methods in molecular biology recombineering Ólafur s. andrésson 13th october 2005
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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 recombinationtechnology 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 recombinationis 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 systemsnot dependent on recA:
λ phage Red functions: Exo, Beta, Gam.
Rac prophage functions: RecE and RecT.
According to this model Red-mediated homologousrecombination should be more efficient withhomologous 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.
stýrill
P. patulum chromosome
Plasmid DNA
Amplification of fragments:
Recombination in yeast
6-MSAS gene
Cloning PKS orfs – removal of introns
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 RecA-independentrecombinationof dsDNA atreplication fork.
Model for RecA-independentrecombination of dsDNA cassette:
Two replication forks.
Retrieval / cloning by gap-repair
GalK allows1) selectionand2) counter-selection.
1) ∆galK host
2) DOG2-deoxygalactoseselects againstGalK+ cells
BAC trimming using GalK system
Selective marker can be targeted to anysequence in E. coli by recombineering.
LAB 21st October:
1) Insertion of KanR cassette at endof E. coli lig gene (encoding DNA ligase).
Producing deletion of carboxy-terminal domain of ligsase enzyme.
2) Insertion of ZeoR cassette at endof 9 kb lichen PKS gene in 15 kb plasmid.
Adding a 6Xhis tail on PKS enzyme to beable to detect protein in transformed host.