Strain & Vector for use
Wei ZHENG8th, Jul, 2008
Lambda InCha simple E.Coli plasmid-chromos
ome shuttle system
Advantages No risk of >1 copy of target gene in our machine Stable integration of a single copy of the plasmid fr
agment Most ordinary E.Coli strains and a variety of pBR3
22-derived Amp-resistant plasmid can be used λphage as the only specialized vector required
General principle
1. Recombination of the desired plasmid-encoded genes onto lambda InCh
2. Integration of the recombinant lambda InCh into the chromosome at the lambda attachment site
3. Deletion of most of the lambda genes, including the att site from the chromosome of the lysogen
Two homologous region
1. “Near ori” – at the region near pBR322 replicative origin
2. A fragment of the bla gene of pBR322
pBR322
pUC18
How to select the lysogens?
1. Low frequency transducing (LFT) lysate: - typically10-3 of the population are recomb
inant phages, having complete bla gene (Ampr)
2. Heat-inducible cI857 repressor: - selection of the temperature- independen
t derivatives at 42oC
E.Coli Strains
E.Coli strain BBa_V1001 DH5a Genotype F-φ80dlacZΔM15 Δ(lacZYA-argF)U169 deo
R, recA1 endA1 hsdR17(rk- mk+ phoA supE44 λ- thi-1 gyrA96 relA1
Strain: BBa_V1001 DH5a
Vector: -λInCh2 - pUC18
More Information Gallery of λInCh Homology Maps http://beck2.med.harvard.edu/resources/InCh/lambda_InCh_frames.htm λInCh pagehttp://beck2.med.harvard.edu/resources/InCh/lambda_InCh_frames.htm Manual
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