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

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

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

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

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

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)

R6K oriV R6K pir Host-dependent conditional replication

The cloning process (1)

Examples of the types of cuts made by restriction enzymes

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

pUC19, a widely used cloning vector

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

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

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

The cloning process (1)

The cloning process (2)

Transformation mechanisms

Isolation of plasmid DNA

Electrophoresis of DNA cut by restriction enzymes

Pulsed field gel electrophoresis (PFGE)

Spot (or dot) blot

Southern blot

Original DNA sequencing procedure

Sequencing using an automated DNA sequencing machine

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

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

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

Expression vector, high copy number, non-mobilizable

Cosmid vector, low copy number

Expression vector, 6xH fusion, high copy number

Lambda vector, cDNA libraries