VECTORS IN

MOLECULAR
BIOLOGY
RENJINI.MR
MSC BOTANY
PLASMIDS

. • A plasmid is a small, circular, extrachromosomal double stranded DNA that has the
capacity to replicate independently.
• Discovered by Laderberg in 1952.
• It naturally occur in bacteria, however sometimes present in archaea and eukaryotes
. • The genes carried in plasmid benefit the survival of the organism by providing them
with genetic advantages like antibiotic resistance etc. under certain situation or
particular conditions.
• They provide mechanism for horizontal gene transfer within a population of microbes
and thus provide a selective advantage under a given environmental state.
 PROPERTIES

• Specific to one or a few particular bacteria.

• Replicate independently and code for their own transfer.
• Do not cause damage to cells and generally are beneficial, do not have extracellular forms and
exist inside cells simply as free and typically circular DNA.
• Size ranges from 1 kbp to several mbp.
• Number of plasmids in an individual cell may vary, ranging from one to several hundreds,
denoted by copy number.
• Genes carried by plasmid encodes traits for antibiotic resistance or resistance to heavy metal.
• Some produces virulence factor that help in defence or nutrient utilization.
• Plasmids can also provide bacteria with the ability to fix nitrogen.
• Some also exhibits properties like sulphur utilization, hydrocarbon degradation, drug resistance
etc.
ELEMENTS OF PLASMIDS •
.
Origin of replication: it is the DNA sequence which directs initiation of
plasmid replication by recruiting bacterial transcriptional machinery.
• Antibiotic resistance gene: these genes allows for selection of plasmid
containing bacteria by providing a survival advantage to the bacterial host.
• Multiple cloning site: this is the short segment containing several
restriction enzyme sites, enabling easy insertion of foreign DNA.
• Promoter region: it drives the transcription of the foreign insert.
• Selectable marker: it is used to select for cells that has successfully
taken up the plasmid for the purpose of expressing the insert.
• Primer binding site: it is the site for binding of short single stranded
DNA sequence, used as an initiation point for PCR amplification or
sequencing of the plasmid.
PLASMID REPLICATION
Plasmids replicate autonomously because they have their own replication origins.

• Most plasmids in gram-negative bacteria replicate in a manner similar to the replication

of bacterial chromosome involving initiation at the replication origin site and bidirectional
replication around the DNA circle giving a theta (Ө) intermediate.

• Most plasmids of gram-positive bacteria replicate by a rolling circle mechanism

MODE OF PLASMID TRANSFER The genetic information encoded in a plasmid of bacteria

is transferred across a broad range of microorganism via-

Transformation: requires competent cells which are ready to accept extracellular plasmid
and further stable replication inside host cell.

Transduction: plasmid mediated gene transfer through bacteriophages.(can be

generalised or specialised)

Conjugation: transfer through cell to cell contact of donor and recipient cell, requires DNA
metabolism of donor cell.

F-PLASMID (fertility plasmid) contain ‘tra’ gene, capable of conjugation. R-PLASMID

(resistance plasmid) contain genes that provide resistance against antibiotics or poisons.
Col PLASMID contain genes that codes for bacteriocins (proteins that can kill bacteria)
pBR322
• It is an artificial plasmid.
• It is a gene cloning vector for E.coli.
• Constructed from two plasmids pSC101 and ColEI and a transposon
Tn3.
• p indicates it’s a plasmid
• BR-F.Boliver and Rodriguez.
• 322- specific number to distinguish from others.
• Consists of 4363 base pairs.
• Has 528 restriction sites for 66 restriction enzymes.
• Two selectable markes- tetracycline resistance gene and amplicin
resistant gene.
• The amplicin resistance determinant is the derivative of Tn3
• The tetracycline resistance is a derivative of pSC101 of R-65 plasmid.
• The copy number of pBR 322 is 15.It can be increased upto 3000 by
adding cloramphenicol to the bacterial culture.
• It is used as a base plasmid for the invitro construction of plasmid
vectors such as pUC8,pUC9,…
• Use- it is being used to introduce desired gene into E-coli cells
• Eg- somatostatin gene of man is introduced into Ecoli through pBR322.
COSMIDS
• 1.Cosmids are plasmids, medium sized cloning vectors.

