Part:BBa_K639000:Experience

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Applications of BBa_K639000

This brick was used as a positive control in experiments for testing rrnB P1 promoter (BBa_K639003). The promoter was inserted upstream of the brick (Link to biobrick) so that it would transcribe LacI and repress mCherry production.

To test our [http://2011.igem.org/Team:NTNU_Trondheim/rrnB+LacI+pLac+mCherry stress sensor], pre-cultures of the construct with, and without the rrnB P1 promoter were grown ON, pelleted and resuspended in M9 medium. The cultures were inoculated 1% in LB and LB+IPTG. IPTG will induce pLac, by repressing lacI's inhibition.

Cultures were grown in flasks in a shaking incubator at 37C for 3,5 hours, and 3 parallels of 100 µL from each flask were sampled to a 96 well fluorometer plate. Fluorescence was measured at ex: 584 em: 620, as well as OD600. Data from the experiment is shown in figure 1, as fluorescence divided by OD600.

Looking at the difference between samples with (+P) and without promoter (-P) in LB and LB+IPTG, it is clear that the rrnB P1 promoter does produce lacI. The fluorescence/OD value of +P in LB is lower than -P in LB, indicating production of lacI. When it is induced by IPTG, inhibiting lacI, the level rises to approximately the same as -P, indicating a nullifying effect of the lacI produced.

In future work with this construct, one could try to increase rrnB P1's strength by maybe tweaking the UTR, or try to lower pLac's strength, to give less leakage. A combination of both strategies would probably give the best result.

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