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Clonación y vectores plasmídicos

Clonación y vectores plasmídicos. Victor de Lorenzo Centro Nacional de Biotecnología CSIC, Madrid (Spain). Plasmids • Extrachromosomal DNA elements • Circular plasmids • Linear plasmids • Megaplasmids. PLASMIDS IN NATURE. • ccc DNA, extrachomosomal. • resistances.

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Clonación y vectores plasmídicos

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  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 • ccc DNA, extrachomosomal • resistances • metabolic properties • pathogenicity island • symbiotic plasmids • self-transmissible • others

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

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

  8. ENGINEERED HOSTS FOR DESIGNED PLASMIDS Maintenance of R6K plasmids Biparental matings E. coli CC118lpir E. coli S17-1lpir E. coli S17-1lpir E. coli SM10lpir Expression with the T7 promoter E. coli BL21(DE3)

  9. Host-dependent conditional replication R6K pir l R6K oriV

  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) DM15 lacZ Blue/White colour in Media with Xgal!

  15. MULTIPLE CLONING SITE (MCS) KpnI ApaI XhoI SalI ClaI HindIII EcoRV EcoRI ..GGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCT.. (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

  26. Original DNA sequencing procedure

  27. Sequencing using an automated DNA sequencing machine

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

  29. 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

  35. Lambda vector, cDNA libraries

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