Clonación y vectores plasmídicos

Victor de LorenzoCentro 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 originrep: replication machinerycop: copy controlseg: segregationmob: mobilizaton sitetra: conjugation genesoriT: 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 pSC101

p15a

high

13-40

low

1

3-7

10-12

E. coli

Pi protein (E. coli pir)

E. coli

Broad host range

Broad host range

E. coli

INCOMPATIBILITY GROUPS

ENGINEERED HOSTS FOR DESIGNED PLASMIDS

E. coli S17-1pir

Biparental matings

E. coli SM10pir

E. coli CC118pir

Maintenance of R6K plasmids

E. coli S17-1pir

Expression with the T7 promoter

E. coli BL21(DE3)

R6K oriV

R6K pir

Host-dependent conditionalreplication

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 inMedia with Xgal!

..GGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCT..

KpnI ApaI XhoI SalI ClaI HindIII EcoRV EcoRI

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

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, 6xH fusion, high copy number

Lambda vector, cDNA libraries