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Methods in Molecular Biology

Methods in Molecular Biology

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Methods in Molecular Biology

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  1. Methods in Molecular Biology Recombineering Ólafur S. Andrésson 13th October 2005

  2. 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/

  3. 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.

  4. Phage recombination systems not dependent on recA: λ phage Red functions: Exo, Beta, Gam. Rac prophage functions: RecE and RecT.

  5. 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.

  6. λ phage Red functions: Exo, Beta, Gam. Gaminhibits 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.

  7. λ Exo: 5´to 3´dsDNA-dependent exonuclease. λ Beta: ssDNA-binding and anneals complementary strands.

  8. In vivo cloning by Gap-Repair in E. coli. Originally done in recBC sbC strains – enhanced by Red or RecET (Rac) systems.

  9. Cloning PKS orfs – removal of introns 6-MSAS gene P. patulum chromosome stýrill 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.

  10. Comparison of standard genetic Engineering and recombineering.

  11. Subcloning from BACs by gap-repair

  12. Recombination of ssDNA needs only Beta

  13. Model for RecA- independent recombination of dsDNA at replication fork.

  14. Model for RecA-independent recombination of dsDNA cassette: Two replication forks.

  15. Retrieval / cloning by gap-repair

  16. GalK allows 1) selection and 2) counter- selection. 1) ∆galK host 2) DOG 2-deoxygalactose selects against GalK+ cells

  17. BAC trimming using GalK system

  18. Selective marker can be targeted to any sequence in E. coli by recombineering.

  19. 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.