Vectors in recombinant dna technology pBR322

biology ans features of vectors used in rdna technology ,artificial vectors,pBR322, puc plasmids

1. TYPES, BIOLOGY AND SALIENT FEATURES OF VECTORS IN RECOMBINANT DNA TECHNOLOGY

2. contents Introduction Plasmids Types-Natural and Artificial plasmids pB R 322 as cloning vector

3. Introduction   vectors should have following characteristics: Capable of replicating inside the host. Have compatible restriction site for insertion of DNA molecule (insert). Capable of autonomous replication inside the host ( ori site). Smaller in size and able to incorporate larger insert size. Have a selectable marker for screening of recombinant organism.

4. Plasmids : Plasmids are naturally occurring extra chromosomal double-stranded circular DNA molecules which can autonomously replicate inside bacterial cells. Plasmids range in size from about 1.0 kb to over 250 kb.

5. Types of Plasmids   Resistance or R plasmids . Eg : RP4 plasmid, which is commonly found in Pseudomonas and in many other bacteria. Fertility or F plasmids . Eg : F plasmid of E. coli . Col plasmids . Eg : ColE1 of E. coli . Degradative plasmids . Eg TOL of Pseudomonas putida . Virulence plasmids Eg : Ti plasmids of Agrobacterium tumefaciens   Cryptic Plasmids

6. Based on the origin or source of plasmids, they have been divided into two major classes: such as Natural plasmids : They occur naturally in prokaryotes or eukaryotes. Example : ColE1. Artificial plasmids : They are constructed in-vitro by re-combining selected segments of two or more other plasmids (natural or artificial). Example: pBR322

7. Natural Plasmids RSF1010 It is a naturally occurring plasmid isolated from E.coli K-12. This plasmid has broad host range in gram negative bacteria. ColE1 It is a naturally occurring multicopy plasmid obtained from E.coli ( copy number is around 40). The size of this natural plasmid is 6646bp.

8. Properties of an ideal vector A good vector should have the following characteristics : Autonomously replicating i.e. should have ori (origin of replication) region. Contain at least one selectable marker e. g . gene for antibiotic resistance . May contain a scorable marker (β- galactosidase , green fluorescent protein etc.) Presence of unique restriction enzyme site. Have multiple cloning sites. Preferably small in size and easy to handle. Relaxed control of replication to obtain multiple copies. Presence of appropriate regulatory elements for expression of foreign gene. High copy number

9. Artificial Plasmids Artificial plasmids vectors are classified into two broad types based on their use : Cloning vector Eg . pUC18 , pUC19, pBluescript vectors etc Expression vector shuttle vector

10. Important features of a cloning vector used to carry DNA molecules are as follows   Stability in host cell Ability to control their own replication Small size Multiple cloning sites Should not be transferred by conjugation Selectable make gene

11. pBR322 created in 1977 in the laboratory of Herbert Boyer at the University of California San Francisco . The p stands for " plasmid " and BR for " Bolivar " and " Rodriguez ", researchers who constructed it. 4361 base pairs in length. Plasmid pBR322

12. How pBR322 acts as cloning vector Preparation of vector DNA Preparation of DNA to be cloned Creation of recombinant DNA by ligation Introduction of recombinant DNA into host organism

13. Selection of host cells/ organism containing vector sequences

14. pUC plasmids: pUC plasmids are small, high copy number plasmids of size 2686bp .