(r2) Barrick Lab :: ProtocolsGeneGorgingMarker

---+++ Gene Gorging Neutral Markers

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.

---++++ Transform Gene-Gorging and Donor Plasmid

   1 Prepare [[ProceduresMakingElectrocompetentCells][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 pJEB12 (for Ara<sup>+</sup>) or pJEB for Mal<sup>-</sup>(the _donor_ plasmid). 
   1 Plate on LB + 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.

---++++ Induce Gene-Gorging

   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.

---++++ Patch for Plasmid Loss

   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 x 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 success. 

---+++ 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: r2 - 2008-04-21 - 02:27:27 - Main.JeffreyBarrick