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.

1. Plate on LB + 20 µg/ml Chloramphenicol (Cam) + Kan.
2. 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).
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Plate Possible Recombinants to Single Colonies

1. Plate 200 µl of a 10⁴ 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 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
2. 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
2. 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 ddH₂O in each well with a multichannel pipetteman. Mix by pipetting up and down several times.
2. Use 2 µl from each well as template for a 20 µl 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 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Plate to Single Colonies

1. Plate 100 µl of a 10⁶ 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.
2. 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.
2. 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.
2. 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.
2. Sean Sleight's Detailed Procedure.

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 µl of pACBSR (the gene-gorging plasmid) and 1 µl of pJEB12 (for Ara⁺) or pJEB for Mal^-(the donor plasmid).
3. Plate on LB + 20 µg/ml Chloramphenicol (Cam) + Kan.
4. 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).
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Plate Possible Recombinants to Single Colonies

1. Plate 200 µl of a 10⁴ 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 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
2. 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
2. 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 ddH₂O in each well with a multichannel pipetteman. Mix by pipetting up and down several times.
2. Use 2 µl from each well as template for a 20 µl 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 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Plate to Single Colonies

1. Plate 100 µl of a 10⁶ 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.
2. Grow plates overnight at 37°C.

Patch for Plasmid Loss

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.
2. 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.
2. Sean Sleight's Detailed Procedure.

Gene Gorging Neutral Markers

This procedure has been used to create defined Ara⁺ revertants of Escherichia coli B (REL606) and Mal^-derivatives of Escherichia coli K12 (Keio collection strains) with the_malF_ Keio deletion.

Transform Gene-Gorging and Donor Plasmid

1. Transform with 1 µl of pACBSR (the gene-gorging plasmid) and 1 µl of pJEB12 (for Ara⁺) or pJEB for Mal^-(the donor plasmid).
2. Plate on LB + 20 µg/ml Chloramphenicol (Cam) + Kan.
3. 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).
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Plate Possible Recombinants to Single Colonies

1. Plate 200 µl of a 10⁴ 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 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
2. 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
2. 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 ddH₂O in each well with a multichannel pipetteman. Mix by pipetting up and down several times.
2. Use 2 µl from each well as template for a 20 µl 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 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Plate to Single Colonies

1. Plate 100 µl of a 10⁶ 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.
2. 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.
2. 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.
2. 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.
2. Sean Sleight's Detailed Procedure.

Gene Gorging Neutral Markers

This procedure has been used to create defined Ara⁺ revertants of Escherichia coli B (REL606) and Mal^-derivatives of Escherichia coli K12 (Keio collection strains) with the_malF_ Keio deletion.

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 µl of pACBSR (the gene-gorging plasmid) and 1 µl of pJEB12 (for Ara⁺) or pJEB for Mal^-(the donor plasmid).
3. Plate on LB + 20 µg/ml Chloramphenicol (Cam) + Kan.
4. 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).
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Plate Possible Recombinants to Single Colonies

1. Plate 200 µl of a 10⁴ 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 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
2. 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
2. 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 ddH₂O in each well with a multichannel pipetteman. Mix by pipetting up and down several times.
2. Use 2 µl from each well as template for a 20 µl 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 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Plate to Single Colonies

1. Plate 100 µl of a 10⁶ 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.
2. 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.
2. 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.
2. 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.
2. Sean Sleight's Detailed Procedure.