ProtocolsGeneGorgingMarker < Lab

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---+ 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<sup>+</sup> and Ara<sup>–</sup> versions of Escherichia coli B (REL606) and Mal<sup>-</sup> 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: [[ProtocolsAraMarker][Ara Marker]].

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---++ Materials
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| *Plasmid* | *Marker* |
| pJEB11 | &rarr;Ara<sup>–</sup> |
| pJEB12 | &rarr;Ara<sup>+</sup> |
| pJEB15 | &rarr;Mal<sup>–</sup> |
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   * Concentrations of antibiotics in all media are 34 µg/L Chloramphenicol (Cam) and ???? Kanamycin (Kan). See also [[ProtocolsAntibioticStockSolutions][Antibiotic Stock Solutions]]
   * [[ProtocolsElectrocompetentCells][Electrocompetent cells]] of the parent strain.
   * pACBSR (the _gene-gorging_ plasmid)
   * Donor Plasmid (contains desired mutation). See table.
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---++Day 1: Transform Gene-Gorging and Donor Plasmid

   1 Transform electrocompetent cells of your parent strain with 1 µl of pACBSR and 1 µl of the donor plasmid for the desired marker change.
   1 Plate on LB + Cam + Kan. 
   1 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 &lambda; Red genes from the gene-gorging plasmid. 
   1 Grow cultures overnight at 37°C, shaking at 120 rpm.

---++ Day 3: Screen or Select for Desired Mutation

   1  Make a 10<sup>4</sup>-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.
   1 If you are gene gorging the – version of the the marker you must *screen* for successful mutations. Plate 200 µl of a 10<sup>4</sup> 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.<br>
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 10<sup>2</sup> dilution on minimal arabinose (MA) or minimal maltose (MM).
   1 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
   1 Grow cultures overnight at 37°C, shaking at 120 rpm.
   1 Grow plates overnight at 37°C.

---++ Day 5: Plate to Single Colonies

   1  Plate 100 µl of a 10<sup>6</sup> 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.
   1 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 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic. 
   1 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.
   1 Grow cultures overnight at 37°C and archive 2 &times; 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.
   1 Sean Sleight's Detailed Procedure.
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Topic revision: r6 - 2010-05-08 - JeffreyBarrick