Vector construction

Prepared By :- Umang Vanani

CONSTRUCTION OF VECTOR

WHY IS A VECTOR REQUIRED ?

During cloning we transfer a particular gene which

has a desired characteristic which we want. For that, we

require a vector(vehicle) which helps us to transfer the

gene to the bacteria.

WHAT IS A VECTOR ?

A vector is a small piece of DNA, taken from a virus, a

plasmid, or the cell of a higher organism, that can be stably

maintained in an organism, and into which a foreign DNA

fragment can be inserted for cloning purposes.

TYPES OF VECTOR

1. CLONING VECTOR

2. EXPRESSION VECTOR

EXPRESSION VECTOR

The vector design for expression of production protein

specified by the DNA insert is called EXPRESSION

VECTOR.

EXPRESSION VECTOR

TWO TYPE :-

1. PROKARYOTIC EXPRESSION VECTOR

Bacterial expression system (e.g. E.coli)

2. EUKARYOTIC EXPRESSION VECTOR

Yeast expression system (e.g. S.cervesiae)

Viral expression system (e.g. Baculovirus)

Mammalian cell expression system

CLONING VECTORS

A cloning vector is a DNA molecule in which foreign DNA

can be inserted or integrated and which is further capable

of replicating within host cell to produce multiple clone of

recombinant DNA.

CLONING VECTORS

TYPES :-

● PLASMID

● BACTERIOPHAGE

● COSMID

● BACTERIAL ARTIFICIAL CHROMOSOME

● YEAST ARTIFICIAL CHROMOSOME

● HUMAN ARTIFICIAL CHROMOSOME

PROKARYOTIC EXPRESSION VECTOR

ADVANTAGES :-

● Gene expression can be easily contolled.

● Easy to grow with high yields.

● Easy to manipulate.

● product can be designed for secretion into the growth

media.

PLASMID

● Plasmid vectors can be

used for cloning of DNA

fragments of size generally

ranging from 0.1 to 10 kb.

● The most common

examples of plasmid

cloning vectors are

pUC18/19, pBluescript,

pGEX, pET series etc.

PLASMID VECTOR pBR 322

CONTAIN

● Ampicillin resistance

gene.

● Tetracycline resistance

gene.

● origin of replication.

● Eco RI site.

BACTERIOPHAGE

● Bacteriophage is a genetically

complex but very extensively

studied virus of E.coli.

● The DNA of phage, is a linear

duplex molecule of 48502 bp

(~49kb) in length.

BACTERIOPHAGE

● The DNA isolated from virus particles is a double

stranded linear molecule with short complementary

single stranded projections of 12 nucleotides its 5’ ends.

● These cohesive termini, also referred to as COS site,

allow the be DNA to be circularized after infection of the

host cell.

COSMID

● Cosmid vectors (5-7 kb) are

hybrid between plasmids and

phages. They contain antibiotic

genes and replication origin

from plasmids and ‘cos’ sites

from phages which are

necessary for packaging of

DNA.

● These vectors can clone larger

fragments of size ranging from

35 to 50 kb.

LAMBDA PHAGE

● Lambda phage can also be

used as vector for cloning

gene of interest of size

ranging from 8-25 kb

fragments.

LAMBDA PHAGE

● This is based on lambda phage genome which is 48,502

bp with a central 33% (stuffer region) which is not critical

for lytic growth and can be replaced with 8-25 kb gene

inserts e.g. gt11, charon phages, lambda ZAP vectors.

Lambda vectors can be introduced into cells at a very high

efficiency.

BACTERIAL ARTIFICIAL CHROMOSOME

● BAC vectors contain

sequences from the E. coli

F plasmid and they have

the ability to clone up to

75 - 200 kb fragments.

YEAST ARTIFICIAL CHROMOSOME

● YAC vectors contain sequences

required to replicate and

maintain chromosome in

budding yeast and contain a

yeast origin of replication, a

centromere, and a telomere at

each end.

● These vectors are able to clone

very large fragments of >2,000

kb inserts (2 Mb).

CONSTRUCTION OF VECTOR

● The first requirement for construction of vector is an

extrachromosomal self replicating circular plasmid.

● A restriction enzyme is required to cut the restriction site

where the desired gene is attached.

● A DNA Ligase is required to join the gene in plasmid at

restriction site.

VECTOR

Four common properties of vector

1. Ability to promote autonomous replication.

2. Contain a genetic marker(usually dominant) for

selection.

3. Unique restriction sites to facilitate cloning to insert

DNA.

4. Minimum amount of non-essential DNA to optimize

cloning.

ORIGIN OF REPLICATION

● Origin of replication is a DNA

segment recognized by the

cellular DNA replication

enzyme.

● Without replication origin,

DNA can not be replicated in

the cell.

SELECTIVE MARKER

● Selective marker is required for

maintenance of plasmid in the

cell.

● Because of the presence of the

selective marker, the plasmid

becomes useful for the cell.

SELECTIVE MARKER

● Under selective conditions, only cells that contain

plasmid with selectable marker can survive.

● Gene that confers resistance to various antibiotic are

used.

● Genes that make cells resistance to ampicillin,

neomycin or chloramphenicol are used.

RESTRICTION ENZYME

Mostly Endonuclease is used for cutting of particular

selected gene from the DNA and cut the same sequence in

the plasmid for the attachment.

Two types of cut is done by the enzyme “blunt cut” and

“sticky cut”.

sticky cut is very specific and unique.

INJECTION INTO CELL

● Applying small heat shock or small electric shock to the

cell creates small pores in the cell membrane.

● Through these small pores, vector is injected in the cell.

CHOICE OF EXPRESSION VECTORS

● Strong promoter

● Intact ORF

● Ribosomal binding site

● Termination sequence

CHOICE OF CLONING VECTORS

● A vector should be small and easily prepared.

● Capable of autonomous replication in the host cell.

● It should have one target site only for any restriction

enzyme(that has been used in to make DNA fragments.

● The vector should show Antibiotic resistance.

● The vector must have a marker permitting recognition.

Maximum DNA insert accommodate by

different cloning vectors…

Types Size of cloned DNA(kb)

Plasmid 20

Lambda Phage 25

Cosmid 45

BAC 300

YAC 1000

APPLICATION OF VECTOR

•The construction of expression libraries.

•The analysis of gene function at protein level.

•The commercial production of proteins.

•The production of antibodies.

•For in vivo studies of the protein.

EXPRESSION VECTOR

APPLICATION OF VECTOR

•To amplify the clones of desired DNA segment

•Allow in vitro production of RNA copies of desired DNA

insert.

•Generates single stranded copies of DNA insert

•Construction of genomic DNA libraries and cDNA libraries.

CLONING VECTOR