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Team:XMU-China/Result
From 2011.igem.org
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summary
Lux pR strength testing devices with mutation at position 3,5 and 3/5
Four lux pR strength testing devices (BBa_K658016 BBa_K658017 BBa_K658018 BBa_K658019) were first cloned into plasmid pSB1A2 respectively, followed by transformation into E.coli strain BL21. Fluorescence was measured when cell growth reached a steady state (around 20h). The results are shown in following figures:
Figure 4, figure 5 and figure 6 illustrate that mutated promoters lux pR-3 (BBa_K658006) and lux R-5 (BBa_K658007) dramatically increased the fluorescence intensity at steady state compared with wild type promoter lux pR (R0062), while mutated promoter lux pR-3/5 (BBa_K658008) gave an even weaker expression of GFP than promoter lux pR (R0062). It might be explained that the mutagenesis at position 3 and position 5 of the sequence of lux pR (R0062) changed the binding strength between promoter lux pR and protein luxR.
As is shown in figure 5, promoter lux pR-3 has the highest strength of the four. Mutation at position 3 might lower the threshold for the binding reaction between LuxR/AHL protein complex and promoter lux pR, which starts the Quorum Sensing system at a relatively earlier period with a lower cell density compared with circuits regulated by wild type promoter lux pR (BBa_R0062). The earlier the QS system is started, the more GFP might be produced, leading to a higher fluorescence intensity at steady state.
population-control devices with RBS of different strength
The performance of iccdB0.6 was tested by measuring its steady-state cell density and comparing it with other population control devices.
population-control devices with mutation at position 3,5 and 3/5
Figure 3 and Figure 4 illustrate that the population-control device iccdB-3 programs a relatively lower steady-state cell density compared with iccdB0.6. This matched the result of the test on four lux pR promoters’ strength in our IR-GFP device (BBa_K658016). The strength of lux pR promoters were defined as follows:
As is shown in figure 5, promoter lux pR-3 has the highest strength of the four. It is probable that mutation at position 3 lowers the threshold for the binding reaction between LuxR/AHL protein complex and promoter lux pR, which starts the Quorum Sensing system at a relatively earlier period with a lower cell density compared with circuits regulated by wild type promoter lux pR (BBa_R0062).
Once the QS system is started, downstream killer protein expresses. The viable cell density reaches a steady state when cell growth rate equals to its death rate. Generally, steady-state cell density seems to fluctuate at the cell density when QS is started. Thus, the higher strength a promoter has, the earlier the population-control device is started, leading to a lower steady-state cell density.
The earlier the QS system is started, the more GFP might be produced, leading to a higher fluorescence intensity at steady state.
iccdB->GFP
