---+ Gene Gorging Evolved Alleles This procedure can be used to directly create unmarked mutations in the _E. coli_ genome. ---++++ Transform Gene-Gorging and Donor Plasmid 1 Prepare [[ProtocolsElectrocompetentCells][electrocompetent cells]] of the strain in which you wish to change the sugar marker. 1 Transform with 1 µl of pACBSR (the _gene-gorging_ plasmid) and 1 µl of pJEB11 (for Ara<sup>–</sup>), pJEB12 (for Ara<sup>+</sup>), or pJEB15 for Mal<sup>–</sup>(the _donor_ plasmid). 1 Plate on LB + 20 µg/ml Chloramphenicol (Cam) + Kan. 1 Grow plates overnight at 37°C. ---++++ Induce Gene-Gorging 1 Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 µg/ml Chloramphenicol (Cam). 1 Grow cultures overnight at 37°C, shaking at 120 rpm. ---++++ Plate Possible Recombinants to Single Colonies 1 Plate 200 µl of a 10<sup>4</sup> dilution of each culture on LB + Cam. The dilution can be made with two 100 µl transfers through dilution tubes containing 10 ml of saline. Alternately, plate 100 µl of a 10<sup>2</sup> dilution on minimal arabinose (MA) or minimal maltose (MM). 1 Grow plates overnight at 37°C. ---++++ Patch Individual Colonies 1 Patch 96 colonies for each trial of each strain on LB plates (no antibiotic) using the 96-well plate template 1 Grow plates overnight at 37°C. ---++++ Screen for Desired Mutation Perform a PCR-FLP or PCR-RFLP test for the desired mutation: 1 Pick each patch from the plates into a 96-well plate containing 100 µl of ddH<sub>2</sub>O in each well with a multichannel pipetteman. Mix by pipetting up and down several times. 1 Use 2 µl from each well as template for a 20 µl PCR reaction. 1 Load gel with the multichannel pipetteman. *Alternately, you can screen for Ara<sup>+/–</sup> or Mal<sup>+/–</sup> phenotypes by streaking on TA plates.* ---++++ Select for Gene-Gorging Plasmid Loss 1 Pick cells from the patch which passed the PCR screen into 5 ml of LB medium. Give each picked colony an isolate designation. (Colonies from the same plate are not independent isolates). 1 Grow cultures overnight at 37°C, shaking at 120 rpm. 1 Grow plates overnight at 37°C. ---++++ Plate to Single Colonies 1 Plate 100 µl of a 10<sup>6</sup> dilution of each culture on LB (no antibiotic). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline. 1 Grow plates overnight at 37°C. ---++++ Patch for Plasmid Loss 1 Patch 6-12 colonies from each plate on three LB plates with 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic. 1 Grow plates overnight at 37°C. ---++++ Grow Culture to Archive 1 Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids. 1 Grow cultures overnight at 37°C in LB (no antibiotic) and archive 2 x 1 ml frozen copies in 10% glycerol. ---+++ Sources 1 Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. _Gene_ *311*, 153-163. 1 Sean Sleight's Detailed Procedure.
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Topic revision: r4 - 2024-01-12 - JeffreyBarrick