopaline,
succinamopine and leucinopine.

•The plasmid has 196 genes that code for 195 proteins. There are
no one structural RNA. The plasmid is 206,479 nucleotides long;
the GC content is 56% and 81% of the material is coding genes.
There are no pseudo genes.
• Important in the creation of transgenic plants.
1)Ti plasmid:
2) Col plasmid:

Any plasmid that carries genetic information for the

production of a colicin; Involved in killing the other harmful
strains of bacteria also called bacteriocinogenic plasmid.
They produce proteins that are sensitive to bacteria and
inhibit its essential processes producing a clear area called
lacuna.

3)Virulence Plasmid: Virulence plasmid turns the

bacterium into a pathogen. So they are responsible for
carrying the genes which cause diseases.
 Types of Plasmid Vectors
4)F Plasmid:
The Fertility factor allows genes to be transferred from one
bacterium to another by conjugation.
F plasmid a conjugative plasmid found in F+ (male) bacterial cells
that leads with high frequency to its transfer.
A cell possessing the F plasmid (F+, male) can form a conjugation
bridge (F pilus) to a cell lacking the F plasmid (F−, female), through
which genetic material may pass from one cell to another.

71
 What are the types of plasmid
vectors?
5)R (Resistance) Plasmid:
• R plasmid is a conjugative factor in bacterial cells.
Promotes resistance to agents such as antibiotics, metal ions, ultraviolet
radiation, and bacteriophage.
•
Many R-factors can pass from one bacterium to another and through which
antibiotic resistance spreads between bacterial species, genera and even
families.
RP1, a plasmid that encodes resistance to ampicillin, tetracycline and kanamycin
originated in a species of Pseudomonas, but also in bacteria such as Escherichia
coli.

72
 What are the types of plasmid
vectors?
6)Degradative Plasmid:
These plasmid are types of plasmids present in certain bacteria’s
such as Pseudomonas putida which impart the ability of
degrade xenobiotic compounds.
For example: salicylic acid, 2-4D etc.

There are 3 such plasmids:

• CAM plasmid- which degrades camphor.
• XYL, - , xylene.
• NAH, - , naphthalene.

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 Types of plasmid Vectors
7)Yeast plasmids:
Yeast naturally harbors plasmids.
These are 2µm plasmid - small circular plasmid often used for genetic
engineering of yeast and linear pGKL plasmids from kluyveromyces lactis that
are responsible for killer phenotype. Yeast cloning vectors include:

