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Assay by UT-Tokyo iGEM 2014

Figure 1 The growth curve of the cells which express sigma factors and do not. Arabinose was added 3 hours after the inoculation. "None" means pSB1C3 plasmid without insert sequence. Error bar shows average ± SE. N = 5.

However, as seen in Figure 1, we found that ecf20 had repressive influence on growth of E. coli. This result, which did not meet that of the previous research, may be caused by the difference of strain of E.coli or the medium used in the measurement. We used MG1655 and M9 medium, while the previous researchers used DH10b and LB medium. Although we found that ecf20 repressed the growth of E. coli, the influence may not be so large as to change general behaviour of our device. In the presence of ecf20, E. coli's growth rate became slower and the maximum concentration became lower a little, but not critically.

Figure 2 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 2, 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.

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