• 2. Cosmid vector are developed by combining the features of plasmid vector and
bacteriophage vector.

• 3. The first cosmid vector was described by Collins in 1978.

• 4. They are capable of incorporating the bacteriophage λ DNA segment. This DNA
segment contains cohesive terminal sites (cos sites). 5. Cos sites are necessary for
efficient packaging of DNA into λ phage particles.

• 6. Large DNA fragments of size varying from 35 to 45 kb can be cloned.

• 7. They are also packaged into λ. This permits the foreign DNA fragment or genes to be
introduced into the host organism by the mechanism of transduction.

• Advantages of using cosmids as vectors:

• i. They have high transformation efficiency and are capable of producing a large number
of clones from a small quantity of DNA.

• Ii. Also, they can carry up to 45 kb of insert compared to 25 kb carried by plasmids and λ.

• Disadvantages of using cosmids as vectors

• i. Cosmids cannot accept more than 50 kb of the insert.

 PROPERTIES OF COSMID VECTOR

• . Contains Features of plasmid •

• Origin of replication
• • Multiple cloning site
• • Selectable marker Contains Features of lambda phage • Only cohesive site or cos site region.
• • A linear DNA molecule, has a 12 base long , single stranded complementary overhang at both
ends. Which emerge during the packaging process into phage particle through splitting of cos site. •
• • A cosmid vector may have one or two cos site.

Uses: ••
• Cosmid vector are used in construction of genomic libraries. • Cosmid vector have cloning capacity
up to 45 kbp.
Cloning of foreign DNA in cosmid vector involves the following steps:

1. The cosmid is opened at its unique restriction site and new DNA fragments inserted –Ligation
of foreign DNA.
• 2. Making a concatemeric DNA.
• 3. In vitro packaging to introduce the DNA into phage head to form the matured phage particle.
• 4. Introduction of the cloned DNA into E. coli by transduction
• 5. After their entry into host cell the cosmid are maintained as plasmid. A concatemer is a long
continuous DNA molecule that contains multiple copies of the same DNA sequence linked in
ser.ies.
PHAGEMIDS

• A phagemid or phasmid is a DNA-based cloning vector, which has both

bacteriophage and plasmid properties.
• These vectors carry, in addition to the origin of plasmid replication, an origin of
replication derived from bacteriophage.
• Unlike commonly used plasmids, phagemid vectors differ by having the ability
to be packaged into the capsid of a bacteriophage, due to their having a
genetic sequence that signals for packaging.
• Phagemids are used in a variety of biotechnology applications; for example,
they can be used in a molecular biology technique called “phage display”
• A phagemid (plasmid + phage) is a plasmid that contains an f1 origin of
replication from an f1 phage.
• It can be used as a type of cloning vector in combination with filamentous
phage M13. A phagemid can be replicated as a plasmid, and also be packaged
as single stranded DNA in viral particles.
• Phagemids contain an origin of replication (ori) for double stranded replication,
as well as an f1 ori to enable single stranded replication and packaging into
phage particles.
• Many commonly used plasmids contain an f1 ori and are thus phagemids.
• Similarly to a plasmid, a phagemid can be used to clone DNA fragments and be introduced into a bacterial host by a range of techniques, such as transformation and
electroporation.
• However, infection of a bacterial host containing a phagemid with a ‘helper’ phage, for example VCSM13 or M13K07, provides the necessary viral components to
enable single stranded DNA replication and packaging of the phagemid DNA into phage particles.
• The ‘helper’ phage infects the bacterial host by first attaching to the host cell’s pilus and then, after attachment, transporting the phage genome into the cytoplasm
of the host cell.
• Inside the cell, the phage genome triggers production of single stranded phagemid DNA in the cytoplasm. This phagemid DNA is then packaged into phage particles.
• The phage particles containing ssDNA are released from the bacterial host cell into the extracellular environment.
YEAST ARTIFICIAL CHROMOSOME
 YEAST ARTIFICIAL CHROMOSOME

