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Assay by UT-Tokyo iGEM 2014

Figure 1 GFP fluorescence reporting the activities of various combinations of sigma factors and promoters. GFP was activated by 488 nm excitation laser and the activity was measured by 514 nm emission laser. Error bar shows average ± SE. N = 3.

As seen in Figure 1, ecf20 and ecf11 activated only their corresponding promoters. Leak from the promoters was also not observed. The result of the sample whose sigma factor was not induced by arabinose (ara-), which had little difference from the one induced by arabinose (ara+), may be caused by the leak of pBAD (BBa_I13453). The amount of the sigma factor leaked from pBAD may be over the switch-point of sigmoid curve indicating relation between sigma factor and its corresponding promoter. Thus, even if the amount of sigma factor changes by arabinose induction, corresponding activity of promoter may not change.

[http://2016.igem.org/Team:UT-Tokyo/Project#intro UT-Tokyo iGEM 2016]

In our project the concentration of sigma factors must be tuned so that while it must be high enough to activate the sigma promoter activity, it should not be too high that it cannot be repressed by anti sigma factors.

The approach that was taken in 2014 to tune the expression of ECF sigma factors was to tune the promoter that transcribes the genes coding for the ECF sigma factors. However, they reported that due to the possible leak from the PBAD promoter, the sigma expression could not be tuned appropriately.

We thus decided to test constitutive promoters of different strengths for generating the sigma factors and as well take another approach to tuning the induction of the sigma promoters, that is, by expressing the sigma factors on a plasmids of different copy number.

Measurement of sigma 11 promoter activity under different sigma11 generating conditions.Strong promoter refers to BBa_J23101, while weak promoter refers to BBa_J23109. High copy plasmid refers to pSB1C3 (copy number of 100-300 per cell) and low copy plasmid refers to pSB3K3 (copy number of 10-12 per cell). Graph illustrates that there is a very significant difference( ** indicates P value 0.001 to 0.01) between different conditions. Data represents an average of 3 samples. Error bars show standard deviation.

The results above indicate that it is possible to tune the expression of the sigma 11 promoter by tuning levels of sigma11. The first approach is to change the expression level of promoter generating the sigma factors, and the second approach is to change the copy number of plasmid in which the sigma factors are expressed.

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