Results - Red Light Sensor Characterization

Objective

Characterize the red light sensor system using Cph8 with its promoter pOmpc. Reminder: in darkness, the EnvZ histidine kinase of Cph8 phosphorylates endogenous OmpR, a transcription factor which activates transcription from the OmpC promoter. Under red light conditions, the activity of the EnvZ histidine kinase domain is inhibited, impeaching transcription from the downstream sequence.

Conception

The following construction was designed:

In order to mimic the real behavior of the red light sensor system in the E. calculus design (https://2013.igem.org/Team:INSA_Toulouse), Cph8 gene was placed under the control of the pTet promoter, the general inducer system. The transformed strain was supposed to express the modified RFP while in the dark. In red light conditions, expression was supposedly promoted, except when aTc was added in the media.

Result

We obtain the previous construction to characterize the red light sensor system.

Used parts are available here:
- Strong promoter and strong rbs
- Ho1
- PcyA
- TetR
- pOmpC promoter
- rfp
- ptet
- Cph8

Discussion

We succeeded in cloning the construction in pSB1C3, but did not characterize it due to lack of time. The final biobrick must be used in an E. coli deficient in EnvZ. The delta EnvZ strain was obtained from the Paris Bettencourt team and has been already described in many other iGEM projects.

http://www.nature.com/nature/journal/v438/n7067/abs/nature04405.html