---+ Gene Gorging Marker Mutations This procedure has been used to create defined Ara<sup>+</sup> revertants of Escherichia coli B (REL606) and Mal<sup>-</sup> derivatives of Escherichia coli K12 (Keio collection strains) with the _malF_ Keio deletion. ---++ General Notes * Concentrations of antibiotics in all media are 34 µg/L Chloramphenicol (Cam) and Kanamycin (Kan). See also [[ProceduresAntibioticStockSolutions][Antibiotic Stock Solutions]] * For a more detailed description of genetic markers see also: [[ProceduresAraMarker][Ara Marker]]. ---++Transform Gene-Gorging and Donor Plasmid <div style="float: right; display:inline; margin:10px;"> %TABLE{valign="bottom"}% | *Plasmid* | *Marker* | | pJEB11 | →Ara<sup>–</sup> | | pJEB12 | →Ara<sup>+</sup> | | pJEB15 | →Mal<sup>–</sup> | </div> 1 Prepare [[ProtocolsElectrocompetentCells][electrocompetent cells]] of the strain in which you wish to change the genetic marker. 1 Transform with 1 µl of pACBSR (the _gene-gorging_ plasmid) and 1 µl of the donor plasmid for the desired marker change (see table). 1 Plate on LB + Cam + Kan. 1 Grow plates overnight at 37°C. <div style="clear:both;"></div> ---++ 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. ---++ Screen for Desired Mutation 1 Plate 200 µl of a 10<sup>4</sup> dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe) containing 20 µg/ml Chloramphenicol (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. ---++ Select for Gene-Gorging Plasmid Loss Expected results are 0-10 white (Ara+) or red (Mal-) colonies per 200 colonies of the other color. 1 Pick one colony from each plate 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 tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). 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 (or TA/TM) plates with 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic. 1 Grow plates overnight at 37°C. ---++ Save a Freezer Stock of the New Strain 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 and archive 2 × 1 ml frozen copies in 10% glycerol. Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures one success in our experience. ---++ References 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: r5 - 2010-05-07 - JeffreyBarrick