"
Team:NTNU Trondheim/stress-sensor
From 2011.igem.org
(→Stress sensor characterization) |
(→Stress sensor characterization) |
||
| Line 6: | Line 6: | ||
| - | To test our [https://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, LB+IPTG, M9 and M9+IPTG. M9 is a minimal medium, lacking amino-acids. M9 was used because ppGpp is mainly produced in the stringent response during amino-acid starvation. | + | To test our [https://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, LB+IPTG, M9 and M9+IPTG. IPTG will induce pLac, by inhibiting lacI's inhibition. M9 is a minimal medium, lacking amino-acids. M9 was used because ppGpp is mainly produced in the stringent response during amino-acid starvation. |
| Line 13: | Line 13: | ||
[[File:Stress-sensorM9.jpg]] | [[File:Stress-sensorM9.jpg]] | ||
| + | |||
| + | |||
| + | As shown in figure 1, the cells do produce a substantial amount of mCherry even when they are not stressed (LB, and LB+IPTG). This is possibly due to the rrnB P1 promoter not being strong enough to produce sufficient amounts of lacI to inhibit pLac's expression of mCherry. | ||
| + | |||
| + | 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. | ||
| + | |||
| + | |||
{{:Team:NTNU_Trondheim/NTNU_footer}} | {{:Team:NTNU_Trondheim/NTNU_footer}} | ||
Revision as of 11:22, 24 August 2011
Stress sensor characterization
To test our 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, LB+IPTG, M9 and M9+IPTG. IPTG will induce pLac, by inhibiting lacI's inhibition. M9 is a minimal medium, lacking amino-acids. M9 was used because ppGpp is mainly produced in the stringent response during amino-acid starvation.
Cultures were grown in flasks in a shaking incubator at 37C for 3,5 hours, and 3 parallells 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.
As shown in figure 1, the cells do produce a substantial amount of mCherry even when they are not stressed (LB, and LB+IPTG). This is possibly due to the rrnB P1 promoter not being strong enough to produce sufficient amounts of lacI to inhibit pLac's expression of mCherry.
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.
