Synthetic construction of vectors

Synthetic construction of vectors
Introduction, enzymes in genetic engineering, linkers, adaptors...
Construction of pBR322
pBR322 derivatives (pUC series)
Modular vectors (pZ series, BioBricks and pSEVA)
Yeast artificial chromosomes
Pseudomonas putida superbug

1. Synthetic construction of vectors Darshan

2. Contents Introduction Components of vectors Tools in synthetic construction pBR322 construction pBR322 derivatives Modular vectors Superbug Cosmids YAC

3. Introduction Naturally occurring plasmids- phenotypes…. ColE1 and RSF1010 Limitations: no. of selection markers and recognition sites for restriction enzymes pBR322

4. Components of vectors Ori Recognized by the cellular replication machinery Define the number of copies of a given plasmid in the cell

5. Selection marker Any gene allowing a selective advantage to the positive transformants Antibiotic resistance Auxotrophy , etc.. Multiple cloning site (MCS) To facilitate cloning of desired DNA, containing several sites recognized by different restriction enzymes

6. Promoters To drive transcription of vector’s transgene as well as other genes that encode certain phenotypic characteristics Ribosome binding sites Termination sequence

7. Ideal vector Small size (low mol wt.) Having multiple selection markers Having unique sites for a large number of restriction enzymes, preferably genes with readily scorable phenotypes Copy no- cloning Promoters- expression

8. Tools in synthetic construction Enzymes Restriction enzymes ( typeII being used the most) Polymerases Ligases

9. Linkers Blunt ligation Contain sites for one or more REs Can be ligated to both ends of DNA and then digested

10. Adaptors Preformed cohesive ends

11. pBR322 construction Why construction? Natural plasmids have limited genetic markers for selecting transformants Contains the Ap R and Tc R genes of RSF1010 and pSC101, respectively combined with replication elements of pMB1

12. R7268 Ap R R1 drd 19 Ap R Cm R Sm R Su R Km R pMB1 pMB3 Ap R ColE1 pSF2124 Ap R pSC101 Tc R pMB9 Tc R pBR312 Ap R Tc R pMB8 pBR313 Ap R Tc R pBR322 Ap R Tc R 1 5 2 8 7 4 3 6 6

13. pBR322 derivatives To fulfill special purpose cloning needs Early works- insertion of additional unique restriction sites and selectable markers Eg . Cm R in pBR325 with unique EcoRI site

14. pUC plasmids Incorporate a DNA sequence that permits rapid visualization of DNA insert MCS in lacZ (encodes promoter and α -peptide to mediate complementation) pUC7 is descended from pBR322

15. Modular vectors

16. Superbug

17. Cosmids Plasmids that can be packaged into bacteriophage λ particles

18. YAC construction