methods in molecular biology recombineering Ólafur s. andrésson 13th october 2005

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.