74
Fosmid Vectors:
These are similar to cosmids but are based on
the bacterial F-plasmid. The cloning vector is
limited, as a host (usually E. coli) can only
contain one fosmid molecule. Fosmids are 40 kb
of random genomic DNA. Fosmid library is
prepared from a genome of target organism and
cloned into a fosmid vector.
Low copy number offers higher stability than
comparable high copy number cosmids. Fosmid
system may be useful for constructing stable
libraries from complex genomes.
pUC19 plasmid
Plasmid pUC19 is 2686 bp long and contains an
ampicillin resistance (Ampr) gene, a regulatable segment
of â- galactosidase gene (lacZ) of the lactose operon of
E. coli, lac I gene that produces a repressor protein that
regulates the expression of lacZ gene, a short sequence
with multiple cloning sites (EcoRI, SacI, KpnI, XmaI, SmaI,
BamHI, XbaI, SalI, HincII, AccI, BspMI, PstI, SphI and
HindIII) and the origin of replication from pBR322. The
presence of lac Z and lacI genes allows to select the
recombinants based on the â- galactosidase production
in the presence of isopropyl- â - D-thiogalactopyranoside
(IPTG), an inducer of the lac operon. (UC in pUC stands
for University of California).
 What is importance of Plasmids?
Easy to work with - Plasmids are a convenient size (generally
1,000-20,000 base pairs). With current cloning technology, it is easy
to create and modify plasmids containing the genetic element that
you are interested in.
Self-replicating - Once you have constructed a plasmid, you
can make an endless number of copies of the plasmid by
growing the plasmid in bacteria.
Stable - Plasmids are stable long-term either as purified DNA or
within bacteria (as glycerol stocks).
Functional in many species and can useful for a diverse set of
applications - Plasmids can drive gene expression in a wide variety
of organisms, including plants, worms, mice and even cultured
human cells.
used to understand gene function,
they can also be used to investigate promoters, small RNAs, or
other genetic elements.
 Bacteriophage Vectors
• Derivatives of phage have been developed as
cloning vectors since the early days of gene
technology. The phage derivatives are
considered to be the most suitable cloning
vehicles for cloning genomic eukaryotic DNA
because of the following advantages over the
plasmids.
• Thousands of phage plaques can be obtained
in a single petridish.
• Selection by DNA-DNA hybridisation is
possible
• In vitro packaging into empty phage head is
possible thus increasing phage infectivity
• Size selection of the packaged DNA is possible
• Millions of independently cloned virus particle
can be constituted to form a gene library.
 Viral Vectors/Bacteriophage Vectors
A viral vector is a virus which has been modified in a laboratory
environment for purpose of introducing genetic material into a
cell.
To form a viral vector, remove the genes in the virus that cause
disease. Then replace those genes with genes encoding the
desired effect (for instance, insulin production in the case of
diabetics). This procedure must be done in such a way that the
genes which allow the virus to insert its genome into its host's
genome are left intact.
Viruses are highly evolved natural vectors for the transfer of
foreign genetic information into cells. But to improve safety,
they need to be replication defective.
So the viruses can be used as vehicles to carry 'good' genes into a
human cell.
Development of the viral vector dates to 1960s, when a number of
researchers recognized that since viruses operate by inserting
genetic material into cells, surely researchers could harness this
trait by modifying viruses to change the genetic material they
are inserting.

The process of delivering genetic material with the use of a virus is

known as transduction with the first successful attempt occurring
in 1968 with plant cells.
Key properties for a viral vector

Safety Cell type

specificity

Low toxicity
Identification

Stability
 Viral vector should be modified in such a way
as to minimize the risk of handlingthem.
Usually involves the deletion of a part of the
Safety viral genome critical for viral replication

viral vector should have a minimal effect

on the physiology of the cell it infects.
Low toxicity
Some viruses are genetically unstable & can
rapidly rearrange their genomes.
This is detrimental to predictability and
reproducibility of work conducted using a viral
Stability vector and is avoided in their design.
 Most viral vectors are engineered to infect as
wide a range of cell types as possible.
viral receptor can be modified to target the
Cell type virus to a specific kind of cell. Viruses modified
in this manner are said to be pseudo typed.
specificity

Viral vectors are often given certain genes

that help identify which cells took up the viral
genes. These genes are called Markers
Identification
Lamda Bacteriophage is a genetically complex
but very extensively studied virus of E. coli. The
DNA of phage, in the form in which it is isolated
from the phage particle is a linear duplex
molecule of 48502 bp (~49kb) in length. The
DNA isolated from virus particles is a double
stranded linear molecule with short
complementary single stranded projections of
12 nucleotides at its 5’ ends. These cohesive
termini, also referred to as cos sites, allow the
DNA to be circularized after infection of the host
cell.
Lamda Phage Lytic Cycle
Types of phage vectors
Wild type phage DNA itself cannot be used as a vector
since it contains too many restriction sites. Further,
these sites are often located within the essential
regions for phage's growth and development. From
these wild phages, derivatives with single target sites
and two target sites have been synthesized. Phage
vectors which contain single site for the insertion of
foreign DNA have been designated as insertional
vectors; vectors with two cleavage sites, which allow
foreign DNA to be substituted for the DNA sequences
between those sites, are known as replacement
vectors.
• Apparently if too much non-essential DNA is
deleted from the genome it cannot be
packaged into phage particles efficiently. For
both types of vector, the final recombinant
genome must be between 39 and 52 kb of the
wild type phage genome, if they are to be
packaged into infectious particles. Insertion
vectors must therefore be at least 39 kb in
length to maintain their viability. This places
an upper limit of about 12 kb for the size of
foreign DNA fragments which can be inserted.
Replacement vectors have a larger capacity
because the entire non-essential region can be
replaced, allowing the cloning of the fragments
upto 22 kb. Several types of vectors have been
developed which allow direct screening for
recombinant phages and are useful for cloning
specific DNA fragments. A list of phage vectors
with their characteristics is given below.
• Cosmids
• Plasmids containing phage cos sites are known as
cosmids. Cosmids can be used to clone large
fragments of DNA by exploiting the phage in vitro
packaging system. Since cosmids have advantages
of both plasmids and phage vectors they can be
delivered to the host by the more efficient
infection procedures rather than by
transformation. Cloning with cosmid vectors has
widened the scope of plasmid cloning in the
following ways.
• The infectivity of plasmid DNA packaged in phage
head is at least three orders of magnitude higher
than that of pure plasmids DNA.
• The process almost exclusively yields hybrid
clones so that a subsequent selection for
recombinant DNA becomes unnecessary.
• In contrast to normal plasmid transformations,
the system strongly selects for clones containing
large DNA inserts. It is therefore, particularly well
suited for generating genomic libraries.
COSMID
The following table provides a list cosmid vectors and their structural features.

