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Team:Calgary Wetware/Results
From 2008.igem.org
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| + | In the image above, the following experiment was undertaken: Is the entire Biobricked ColicinE2 operon (under control of the SOS promoter) able to produce the cytotoxic colicinE2 when induced in ''E. coli''?? Because expression of the ColE2 operon necessitates the activation of the SOS promoter (pSOS) brought on by the induction of the SOS response within ''E. coli'', this state of crisis was initiated in the cells harboring this part using the DNA cross-linking agent Mitomycin C. This agent, however is not sufficient to allow for the expression of the ColE2 genes, as RecA (induced by the SOS response) must first cleave the lexA repressors binding to the operator region of pSOS. Unfortunately, as TOP10 ''E. coli'' (Our lab's little bugger of choice!) are mutants for RecA (i.e., do not express a functional RecA protein), the BL21 strain of ''E.coli'' (producing functional RecA protein) was transformed with the ColE2 part and induced with Mytomycin C. A portion the Mytomycin C induced BL21 culture was spotted on a lawn of growing ''E. coli'' constitutively expressing RFP (for visual effect) and zones of clearing were visualized. Finally, to ensure that it was ColicinE2 that was killing the lawn of RFP-bacteria and not some remnant Mitomycin C from the induced cultures, we spotted Mitomycin C dissolved in LB onto a quadrant of the plate and saw no clearing, indicating that we were able to induce ColE2 production using Mytomycin C and that ColE2 was capable of killing ''E. coli''!! | ||
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Revision as of 03:36, 30 October 2008
| Home | The Team | The Project | Notebook | Protocols | Results | Beyond the Lab | [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Calgary_Wetware Parts] |
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The above image depicts the results obtained when testing a couple of intermediates of System #1 (i.e. AHL production by Bad Guy #1 and signal interception by our Champion Cell). Basically, we grew a lawn of our Champion cells containing an intermediate (F2620+I13500) in tandem with our Bad Guy #1 (containing the J23039 part) in order to see whether or not our culture of Bad Guy #1 was in fact producting AHL to which our champion cells could respond. A positive AHL control was used (AHL dissolved in Methanol and Ethyl Acetate). As can been seen by the very nice green halos, our Champion cells are able to respond to AHL by producing GFP (as seen in the control quadrants) and our Bad Guy #1 cells are making AHL to which our Champion cell is also responding!
In the image above, the following experiment was undertaken: Is the entire Biobricked ColicinE2 operon (under control of the SOS promoter) able to produce the cytotoxic colicinE2 when induced in E. coli?? Because expression of the ColE2 operon necessitates the activation of the SOS promoter (pSOS) brought on by the induction of the SOS response within E. coli, this state of crisis was initiated in the cells harboring this part using the DNA cross-linking agent Mitomycin C. This agent, however is not sufficient to allow for the expression of the ColE2 genes, as RecA (induced by the SOS response) must first cleave the lexA repressors binding to the operator region of pSOS. Unfortunately, as TOP10 E. coli (Our lab's little bugger of choice!) are mutants for RecA (i.e., do not express a functional RecA protein), the BL21 strain of E.coli (producing functional RecA protein) was transformed with the ColE2 part and induced with Mytomycin C. A portion the Mytomycin C induced BL21 culture was spotted on a lawn of growing E. coli constitutively expressing RFP (for visual effect) and zones of clearing were visualized. Finally, to ensure that it was ColicinE2 that was killing the lawn of RFP-bacteria and not some remnant Mitomycin C from the induced cultures, we spotted Mitomycin C dissolved in LB onto a quadrant of the plate and saw no clearing, indicating that we were able to induce ColE2 production using Mytomycin C and that ColE2 was capable of killing E. coli!!
