Derivatives of pBR322

This presentation is about construction of plasmid vector pBR322 and it's different derivatives.

1. DERIVATIVES OF pBR322 By Ankur Sharma

2. Vector pBR322 This artificial vector was constructed by Bolivar and Rodriguez in 1977 . It is one of the most popular and commercially used early purpose built plasmid based vector.

3. Its size is 4361bp(Watson 1988) Contain two antibiotic resistance genes: – Ampicillin resistance gene ( Ap R ) Tetracycline resistance gene ( Tc R ) These genes were taken from plasmid RSF2124 and pSC101 respectively. Origin of replication from plasmid pMB1(ColE1 like plasmid) Basics

4. Its total genome has been sequenced in 1979. It contain unique restriction sites for >40 different restriction endonucleases . Out of these, 11 are present within tetracycline resistant gene( Tc R ) two for Cla I and Hind III within the promoter of that gene. Ampicillin resistant Ap R gene contain unique cleavage sites for 6 restriction enzymes If any of these 19 R.E. is used for inserting the desired DNA fragment then it will result in insertional inactivation of that particular gene(either Ap or Tc ) Cont….

5. Use of any RE out of 19 different RE will result in insertional inactivation of that particular antibiotic resistant gene and it will no longer remain functional. For eg . If it is inserted in Tc R gene then the cells containing it will be resistant to ampicillin but sensitive to tetracycline. Transformed cells are selected on the basis of their Ap R and then replica plated onto a Tetracyclin containing media. Selection of transformed cells and the cells containing rDNA :-

6. Cells containing DNA insert in their Tc R gene are sensitive to it so unable to grow in its presence and only those cells containing recircularized vector will survive on this medium and the cells containing r-DNA can be recovered from master plate. Importance :- Model system for study of prokaryotic transcription and translation. Investigation of the effects of topological changes on DNA conformation. Cont….

7. L’[; pBR322 construction Plasmid R7268 (R1) was isolated Variant of this plasmid R1drd19 isolated Tansposon Tn3 was transported to pMB1 to form pMB3

8. Due to EcoR1 rearrangement formation of pMB8 (carries colicin immunity) combination of EcoR1 fragments from pSC101 with pMB8 (opened at its unique EcoR 1 site) to generate pMB9 *pMB9 code for both tetracycline resistance and colicin immunity Cont….

9. Transfer of Tn3 of R1drd19 to ColE1 to form pSF2124 Transport of Tn3 from pSF2124 to pMB9 to form pBR312 EcoR 1 rearrangement results in formation of pBR 313 Isolation of two fragments and their ligation to form pBR322 Cont….

10. k Generation of plasmid pBR322 from pBR313

11. The phenotype of plasmid pBR313 is Ap R Tc R Col imm It contain three restriction sites of R.E. Pst l , one of them is located within ampicillin resistant gene. In order to eliminate the two other restriction sites and to maintain the Ap R in functional state two new plasmid had to be constructed. These are- pBR318 pBR320 Cont ….

12. Construction of pBR318:- pBR313 was digested with Pst l E.Coli strain RRl was transformed with these three pBR313 Pst l fragments and selected for tetracycline resistance Two small fragments lost. pBR318 (tetracycline-resistant but sensitive to ampicilline )

13. Construction of pBR320 :- pBR313 was digested with Eco Rll Bacteria were transformed with unligated Eco Rll fragment of pBR313 Ampicillin resistant cells were selected and screened for tetracycline and colicin sensitivity Plasmid pBR320(containing R.S. for Pst l within amp. Resistant gene.)

14. Formation of pBR322 from pBR318 and pBR320 :- pBR318 digested with Pst l and Hpa l simultaneously Two fragments – 1.95Da and 2.2×10 6 Da Tetracycline resistance gene and a part of amp. Resistance gene. pBR320 Digested with Pst l and Hinc ll 3 fragments largest Origin of replication and remaining part of amp. Resistance gene.

15. Transformation of mixture in E.Coli strain RRl pBR322 Cont….

16. Construction of pBR327 from pBR322 :-

17. pBR322 contain 6 restriction sites for Eco Rll and a long non essential region flanked by two Eco Rll cleavage sites at position 1442 and 2502 (between origin of replication and tetracycline resistance gene) partial digestion with Eco Rll The two Eco Rll 1442(CCTGG) and 2502(CCAGG) differ so resulting non complementary ends had to be trimmed with nuclease S1 Gel electrophoresis Desired 3.3 Kb DNA fragment isolated, circularized with T4 DNA ligase transformation Cont….

18. Isolation of pBR327 Advantages :- Small size(1089bp less than pBR322) It is a EK2 vector and lacks mobilization function. Cont… .

19. pUC18/19 vector :- Size – 2.69 Kb They are identical but differ in orientation of MCS (opposite) pUC18/19 contain : 1. pMB1 replicon rep for replication Source – pBR322

20. 2. Bla gene code for beta lactamase Resistance to ampicillin High copy no. due to lack of rop gene and single point mutation in this gene. encode promoter and α - peptide of β - galactosidase 3. lacZ ’ sequence Cont….

21. i 4. MCS(Multiple Cloning Site) or Poly linker sequence Short DNA sequence (2.8Kb in pUC19) carrying R.S. for different R.E. Use of MCS increase the no. of potential cloning strategies available by extending the range of enzymes. The usual site for insertion is between initiator ATG codon and codon 7 Cont….

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23. Selection of recombinant cells For selection of recombinant cells blue-white screening is used. Colonies are cultured on medium containing X-gal (5-Bromo-4-chloro-3-indolyl- β -D- galactoside ) and IPTG is used as inducer.

24. pUC 118/119 Phagmid based ds DNA vectors which are derivatives of pUC 18/19. Size – 3162bp Constructed by insertion of intergenic region of M13 phage DNA into pUC18/19. Infection by helper phage M13K07 induce the production of vector as ssDNA .

25. Function :- Cloning, sequencing, oligo -nucleotide directed mutagenesis, strand specific probe synthesis. Host - E.coli JM103, JM105, JM110.

26. se Some other important vectors :- pAT153 :- It is a dsDNA plasmid based vector Size – 3658bp Constructed by deletion of two Hae ll fragments (positions 1644-2349) Immobilization due to deletion of bom site. High copy number – 2-4 fold greater than pBR322 and pBR327.

27. Bluescript M13 :- Size – 2.96 kb Origin of replication from bacteriophage M13 which is inserted in opposite direction. Derived from pUC plasmid having promoters for bacterophage T3 and T7 Use in generation of ssDNA (in vivo) or RNA(in vitro).

28. pGEM-3/4 :- Contain SP6 and T7 promoter flanking the cloning site. In pGEM-4 the orientation of MCS is opposite to that of pGEM-3

29. pGEM3Z :- pGEM3Z and pGEM4Z carry lac Z gene Hinc ll fragment encoding α -peptide and the same MCS that of pGEM3 and 4. The promoter for T7 and SP6 RNA Poly. Are present at opposite ends of MCS in the vector.

30. ` References :- Principles of Gene Manipulation and Genomics – S.B. Primrose and R.M. Twyman (seventh edition) From Genes to Clones – Ernst-L Winnacker A Practical Guide To Molecular Cloning – Bernard Perbal (second edition)

31. Thank you…..