Team:XMU-China/Result

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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:

File:XMU China 4beta.jpg
Figure 4 Promoters strength relative to lux pR-3 (BBa_K658006).
File:XMU China 5beta.jpg
Figure 5 Efficiency of promoter lux pR (BBa_R0062) and its three mutants.
File:XMU China 6beta.jpg
Figure 6: Fluorescence of four IR-GFP devices at 20h. 5, 3, 3/5, N represent for IR-3-GFP (BBa_K658017), IR-5-GFP (BBa_K658018), IR-3/5-GFP (BBa_K658019) and IR-GFP (BBa_K658016) respectively.

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.

File:XMU China 32.jpg
Figure 2 Experimentally measured densities of BL21’s cells without population-control circuit and BL21’s cells with four population-control circuits respectively at steady state.


File:XMU China 33.jpg
Figure 3 Experimentally measured steady-state cell density of iccdB0.6(BBa_K658003) and iccdB-3 (BBa_K658009).


File:XMU China 34.jpg
Figure 4 Average of experimentally measured cell densities of BL21’s cells with iccdB0.6 (BBa_K658003) and its mutant iccdB-3 (BBa_K658009).


population-control devices with mutation at position 3,5 and 3/5

File:XMU China 34.jpg
Figure 4 Average of experimentally measured cell densities of BL21’s cells with iccdB0.6 (BBa_K658003) and its mutant iccdB-3 (BBa_K658009).



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:

File:XMU China 35.jpg
Figure 5 Efficiency of promoter lux pR (R0062) and its 3 mutants.


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

File:XMU China 37.jpg
Figure 2 Experimentally measured fluorescence at steady state of BL21's cells with iccdB0.6-GFP(BBa_K658020) and BBa_K658021.

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