• Yeast artificial chromosomes (YACs) are genetically engineered

chromosomes derived from the DNA of the yeast.
•  It is a human-engineered DNA molecule used to clone DNA sequences
in yeast cells.  They are the products of a recombinant DNA cloning
methodology to isolate and propagate very large segments of DNA in a
yeast host.
•  By inserting large fragments of DNA, the inserted sequences can be
cloned and physically mapped using a process called chromosome
walking.
•  The amount of DNA that can be cloned into a YAC is, on average,
from 200 to 500 kb.  However, as much as 1 Mb (mega, 106) can be
 STRUCTURE
• Cloning vector consists of two copies of a yeast telomeric sequence
(telomeres are the sequences at the ends of chromosomes), a yeast
centromere, a yeast ars (an autonomously replicating sequence where
DNA replication begins),and appropriate selectable markers.
WORKING PRINCIPLE OF YEAST ARTIFICIAL
CHROMOSOMES
 The yeast artificial chromosome, which is often shortened to YAC, is an artificially
constructed system that can undergo replication. The design of a YAC allows extremely
large segments of genetic material to be inserted. Subsequent rounds of replication
produce many copies of the inserted sequence, in a genetic procedure known as cloning.
 The experimenter introduces some typical elements that are necessary for correct
replication.
 In the case of YACs, the replication origins are the centromeres and telomeres of the
yeast chromosomes, which must be inserted into the DNA being cloned.
 The constructs can be transformed in yeast Spheroplast and are then replicated there.
 In contrast to the vectors, YACs are not circular; they are made of linear DNA.
PROCESS

 YAC vector is initially propagated as circular plasmid inside bacterial host

utilizing bacterial ori sequence.
 The circular plasmid is cut at a specific site using restriction enzymes to
generate a linear chromosome with two telomere sites at terminals.
 The linear chromosome is again digested at a specific site with two arms
with different selection marker.
 The genomic insert is then ligated into YAC vector using DNA ligase enzyme.
 The recombinant vectors are transformed into yeast cells and screened for
the selection markers to obtain recombinant colonies.
 ADVANTAGES OF YEAST ARTIFICIAL
CHROMOSOMES

 Yeast artificial chromosomes (YACs) provide the largest insert capacity of any cloning system.
 Yeast expression vectors, such as YACs, YIPs (yeast integrating plasmids), and YEPs (yeast
episomal plasmids), have advantageous over bacterial artificial chromosomes (BACs). They can be
used to express eukaryotic proteins that require post-translational modification.
 A major advantage of cloning in yeast, a eukaryote, is that many sequences that are unstable,
underrepresented, or absent when cloned into prokaryotic systems, remain stable and intact in YAC
clones.
 It is possible to reintroduce YACs intact into mammalian cells where the introduced mammalian
genes are expressed and used to study the functions of genes in the context of flanking sequences.
USES OF YEAST ARTIFICIAL CHROMOSOMES

