Team:Groningen/project characterisation promoters hybb

From 2011.igem.org


hybB promoter

We have chosen the hybB Cold Shock Promoter (BBa_J45503) as our system's input. A cold shock seemed to be a good input method, as it is easy to cool down the cells and heat them up again, thus allowing for short impulses of lower temperature that would facilitate short-term expression needed for a functional circuit.

After cloning, we encountered problems with the activation of the PhybB promotor cloned upstream gfp, designed to measure the promoter activity (BBa_K607039). Previous teams claim that the part is functional and that it is active in temperatures below 30 degrees. This was not our experience. The construct, despite having proper sequence and being present in the cells during the measurements (done in presence of antibiotic), did not show any activity neither in low temperature nor in anaerobic conditions, which should also activate it (Richard DJ, 1999). After obtaining the negative results, we have also tested two another strains: BW25113 and TOP10. In neither of them the promoter was active.

We believe that the problems with the promoter might be a result of a wrong sequence listed in the Parts Registry. Ecocyc.org entry shows that the promoter is much shorter than the version listed in the Parts Registry. Utilizing the proper, shorter sequence might cause that it will be possible to activate the promoter.