2. Cloning Using Plasmid Vectors

3. Vector = a molecule used as a vehicle to carry foreign DNA into a host cell Simplest vector = plasmid

4. Features of Plasmids Size Functions encoded Structure Nomenclature: R-plasmids ColE1 Now standardised

5. Why are plasmids suitable cloning vectors? Generally do not kill host cell Relatively easy to purify Can be made small

6. Why not use naturally occurring plasmids? Too large No selectable markers Lack of unique recognition sequences for restriction enzymes

7. Replication Plasmid = replicon Requires an origin of replication (oriV) What is required?

8. Functions of the ori region Host range Narrow vs Broad Copy number

9. Features of Plasmid Cloning Vectors Contain an oriV that allows for high copy number, may have narrow (pUC) or broad (R) host ranges Small – why is this an advantage? Selectable Genes Unique restriction sites May have additional features such as mob sites, RNA polymerase promoters, etc.

10. pBR322 1973-1978 Bolivar and Rodriguez derivative 322 4.36 Kb; ~16 copies per cell Narrow host range Encodes resistance to ampicillin and to tetracycline

12. How can we tell if plasmid contains DNA of interest? Insertional inactivation Use BamHI site in Tet r gene for cloning Transform Plate cells on? Amp – why not Tet? Confirm by replica-plating on? Tet – what do you expect to see?

13. Early 1980s – pUC series

14. Features of pUC Plasmids Small Very high copy number No insertional inactivation

15. Blue-White Selection Vector contains first 146 aa of the  galactosidase gene (lacZ) (  peptide) MCS embedded within this region

16. Blue-White Selection Continued Host cell encodes carboxy terminal portion of lacZ Neither host nor plasmid encodes for entire protein Together produce enzyme that can cleave Xgal to produce blue precipitate

17. What if foreign DNA inserted into MCS? Foreign DNA will contain a termination codon in the same reading frame as the  peptide No  peptide therefore no  galactosidase and no blue coloured colonies

18. pGEM series

19. Additional Features Origin of DNA replication from ss filamentous phage such as F1 or M13 Phagemid vector T7 and SP6 promoters

20. www.promega.com

21. Variations on the pGEM theme pGEMT vectors (Hengen, 1995) Make use of unique property of Taq Facilitates easy cloning of PCR products - how? Limitations?