Clonacin y vectores plasmdicos Victor de Lorenzo Centro

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 metabolic properties pathogenicity island symbiotic plasmids • self-transmissible • others

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

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

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

INCOMPATIBILITY GROUPS Col. E 1 p. BR 322, p. UCs, … high E. coli R 6 K p. UTs, p. GP 704, … 13 -40 Pi protein (E. coli lpir) Inc. Q RSF 1010, p. KT 230, … 10 -12 Broad host range Inc. P RK 2, RP 4, p. LAFRs, … 3 -7 Broad host range p. ACYC 177 p 15 a low E. coli 1 E. coli p. SC 101 Inc. FI F factor

ENGINEERED HOSTS FOR DESIGNED PLASMIDS Maintenance of R 6 K plasmids Biparental matings E. coli S 17 -1 lpir E. coli CC 118 lpir E. coli S 17 -1 lpir E. coli SM 10 lpir Expression with the T 7 promoter E. coli BL 21(DE 3)

Host-dependent conditional replication l R 6 K pir R 6 K ori. V

The cloning process (1)

Examples of the types of cuts made by restriction enzymes

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

p. UC 19, a widely used cloning vector

Alpha-complentation of lac. Z (beta -galactosidase) DM 15 lac. Z Blue/White colour in Media with Xgal!

MULTIPLE CLONING SITE (MCS) Kpn. I Apa. I Xho. I Sal. I Cla. I Hind. III Eco. RV Eco. RI . . GGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCT. . (Fragment of the MCS of p. KS)

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

Original DNA sequencing procedure

Sequencing using an automated DNA sequencing machine

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

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, 6 x. H fusion, high copy number

Lambda vector, c. DNA libraries