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 electrocompetent cells of the strain in which you wish to change the sugar marker.
  2. Transform with 1 l of pACBSR (the gene-gorging plasmid) and 1 l of pJEB12 (for Ara+) or pJEB for Mal-(the donor plasmid).
  3. Plate on LB + 20 g/ml Chloramphenicol (Cam) + Kan.
  4. Grow plates overnight at 37C.

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 g/ml Chloramphenicol (Cam).
  2. Grow cultures overnight at 37C, shaking at 120 rpm.

Plate Possible Recombinants to Single Colonies

  1. Plate 200 l of a 104 dilution of each culture on LB + Cam. The dilution can be made with two 100 l transfers through dilution tubes containing 10 ml of saline. Alternately, plate 100 l of a 102 dilution on minimal arabinose (MA) or minimal maltose (MM).
  2. Grow plates overnight at 37C.

Patch Individual Colonies

  1. Patch 96 colonies for each trial of each strain on LB plates (no antibiotic) using the 96-well plate template
  2. Grow plates overnight at 37C.

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 l of ddH2O in each well with a multichannel pipetteman. Mix by pipetting up and down several times.
  2. Use 2 l from each well as template for a 20 l PCR reaction.
  3. 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).
  2. Grow cultures overnight at 37C, shaking at 120 rpm.
  3. Grow plates overnight at 37C.

Plate to Single Colonies

  1. Plate 100 l of a 106 dilution of each culture on LB (no antibiotic). The dilution can be made with three 100 l transfers through dilution tubes containing 10 ml of saline.
  2. Grow plates overnight at 37C.

Patch for Plasmid Loss

  1. Patch 6-12 colonies from each plate on three LB plates with 20 g/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
  2. Grow plates overnight at 37C.

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.
  2. Grow cultures overnight at 37C 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.
  2. Sean Sleight's Detailed Procedure.


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Topic revision: r3 - 22 May 2014 - 14:44:39 - Main.JeffreyBarrick