PRESENTED TO PROF.

RITA SINGH

PRESENTED BY GEETESH M.Sc BIOTECH 1

INTRODUCTION
A vector is a DNA moleculethat has ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned (DNA insert)is inserted for cloning.
A vector must have an origin of DNA replication that function in the host cell. Any extra chromosomal small genome e.g plasmid, phage or virus may be used as vector.

 Cloning vectors: to clone a gene in a vector Expression vectors: allowing the exogenous DNA to be Integration vectors:

TYPES OF VECTORS

inserted, stored, and manipulated mainly at DNA level allowing the exogenous DNA to be inserted, stored, and expressed

 It should be able to replicate autonomously.

PROPERTIES OF VECTORS

A vector should be ideally less than 10 kb in

size. The vector should be easy to isolate and purify. It should be easily introduced into the host cell The vector should have suitable marker genes. A vector should contain unique target sites

TYPES OF CLONING VECTOR

PLASMIDS
COSMID BACTERIOPHAGE

PHAGEMID
PHASMID SHUTTLE

PLASMID VECTOR
1. autonomously replicating independent of hosts

genome.

2. Easily to be isolated from the host cell. (Plasmid

preparation)

3. Selective markers: Selection of cells

(1) Containing vector: one marker is enough (2) Containing ligation products: # twin antibiotic resistance # blue-white screening

4. Contains a multiple cloning site (MCS)

Screening by insertional inactivation of a resistance gene

Ampr pBR322

Tcr

B B

B Tcr

ori

Ampr

X B

Ampr

ori
Ampicillin resistant?

ori

yes

yes yes

Tetracycline resistant? No

Replica plating: transfer of the colonies from one plate to

another using absorbent pad or Velvet

transfer of colonies

+ampicillin

+ ampicillin + tetracycline

these colonies have bacteria with recombinant plasmid

Screening by insertional inactivation of the lacZ gene

Blue white screening

Lac promoter
MCS (Multiple cloning sites)

Ampr

pUC18 (3 kb) ori

lacZ

The insertion of a DNA fragment interrupts the ORF of lacZ gene, resulting in non-functional gene product that can not digest its substrate xgal.

lacZ encode enzyme b-galactosidase lac promoter IPTG X-gal

(substrate of the enzyme)

Blue product

The expression of active b-galactosidase has to be vector dependent for the selection purpose
lacZ: a shortened derivative of lacZ, encoding N-terminal a-peptide of b-galactosidase. Host strain for vectors containing lacZ: contains a mutant gene encoding only the Cterminal portion of b-galactosidase which can then complement the a-peptide to produce the active enzyme

Recreated vector: blue transformants Recombinant plasmid: containing inserted DNA: white transformants
Recreated vector (no insert)

Recombinant plasmid (contain insert)