Vectors in recombinant dna technology pBR322
- 1. TYPES, BIOLOGY AND SALIENT FEATURES OF VECTORS IN RECOMBINANT DNA TECHNOLOGY
- 2. contents • Introduction • Plasmids • Types-Natural and Artificial plasmids • pBR322 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.