Composite

Part:BBa_K3747204:Experience

Designed by: Riemer van der Vliet   Group: iGEM21_Wageningen_UR   (2021-10-21)


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Applications of BBa_K3747204

This biobrick can be used to make an AHL producing strain. The strain produces AHL which binds to LuxR. Upon high cell densities, the concentration of AHL increases, repressing the lux pL promoter. Upon downregulation, less GFP is produced which can be measured using plate reader experiments.

GFP is under control of the lux pL promoter, but here luxI is produced under control of the lux pR promoter. Therefore, in high cell densities, the production of GFP should be downregulated, also when no AHL is added to the medium.

Figure 1 Fluorescence of the reporter-pL-GFP and positive control-pL-GFP strain after growing for 11 hours in 25% AHL rich medium (blue), simulating high cell density or without AHL (green) medium, simulating a low cell density. The fluorescence is corrected by the OD and normalized for the fluorescence of the strain where 0% AHL medium is added.

The results show that conditioned medium rich in AHL represses the production of GFP in the reporter strain. The positive control strain did not show repressed production of GFP in fresh medium compared to AHL rich medium as it produces AHL itself.

Protocol plate reader experiments

Day 1:

  • Make a preculture of a AHL producing strain in LB.

Day 2:

  • Make precultures of the positive control (link naar biobrick PC-pR-GFP), reporter and wild type strain in LB.
  • Wash the preculture of day 1 in the media that will be used for the plate reader experiment and make a new preculture in that medium. As initially M9 media gave growth issues, we used 80% M9 (supplemented with 0.2% glucose) and 20% LB.

Day 3:

  • Spin off the preculture of the posivite control in 80% M9 medium and filtersterilze the supernatant. Spike the medium with 0.2% glucose. This conditioned medium (CM) is rich in AHL. Dilutions of the CM medium were made by adding fresh 80% M9 + 20% LB medium.
  • Wash the LB precultures of the positive control, reporter and wild type strain in 80% M9 + 20% LB medium.
  • A 96 wells plate was prepared with the three strains (inoculated with OD=0.5) in triplicates and with different dilutions of the CM. Blanks were used to correct the OD for background absorption. The fluorescence of the positive control and reporter strain was corrected by substracting the fluorescence of the wildtype strain.

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