clonación y vectores plasmídicos
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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Clonación y vectores plasmídicos
Victor de LorenzoCentro Nacional de Biotecnología
CSIC, Madrid (Spain)
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Plasmids
• Extrachromosomal DNA elements
• Circular plasmids• Linear plasmids• Megaplasmids
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PLASMIDS IN NATURE
• resistances• metabolic properties• pathogenicity island• symbiotic plasmids
• self-transmissible
• others
• ccc DNA, extrachomosomal
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GENES & SITES IN A PLASMID
oriV: replication originrep: replication machinerycop: copy controlseg: segregationmob: mobilizaton sitetra: conjugation genesoriT: origin of transfer
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PLASMIDS: APPLICATIONS
• Cloning vectors
• Gene expression
• Transposon delivery
• Helper plasmids for conjugation
• Knock-outs
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• 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
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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
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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)
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R6K oriV
R6K pir
Host-dependent conditionalreplication
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The cloning process (1)
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Examples of the types of cuts made by restriction enzymes
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General purpose cloning vector, high copy number, non-mobilizable
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pUC19, a widely used cloning vector
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Alpha-complentation of lacZ (beta-galactosidase)
M15 lacZ
Blue/White colour inMedia with Xgal!
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..GGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCT..
KpnI ApaI XhoI SalI ClaI HindIII EcoRV EcoRI
MULTIPLE CLONING SITE (MCS)
(Fragment of the MCS of pKS)
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General purpose & expression vector, high copy number, non-mobilizable
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The cloning process (1)
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The cloning process (2)
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Transformation mechanisms
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Isolation of plasmid DNA
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Electrophoresis of DNA cut by restriction enzymes
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Pulsed field gel electrophoresis (PFGE)
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Spot (or dot) blot
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Southern blot
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Original DNA sequencing procedure
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Original DNA sequencing procedure
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Sequencing using an automated DNA sequencing machine
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General purpose cloning vector, low copy number, non-mobilizable
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General purpose cloning vector, low copy number, non-mobilizable
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General purpose cloning vector, high copy number, non-mobilizable
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Cloning vector for PCR products, high copy number, non-mobilizable
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Expression vector, high copy number, non-mobilizable
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Cosmid vector, low copy number
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Expression vector, 6xH fusion, high copy number
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Lambda vector, cDNA libraries