Cloning vectors

What is a cloning vector?

• Vectors
– Cloning vehicles
– Enabled the replication of cloned fragments
• Cloning vector
– Is a small piece of DNA. Taken from a virus,
bacteria or cell of higher organism
– Stably maintained in the host
– Caries the desired gene of interest
 Features
1. Cloning site
• Point where a gene is inserted or removed
• Multiple cloning site (MCS)
• Restriction site
– That generate same overhangs in vector and DGI
2. Selectable marker
• Used for the selection of positively
transformed cells
• Antibiotic resistance
• More than one selectable marker are also
present in a vector
– E.g. ampicillin and tetracycline
3. Reporter gene
• Are also used for the screening of the
positively transformed clones
• E.g. lacZ a fragment
– Blue-white screening
– Marker genes in frame with or flank the MCS
– Production of fusion protein
4. Expression elements
• Promoter
• Ribosomal binding site
• The desired gene is inserted into a site
– Under control of promoter
– Necessary for the gene expression in host
– The expression is inducible
– T7 and lac promoters
• For some DNA cloning these elements are not
required
– Just to construct a genomic library
– Sub-cloned into an expression vector
 Types of vectors
• Selection of appropriate vector is compulsory
• Depends on
– Size of insert
– Copy number
– Cloning method
• Various vectors are available
1. Plasmids
2. Bacteriophages
3. Cosmids
4. Yeast artificial chromosome (YAC)
5. Bacterial artificial chromosome
 Plasmids
• A double stranded DNA molecule that
replicate independent of the chromosome
• It is not required for the cell growth and
reproduction
• Extra cellular DNA
• These are the first and standard cloning
vectors
• Most commonly used vectors
• Plasmids are used to clone
– DNA insert up to 15 kb in size
• Earliest plasmid
– pBR322 plasmid
– pUC series of plasmids
• Many plasmids have high copy number
– E.g. pUC19 which has a copy number of 500-700
copies per cell
– high copy number is useful as it produces greater
yield of recombinant plasmid for subsequent
manipulation
– low-copy-number plasmids may be preferably
used in certain circumstances
– E.g. when the protein from the cloned gene is toxic
to the cells
• Plasmids may be
– conjugative/transmissible
– Conjugative plasmids mediate DNA transfer
through conjugation and therefore spread rapidly
among the bacterial cells of a population
– Non-conjugative
– Plasmids do not mediate DNA through conjugation

• Plasmids do not combine with the host genetic

material
– But stay independently
• Plasmids can also be categorized on the basis
of the copy number per cell
– Relaxed plasmids
• Multiple number of copies per cell
– Stringent plasmids
• Limited number of copies per cell

• A recombinant plasmid carrying a foreign DNA

is called as chimera
• Some phenotypic traits exhibited by the
plasmid carried genes:
– Antibiotic resistance
– Antibiotic production
– Degradation of aromatic compounds
– Sugar fermentation
– Enterotoxin production
– Heavy metal resistance
– Bacteriocin production
– Induction of plant tumors
 Functions
1. Main function is to carry antibiotic resistance
genes and spread them in whole human and
animal body
– Treatment of various diseases
2. Function to carry genes which are involved in
metabolic activities
– Helpful in digesting pollutants from the
environment
3. Capable of producing bacterial proteins

4. Able to carry certain genes that are involved

in increasing the pathogenicity of bacteria which
causes diseases like anthrax
 Types of plasmids
• Resistance plasmids
– Involved in bacterial conjugation
– Usually carry genes for antibiotic resistance or
poisons
– Code for genes which are responsible for
conjugation pili
– Conjugation pili is used for transfer of R plasmid
from the donor to the recipient strain
– Bacteria become antibiotic resistant
• Degradative plasmid
– This type is capable of degrading or digesting the
dead organic matter from dead animals or plants
– They use this in process of biosynthesis and make
energy and recycle them

