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