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Saturday, December 20, 2008

VECTORS FOR GENE CLONING

Vectors are the DNA molecules onto to which f.DNA can be cloned. There are two types of vectors
1. Cloning Vectors
2. Expression Vectors
Cloning vectors are those to propagate the f.DNA inserts.
Expression vectors are those, if the objective is to obtain the gene product (protein) from the inserted f.DNA. For expression vectors suitable regulatory sequence has to be provided, like Promoter, Repressor,Operator, Terminator, etc
Promoters are those which marks the starting on the gene which helps the binding of the RNA polymerase and there by the expression of the gene. Promoters are of two types Strong promoter & Weak promoter.

Strong Promotes- Which helps in high rate of gene expression
Weak promoter - which low level expression of the gene.

The Vectors:
Different vectors are available
examples:
pBR322:
Features
Size : 4.3kb
Markers:ampr, tetr
Restriction Sites: BamHI,HindIII, EcoRI,SalI,PstI,etc
Ori: Present

Vectors - Cloning Vehicles

Vector is a DNA molecule used as a vehicle to transfer foreign genetic material into the cell. The four major types of vectors, they are plasmids, viruses, cosmids, and artificial chromosomes.
Here are the basic features of an E.coli expression vector which is needed for the cloning and expression of recombinat protein
Vector should have the following features
  • Selectable marker : Anitibiotic resistant genes used for Screening
  • Regulatory gene (repressor)
  • Origin of replication
  • Promoter
  • Transcription terminator
  • Shine-Delgarno sequence
  • Start codon and Stop codon
  • Tags and fusion proteins
  • Protease cleavage site
  • Multiple cloning site
There are many vectors commercially available for various kinds of protein expression and stability studies, strains and vectors can be obtained from Invitrogen or promega, there are so many other manufacturers also provides the strain and vectors.
Some of the examples of E.coli vectors are
pET series from Novagen
pALTER from Promega
pCal-n from Stratagene
pBAD/His from Invitrogen, etc
To see in detail click here or visit respective manufacturers website.