Results - Blue Light Sensor Characterization

Objective

Characterize the blue light sensor system using YF1-FixJ with its promoter FixK2. Reminder: in darkness, YF1-FixJ can bind to the FixK2 promoter and actives the downstream coding sequence whereas with blue light, YF1-FixJ cannot bind to the FixK2 promoter, impeaching transcription.

Conception

The following construction was designed:
YF1 and FixJ genes were under the control of the pTet promoter, the general inducer system to mimic the real behavior of the blue light sensor system in the E. calculus design (https://2013.igem.org/Team:INSA_Toulouse). The transformed strain was supposed to express the modified RFP while in the dark. In blue light conditions, expression was supposedly promoted, except when aTc was added in the media.

Result

We did not obtain the previous construction but managed to construct a biobrick to characterize the blue light sensor system. YF1 and Fix J were placed under the control of a weak promoter. Used parts are available here : - Promoter J23116 (http://parts.igem.org/Part:BBa_J23116) - YF1-FixJ (http://parts.igem.org/Part:BBa_K592016) - Terminator B1010 (http://parts.igem.org/Part:BBa_B1010) - FixK2 promoter (http://parts.igem.org/Part:BBa_K592006) - RFP (http://parts.igem.org/Part:BBa_K081014) The transformed strain was analyzed as follows: Several subcultures were done to ensure that transformed bacteria were placed in light/dark states for at least 3 subcultures (1000) generation. Visually, cultures in darkness didn’t show any differences with these placed in the light. Fluorescence measurements during induction for 18 hours demonstrated the same inconclusive results.

Discussion

Our interpretation of the non-expression of RFP is that the expression of YF1 and FixJ was too low to activate the FixK2 promoter. A biobrick without promoter in front of YF1 and FixJ was also submitted to the registry: YF1-FixJ-terminator-pFixK2-RFP (BBa_K1132020). Adding a strong promoter may improve the system.