Transcript Slide 1

Methods in Molecular Biology
Recombineering
Ólafur S. Andrésson 13th October 2005
Recombineering:
Engineered homologous recombination
of PCR products or oligos in E. coli.
Analogous to homologous recombination
technology in S. cerevisiae and S. pombe.
References (read!): Court, DL, Sawitzke, JA,Thomason, LC.
2002. Genetic engineering using homologous recombination.
Ann. Rev. Genet. 36:361-388.
And chapter from Current Protocols in Molecular Biology.
See also: http://recombineering.ncifcrf.gov/
E. coli homologous recombination
is dependent on recA.
RecBCD generates 3´single strand overhangs.
RecA binds ssDNA and mediated strand invasion.
RecF-pathway similar but more complex.
Acts primarily at replication forks.
Phage recombination systems
not dependent on recA:
λ phage Red functions: Exo, Beta, Gam.
Rac prophage functions: RecE and RecT.
According to this model Red-mediated homologous
recombination should be more efficient with
homologous ends than with end + linear DNA.
Does not appear to be much of a hindrance –
we have used Red system to recombine into plasmid.
λ phage Red functions: Exo, Beta, Gam.
Gam inhibits two nucleases, RecBCD and SbcCD both
involved in double-srand break dependent recombination.
RecBCD and SbcCD destroy linear dsDNA.
Coordinate expression of Exo, Beta and Gam.
λ Exo: 5´to 3´dsDNA-dependent exonuclease.
λ Beta: ssDNA-binding and anneals
complementary strands.
In vivo cloning by Gap-Repair in E. coli.
Originally done in recBC sbC strains –
enhanced by Red or RecET (Rac) systems.
Cloning PKS orfs – removal of introns
6-MSAS gene
P. patulum chromosome
stýril
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Amplification of fragments:
Recombination in yeast
Plasmid DNA
Amplification performed with Taq or Dynazyme with Pfu-Ultra
Has been used to assemble 9-10 kb PKS genes
and remove 5-6 introns at the same time.
Comparison of standard genetic
Engineering and recombineering.
Subcloning from BACs by gap-repair
Recombination of ssDNA needs only Beta
Model for RecAindependent
recombination
of dsDNA at
replication fork.
Model for RecAindependent
recombination of
dsDNA
cassette:
Two replication forks.
Retrieval / cloning by gap-repair
GalK allows
1) selection
and
2) counterselection.
1) ∆galK host
2) DOG
2-deoxygalactose
selects against
GalK+ cells
BAC trimming using GalK system
Selective marker can be targeted to any
sequence in E. coli by recombineering.
LAB 21st October:
1) Insertion of KanR cassette at end
of E. coli lig gene (encoding DNA ligase).
Producing deletion of carboxy-terminal
domain of ligsase enzyme.
2) Insertion of ZeoR cassette at end
of 9 kb lichen PKS gene in 15 kb plasmid.
Adding a 6Xhis tail on PKS enzyme to be
able to detect protein in transformed host.