Table of Contents

Materials

• Antibiotic stock solutions of Chloramphenicol (Cam) and Ampicillin (Amp).
  • Final concentrations of antibiotics in all media (liquid or plates) are 25 µg/ml Cam and 100 µg/ml Amp.
• LB and TA plates with antibiotic. You will need LB + Cam + Amp, TA + Cam, TA + Amp, and TA plates.
  • Note: The protocol refers to TA plates throughout. If you are not using the Ara marker, substitute the proper sugar, e.g. make TM plates if using the maltose marker.
• 20% L-Arabinose stock. Dissolve 10 g in 50 ml of ddH₂0 and filter sterilize or autoclave.
• Electrocompetent cells of the parent strain.
• pACBSR (the gene-gorging plasmid)
• Donor plasmid for desired mutation (see Table).
  • These plasmids were generated by TOPO cloning a ~1000 bp DNA fragment PCR amplified from a strain containing the marker mutation with a meganucelase I-Sce I site added in the primer on one end of the homology region (5′-TAGGGATAACAGGGTAAT) into the vector pCR3.1-derived plasmid. They are pBR322 type plasmids with AmpR.

Day 1: Transform Gene-Gorging and Donor Plasmid

1. Thaw one 1.7 ml microfuge tube of electrocompetent cells for each parent strain on ice.
2. While waiting for this to thaw, place one electroporation cuvette on ice for each parent strain.
3. Add 1 µl of pACBSR and 1 µl of the donor plasmid to each tube of electrocompetent cells on ice. Mix by flicking the tube with your finger. Do not pipette up and down or vortex to mix.
4. Electroporate
5. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 µl of room temperature SOC medium.
6. Grow at 37°C for 1 hr in a shaking incubator to induce antiobiotic expression.
7. Plate 100 µl and 10 µl + 90 µl saline on two separate LB + Cam + Amp plates.
8. Grow plates overnight at 37°C.

Day 2: 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). The arabinose is to induce expression of the λ Red genes from the gene-gorging plasmid. Use arabinose regardless of which sugar marker you are changing.
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Day 3: Screen or Select for Desired Mutation

1. Make a 10⁴-fold dilution of each overnight culture with two 100 µl transfers through dilution tubes containing 10 ml of saline. Note that the cell density achieved after induction is considerably lower than that usually achieved during growth in LB.
2. A. If you are gene gorging the – version of the the marker you must screen for successful mutations. Plate 200 µl of a 10⁴ dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 µg/ml Chloramphenicol (Cam). Successful mutants will be red.
   B. If you are gene gorging the + version of the the marker you could screen for successful mutations as above, but it is easier to select for them by plating 100 µl of a 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
3. Grow plates overnight at 37°C.

Day 4: Screen for Gene-Gorging Plasmid Loss

Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.

1. Pick one colony from each plate into 5 ml of LB medium in a test tube. Give each picked colony an isolate designation. Colonies from the same plate are not necessarily independent isolates, they may share undesired second-dite mutations. Picking individual isolated
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Day 5: Plate to Single Colonies

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

Day 6: Patch for Plasmid Loss

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with Chloramphenicol, with Ampicillin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

Day 7: 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.
2. Grow cultures overnight at 37°C and archive 2 × 1 ml frozen copies in 10% glycerol.

Test for Marker Neutrality

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.

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.
2. Sean Sleight's Detailed Procedure.