Cosmid Size Cleavage sites Size of insertion (kb)

MUA3 4.76 EcoRI/PstI/PvuII/PvuI 40 – 48

pJB8 5.40 BamHI 32 – 45

Homer I 5.40 EcoRI/ClaI 30 – 47

Homer II 6.38 SstI 32 – 44

pJC79 6.40 EcoRI/ClaI/BamH I 32 – 44

 Phasmid
A phasmid can be propagated as a plasmid or lytically
as a phage. Lytic functions of phasmid can be
switched off by propagation in the appropriate
lysogene where the plasmid origin of replication is
used for maintenance. The phasmid may replicate as
phage if propagated in a non-lysogenic strain. In the
case of phasmids based on ë, such as ë1130, the
temperature sensitive gene, cI857 carried by the
vector may be used to switch between replication
modes, simply by growing the host at the permissive
(plasmid mode) or restrictive (phage mode)
temperature.
• 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 Chromosomes
(BAC)
BACs are based on bacterial mini-F
plasmids, which are small pieces of
episomal bacterial DNA that give the
bacteria the ability to initiate
conjugation with adjacent bacteria.
They have a cloning limit of 75-300 kb.
Yeast Artificial Chromosomes (YAC)
YACs are artificial chromosomes that replicate in
yeast cells. They consist of:
Telomeres, which are ends of chromosomes
involved in the replication and stability of linear
DNA.
Origin of replication sequences necessary for the
replication in yeast cells.
A yeast centromere, which is a specialized
chromosomal region where spindle fibers attach
during mitosis.
A selectable marker for identification in yeast cells.
 Ampicillin resistance gene for selective amplification.
Recognition sites for restriction enzymes.
The procedure for making YAC vectors is as follows (see
Appendix D):
The target DNA is partially digested by a restriction
endonuclease, and the YAC vector is cleaved by restriction
enzymes.
The cleaved vector segments are ligated with a digested
DNA fragment to form an artificial chromosome.
Yeast cells are transformed to make a large number of
copies.
They are the largest of the cloning vectors, with a cloning
limit of 100-1000 kb, however they have very low efficiency.
• Shuttle vectors
• Shuttle vectors normally comprise an E. coli plasmid
or part of such plasmid (e.g., pBR 322), ligated in
vitro to a plasmid or virus replicon from another
species. Shuttle vectors can be made, for example,
for E. coli/B. subtilis, E. coli/yeast or E.
coli/mammalian cells. The shuttle vector strategy
permits the exploitation of the many manipulative
procedures, such as amplification, available in E. coli
(or other genetically well characterized species such
as B. subtilis or S. cerevisiae) backgrounds. The
ability to transfer cloned genes across species
boundaries is of potential value in the genetic
manipulations of industrially important species and
this can be achieved by using shuttle vectors.
Expression vectors
In DNA cloning experiments all the genes cloned are
not expressed fully because of weak promoters in
vector DNA. This can be dramatically improved by
placing such genes downstream of strong promoters.
An additional problem in maximizing expression of
cloned genes in E. coli which is frequently encountered
with genes from a heterologous source is that the gene
carries no translation start signal which can be
efficiently recognized by the E. coli translation system.
This problem may arise for heterologous genes
cloned into any host. Thus, even though the gene can
be transcribed from a promoter within the vector,
the resulting mRNA is poorly translated and little or
no protein product will be synthesized. In such cases
alternative strategies available are fusing the gene to
amino terminal region of vector gene that is
efficiently translated in the host or coupling the gene
to a DNA fragment carrying both strong promoter
and a ribosomal binding site. Vectors with this
additional feature are called expression vectors.
Vectors used in rDNA technology should possess …….
a). Autonomous replication b). Small size
c).Possess one or more genetic markers
d). All the above
Plasmids DNA is …….
a). Self replicating b). Double stranded
c). Circular d). All the above
Plasmids are grouped into ……. major types
a). 2 b). 3
c). 4 d). None of the above
Conjugative plasmids have ……. genes
a). Only transfer genes (tra) b). Only mobilizing
genes (mob)
c). Both a and b d). Promiscuous plasmids

