(r1) Barrick Lab :: ProceduresGeneGorgingEvolvedAlleles

---+++ 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 + 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.

---++++ 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 (or TA/TM) 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.
Barrick Lab > ProtocolList > ProceduresGeneGorgingEvolvedAlleles
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JeffreyBarrick
Topic revision: r1 - 2008-06-17 - 20:16:08 - Main.JeffreyBarrick