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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