Non - conjugative plasmids have ……. genes

a). Only transfer genes (tra) b). Only mobilizing
genes (mob)
c). Both a and b d). Promiscuous plasmids

Relaxed plasmids are also called as ………….

a). High copy number plasmids b). Stringent plasmids
c). Low copy number plasmids d). Promiscuous
plasmids
pBR322 plasmid was created by …………………….
a). F. Bolivar b). R. Rodriguez
c). Both a and b d). None of the above
The resistance gene(s) in the pBR322 plasmid is/are…………………….
a). Ampicillin (Ampr) b). Tetracycline (Tetr)
c). Both a and b d). None of the above
The phage vectors that contain two cleavage site and which allow
foreign DNA to be substituted for the DNA sequences between
those sites are designated as
a). Insertional vectors b). Replacement vectors
c). Both a and b d). None of the above
The resistance gene(s) in the pUC19plasmid
is/are…………………….
a). Ampicillin (Ampr) b). Tetracycline (Tetr)
c). Both a and b d). None of the above

The phage vectors that contain single site for the

insertion of foreign DNA are designated as
a). Insertional vectorsb). Replacement vectors
c). Both a and b d). None of the above
Plasmids containing phage cos sites are known as …………………….
a). Cosmids b). Phasmids
c). Both a and b d). None of the above

Phasmids are also called as …………………….

a). Cosmids b). Plasmids
c). Phagemids d). None of the above

Phasmids are hybrids formed between …………………….

a). Plasmids and bacteriophages b). Cosmids and
bacteriophages c). BAC and bacteriophages d). None of the above
Bacterial Artificial Chromosomes (BAC) are …………………….
a). Mini-F plasmids b). Have the ability to
initiate conjugation with adjacent bacteria
c). Have a cloning limit of 75-300 kb d). All the above

Yeast Artificial Chromosomes (YAC) are …………………….

a). Artificial chromosomes that replicate in yeast cells

c). Have ampicillin resistance gene for selective amplification

b). Have recognition sites for restriction enzymes

d). All the above
Stringent plasmids are also called as ………….
a). High copy number plasmids b). Relaxed plasmids
c). Low copy number plasmids d). Promiscuous
plasmids

Plasmids that carry specific sets of genes for the

utilization of unusual metabolites are called as ………….
a). Degradative plasmids b). Relaxed plasmids
c). Stringent plasmids d). Promiscuous plasmids
Plasmids without any apparent functional coding genes
are called as …….
………….
a). Degradative plasmids b). Cryptic plasmids
c). Stringent plasmids d). Promiscuous plasmids

Plasmids capable of promoting their own transfer to a

wide range of host are called as …………………….
a). Degradative plasmids b). Cryptic plasmids
c). Stringent plasmids d). Promiscuous plasmids