Yeast artificial chromosomes (YACs) were originally constructed in order

to study chromosome behavior in mitosis and meiosis without the
complications of manipulating and destabilizing native chromosomes.
YACs representing contiguous stretches of genomic DNA (YAC contigs)
have provided a physical map framework for the human, mouse, and
even Arabidopsis genomes.
YACs are extremely popular for those trying to analyze entire genomes.
LIMITATIONS OF YEAST ARTIFICIAL
CHROMOSOMES
 A problem encountered in constructing and using YAC libraries is that they typically contain clones that
are chimeric, i.e., contain DNA in a single clone from different locations in the genome.
 YAC clones frequently contain deletions, rearrangements, or noncontiguous pieces of the cloned DNA.
As a result, each YAC clone must be carefully analyzed to be sure that no rearrangements of the DNA
have occurred.
 The efficiency of cloning is low (about 1000 clones are obtained per microgram of vector and insert
DNA).
 YACs have been found to be less stable than BACs.
 The yield of YAC DNA isolated from a yeast clone containing a YAC is quite low.  The YAC DNA is only a
few percents of the total DNA in the recombinant yeast cell. It is difficult to obtain even 1 μg of YAC DNA.
 The cloning of YACs is too complicated to be carried out by a lone researcher.
BACTERIAL ARTIFICIAL CHROMOSOME
• .  BAC vectors are plasmids constructed with the replication origin of E. coli F
factor, and so can be maintained in a single copy per cell.
• These vectors can hold DNA fragments of up to 300 kb
•  Recombinant BACs are introduced into E. coli by electroportation
•  Once in the cell, the rBAC replicates like an F factor
 CHARACTERISTIC FEATURES OF BAC
VECTORS
•  The original BAC vector, pBAC108L, is based on a mini-F plasmid, pMBO131 (Figure 1) which
encodes genes essential for self-replication and regulates its copy number inside a cell. The
unidirectional self-replicating genes are oriS and repE while parA and parB maintain copy number to
one or two for each E. coli genome.
•  Multiple cloning sites is present, flanked by “universal promoters” T7 and SP6, all flanked by GC-rich
restriction enzyme sites for insert excision.
•  Presence of cosN and loxP sites(cloned in by bacteriophage l terminase and P1 Cre recombinase,
respectively) permits linearization of the plasmid for convenient restriction mapping.
•  There is a chloramphenicol resistance gene for negative selection of non- transformed bacteria.
•  Vector is 6900 bp in length and is capable of maintaining insert DNA in excess of 300 kilobases (kb)
 DEVELOPMENT OF BAC VECTOR

• Bacterial plasmid is cut within lacZ gene with restriction enzymes.

• Gene of Interest is cut out using restriction enzyme.
• F+ Plasmid and gene of interest ligate.
• BAC is electroporated into E.coli cells.
• Cells are plated on medium.
• White colonies are composed of transformed lacZ cells.
 ADVANTAGES OF BAC VECTORS

•  The large size of BACs help to minimize site of integration effects, a phenomenon which
has been defined as endogenous sequences (such as gene coding regions and distal
regulatory elements) to be disrupted, and to produce potentially undesirable phenotypes in
gene cloning technology.
•  Endogenous gene expression more accurately than other cloning systems.
•  The human genome BACs consist of the full gene structure(which play very important role
in gene regulation). Therefore the human genome BACs will ensure full mRNA processing
and splicing when genes are transcribed, and produce the full complement of protein
isoforms once mRNAs are translated.
• It can be transfected and expressed in mammalian cell lines even if transfection efficiency
and copy numbers are low.
DISADVANTAGES OF BAC VECTORS

 A construct containing a large genomic fragment is likely to contain

non-related genes which may lead to indirect, non-specific gene
expression and unanticipated changes in the cell phenotype.
 Recombinant BAC constructs can be time- consuming and labor-
intensive.
 The large size BAC DNA constructs are more easily degraded and
sheard during manipulation before transfection.
. APPLICATIONS OF BAC VECTORS

 BACs are useful for the construction of genomic libraries but their range of use is vast. It
spans from basic science to economically rewarding industrial research, and fields as prosaic
as animal husbandry.
 In genomic analyses, it helps in determining phylogenetic lineage det between species.
 Helps in study of horizontal gene transfer and since bacterial genes are usually clustered,
the ability of BAC vectors to accommodate large inserts has allowed the study of entire
bacterial pathways.
 By isolating DNA directly from soil or from marine environments, the “metagenomes” of
those organisms which are either uncultureable or are termed viable but uncultureable can be
cloned into BAC vectors and indirectly studied.
• In industrial research fields where BAC vectors are invaluable tools in
cataloguing novel genomes is in the discovery of novel enzymes. Work has been
done on identifying enzymes that are involved in biopolymer hydrolysis or even
radioactive waste management.
•  BAC vectors have been instrumental in studying large double stranded DNA viruses
both from an academic point of view and as a tool to develop improved vaccines.
•  In genomic research, high throughput determination of gains and losses of genetic
material using high resolution BAC arrays and comparative genomic hybridization
(CGH) have been developed into the new tools for translational research in solid
tumors and neurodegenerative disorders.
REFERENCES

• Biotechnology,B D Singh.