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From 2011.igem.org

Project: Creating ΔchiP, ΔchiX, ΔchiXR, ΔlacZ strain

Strain used: W3110

Sex: F- Chromosomal Markers: &lambda-, IN(rrnD-rrnE)1, rph-1

Plasmids: pSLD18, pFHC3220

Short description

To create a strain with 4 gene-knockouts we decided to use Red swap recombineering system. In order for the recombination to work, two plasmids were required: pSLD18, pFHC3220. The first one….. and the second one…… Then, a 3-day cycle of gene knock-out experiments was repeated four times to delete chip, chiX, chiXR and lacZ.

Detailed description

Week 1. PCR of DNA containing kanR and homologous regions to regions encompassing our genes of interest. Transforming E. coli with pFHC3220; Transforming E. coli with linear pieces of DNA and selecting for gene deletions - ΔchiP, ΔchiX, ΔchiXR, ΔlacZ;

Week 2. Growth of E. coli in arabinose minimal medium containing amp and ery. Selecting for loss of kan resistance after first round of knock-out