Gene Gorging Marker Mutations

For competitive fitness assays, one must be able to distinguish two E. coli subpopulations in a mixed culture. One way this can be accomplished is by mutating genes required for sugar fermentation to create a genetic marker (Ara, Mal, Lac, etc.). Plating a version of these strains will produce red colonies and plating the + version will produce pinkish white colonies on tetrazolium indicator agar containing a rich media base plus the sugar of interest (TA, TM, etc).

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

For a more detailed description of the Ara genetic marker, see also: Ara Marker.

Table of Contents

Materials

Plasmid Marker
pJEB11 →Ara
pJEB12 →Ara+
pJEB15 →Mal

  • Antibiotic stock solutions of Chloramphenicol (Cam) and Kanamycin (Kan).
    • Final concentrations of antibiotics in all media (liquid or plates) are 25 g/ml Cam and 50 g/ml Kan.
  • LB and TA plates with antibiotic. You will need LB + Cam + Kan, TA + Cam, TA + Kan, 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).
    • Typically this is a pCR3.1-derived plasmid generated by TOPO cloning a ~1000 bp DNA fragment PCR amplified from a strain containing the marker mutation.

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 l of pACBSR and 1 l 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 l of room temperature SOC medium.
  6. Grow at 37C for 1 hr in a shaking incubator to induce antiobiotic expression.
  7. Plate 100 l and 10 l + 90 l saline on two separate LB + Cam + Kan plates.
  8. Grow plates overnight at 37C.

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 g/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 37C, 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 l 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 l of a 104 dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 g/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 l of a 102 dilution on minimal arabinose (MA) or minimal maltose (MM).
  3. Grow plates overnight at 37C.

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 37C, shaking at 120 rpm.
  3. Grow plates overnight at 37C.

Day 5: Plate to Single Colonies

  1. Plate 100 l 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 l transfers through dilution tubes containing 10 ml of saline.
  2. Grow plates overnight at 37C.

Day 6: Patch for Plasmid Loss

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

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 37C 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.


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Topic revision: r10 - 20 Feb 2013 - 02:17:59 - Main.JeffreyBarrick