Strain & Vector for use

download Strain & Vector for use

of 14

  • date post

    22-Feb-2016
  • Category

    Documents

  • view

    31
  • download

    1

Embed Size (px)

description

Strain & Vector for use. Wei ZHENG 8th, Jul, 2008. Lambda InCh a simple E.Coli plasmid-chromosome shuttle system. Advantages. No risk of >1 copy of target gene in our machine Stable integration of a single copy of the plasmid fragment - PowerPoint PPT Presentation

Transcript of Strain & Vector for use

  • Strain & Vector for useWei ZHENG8th, Jul, 2008

  • Lambda InCha simple E.Coli plasmid-chromosome shuttle system

  • AdvantagesNo risk of >1 copy of target gene in our machineStable integration of a single copy of the plasmid fragmentMost ordinary E.Coli strains and a variety of pBR322-derived Amp-resistant plasmid can be used phage as the only specialized vector required

  • General principleRecombination of the desired plasmid-encoded genes onto lambda InCh

    Integration of the recombinant lambda InCh into the chromosome at the lambda attachment site

    Deletion of most of the lambda genes, including the att site from the chromosome of the lysogen

  • Two homologous regionNear ori at the region near pBR322 replicative origin

    A fragment of the bla gene of pBR322

  • pBR322

  • pUC18

  • How to select the lysogens? Low frequency transducing (LFT) lysate: - typically10-3 of the population are recombinant phages, having complete bla gene (Ampr)

    Heat-inducible cI857 repressor: - selection of the temperature- independent derivatives at 42oC

  • E.Coli Strains

  • E.Coli strainBBa_V1001 DH5aGenotype F-80dlacZM15 (lacZYA-argF)U169 deoR, recA1 endA1 hsdR17(rk- mk+ phoA supE44 - thi-1 gyrA96 relA1

  • Strain: BBa_V1001 DH5a

    Vector: -InCh2 - pUC18

  • More Information Gallery of InCh Homology Maps http://beck2.med.harvard.edu/resources/InCh/lambda_InCh_frames.htmInCh pagehttp://beck2.med.harvard.edu/resources/InCh/lambda_InCh_frames.htmManual