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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
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
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
ColE1
pBR322, pUCs, …
high
E. coli
R6K
pUTs, pGP704, …
13-40
Pi protein (E. coli lpir)
IncQ
RSF1010, pKT230, …
10-12
Broad host range
IncP
RK2, RP4, pLAFRs, …
3-7
Broad host range
pACYC177
p15a
low
E. coli
1
E. coli
pSC101
pSC101
IncFI
F factor
ENGINEERED HOSTS FOR DESIGNED PLASMIDS
Maintenance of R6K plasmids
Biparental matings
E. coli S17-1lpir
E. coli CC118lpir
E. coli S17-1lpir
E. coli SM10lpir
Expression with the T7 promoter
E. coli BL21(DE3)
Host-dependent conditional
replication
l
R6K pir
R6K oriV
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)
DM15 lacZ
Blue/White colour in
Media with Xgal!
MULTIPLE CLONING SITE (MCS)
KpnI
ApaI
XhoI
SalI
ClaI
HindIII EcoRV EcoRI
..GGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCT..
(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