1. Clonación y vectores plasmídicos Victor de Lorenzo Centro Nacional de Biotecnología CSIC, Madrid (Spain)

2. Plasmids Extrachromosomal DNA elements Circular plasmids Linear plasmids Megaplasmids

3. PLASMIDS IN NATURE resistances metabolic properties pathogenicity island symbiotic plasmids self-transmissible others ccc DNA, extrachomosomal

4. GENES & SITES IN A PLASMID oriV: replication origin rep: replication machinery cop: copy control seg: segregation mob: mobilizaton site tra: conjugation genes oriT: origin of transfer

5. PLASMIDS: APPLICATIONS Cloning vectors Gene expression Transposon delivery Helper plasmids for conjugation Knock-outs

6. High, medium, low-copy number Broad host range / shuttle vectors Suicide delivery systems Antibiotic resistances / selection markers Self-transmissible, mobilizable Multiple cloning sites CHARACTERISTICS OF PLASMID VECTORS

7. ColE1 R6K IncFI IncP IncQ pBR322, pUCs, … pUTs, pGP704, … F factor RK2, RP4, pLAFRs, … RSF1010, pKT230, … pACYC177 pSC101 p15a high 13-40 low 1 3-7 10-12 E. coli Pi protein (E. coli pir) E. coli Broad host range E. coli INCOMPATIBILITY GROUPS

8. ENGINEERED HOSTS FOR DESIGNED PLASMIDS E. coli S17-1 pir Biparental matings E. coli SM10 pir E. coli CC118 pir Maintenance of R6K plasmids E. coli S17-1 pir Expression with the T7 promoter E. coli BL21(DE3)

9. R6K oriV R6K pir Host-dependent conditional replication

10. The cloning process (1)

11. Examples of the types of cuts made by restriction enzymes

12. General purpose cloning vector, high copy number, non-mobilizable

13. pUC19, a widely used cloning vector

14. Alpha-complentation of lacZ (beta-galactosidase)  M15 lacZ Blue/White colour in Media with Xgal!

15. ..GGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCT.. KpnIApaIXhoISalIClaIHindIIIEcoRVEcoRI MULTIPLE CLONING SITE (MCS) (Fragment of the MCS of pKS)

16. General purpose & expression vector, high copy number, non-mobilizable

17. The cloning process (1)

18. The cloning process (2)

19. Transformation mechanisms

20. Isolation of plasmid DNA

21. Electrophoresis of DNA cut by restriction enzymes

22. Pulsed field gel electrophoresis (PFGE)

23. Spot (or dot) blot

24. Southern blot

25. Original DNA sequencing procedure

27. Sequencing using an automated DNA sequencing machine

28. General purpose cloning vector, low copy number, non-mobilizable

30. General purpose cloning vector, high copy number, non-mobilizable

31. Cloning vector for PCR products, high copy number, non-mobilizable

32. Expression vector, high copy number, non-mobilizable

33. Cosmid vector, low copy number

34. Expression vector, 6xH fusion, high copy number

37. Lambda vector, cDNA libraries