Download - Clonación y vectores plasmídicos
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