• Fertility plasmid
– These plasmid carry the tra-genes----used in
process of conjugation
– Helpful in transferring genetic material between
bacteria
• Col plasmids
– These plasmids produce such type of antibiotics
which are harmful for the other bacterial strains
– By staying in the host bacterial cell
– The antibiotics produce by these plasmids are
known as colicin
• Virulence plasmids
– Carry the genes which causes diseases
– Make bacteria pathogenic
Plasmids in recombinant DNA technology
• Recombinant DNA technology make use of
plasmids for various purposes
– Drug delivery
– Make use of plasmid to insert the desired drug into
the body
– Causing antibiotic resistance ….used to kill harmful
bacteria from the body
• Recombinant DNA technology uses first time for
the insertion of human insulin
• It is also applicable for the insertion of human
growth hormone in mammalian cells of animals
 Plasmids and gene therapy
• Significant role in gene therapy
• Mostly used for the insertion of therapeutic
genes in the human diseases
– To fight against diseases
• easy to manipulate and replication in
bacterial cell in easy
• They have the ability to target the defected
cell and trigger the therapeutic ability
efficiently
• pBR322
– a plasmid and was one of the first widely used E.
coli cloning vectors
– pBR322 is completely sequenced
– pBR322 is 4361 base pairs in length
– origin of replication of plasmid pMB1
– pBR322 also contains the ampR gene
– encoding the ampicillin resistance protein (source
plasmid RSF2124)
 – the tetR gene
– encoding the tetracycline resistance protein
(source plasmid pSC101)
– the rop (repressor of primer) gene, encoding a
restrictor of plasmid copy number
• If the foreign DNA is inserted into the Amp
resistance gene it is no longer resistance to
Amp
• So the Tet resistant gene is used as marker
• The plasmid has unique restriction sites for
more than forty restriction enzymes
• 11 of these 40 sites lie within the tetR gene
• There are 2 sites for restriction
enzymes HindIII and ClaI within the
promoter of the tetR gene
• There are 6 key restriction sites inside the
ampR gene
• pBR322 is a widely used cloning vehicle
• It has been widely used as a model system for
the study of prokaryotic transcription and
translation
• To study the topological changes on DNA
confirmation
 Cosmids
• Cosmids are hybrid plasmids
– that incorporate a segment of bacteriophage λ
DNA that has the cohesive end site (cos sequence)
– cos sites + plasmid = cosmids
– which contains elements required for packaging
DNA into λ particles
– It is normally used to clone large DNA fragments
between 28 to 45 Kb
– They can replicate as plasmids if they have a
suitable origin of replication
• They also contain a gene for selection such as
antibiotic resistance
– so that the transformed cells can be identified by
plating on a medium containing the antibiotic
– Those cells which did not take up the cosmid
would be unable to grow
• They have several restriction sites
• They can be injected into the bacteria and also
they act as plasmid within a bacterial host
 Cos Sequences
• Lambda DNA contains a recognition sequence
known as Cos sequence or site at each end
• Cos sequences are ~200 base pairs long and
essential for packaging
• When the genome is to be packaged into the
capsid
• It is cleaved at one Cos site and the DNA is inserted
until the second Cos site has entered
• They contain a
– cosN site where DNA is nicked at each strand, 12
bp apart, by terminase
– This causes linearization of the circular cosmid
with two "cohesive" or "sticky ends" of 12bp
– The cosB site holds the terminase while it is
nicking and separating the strands
– The cosQ site of next cosmid is held by the
terminase after the previous cosmid has been
packaged, to prevent degradation by
cellular DNases
• With a cosmid vector of 5kb
– About 32-47 kb foreign DNA inserted
• After packaging in vitro the particle is used to
infect a suitable host
• The recombinant cosmid DNA is injected and
circularizes like phage DNA
– But replicates as normal plasmid without the
expression of phage function
• Transformed cells are selected on the basis g
the resistance marker
ApR
Restriction endonuclease -----------------------------------
target site
Foreign DNA restriction
fragment
Cos site
v

Restriction endonuclease

DNA ligase

v v ----------------
-----------------------------------
Cos site
Cos site
 Packaging in vitro

Transducing particles Absorption + injection

containing cosmid
recombinant DNA
circularizes
after
injection

v
• Cosmids are efficient means of cloning large
DNA fragments
• Because of their capacity for large fragments
of DNA
– Cosmids are particularly used for constructing
libraries of eukaryotic genome fragments
– Partial digestion with a restriction endonuclease
provides suitably large fragments but this is
problematic
• Because two genome fragments may ligate together in
ligation reaction