6533b836fe1ef96bd12a1419

RESEARCH PRODUCT

Vanillin cell sensor

subject

description

Our project for iGEM 2006 consisted of designing a cellular vanillin biosensor. We used an EnvZ -E. coli strain as a chassis, and constructed two different devices: a sensor and an actuator, assembled using OmpR-P as a standardised mediator. The sensor device contained a computation- ally designed vanillin receptor and a synthetic two-component signal transduction protein (Trz). The receptor protein was based on a ribose-binding protein as scaffold. The Trz was built by fusion of the periplasmic and transmembrane domains of a Trg protein with an EnvZ kinase domain. When the receptor complex binds Trg, an allosteric motion is propagated to the cyto- plasmic EnvZ kinase domain, resulting in autophosphorylation and subsequent phosphate transfer to the OmpR transcription factor, which finally induces transcription of the ompC promoter. As actuator, we used a synthetic transcriptional circuit, which implements an OmpR-P band detector having GFP and RFP as an output. We designed this circuit using a synthetic promoter working as an AND gate, which is synergistically activated by cI and CRP. Our constructed Trg-EnvZ fusion and AND promoter will be very useful to future synthetic biology projects. 1 Aims of the project The main goal of this project was to introduce students to synthetic biology through the iGEM competition (1), while trying to do an interesting project that could generate parts to be reused by many future projects in synthetic biology. We chose to design a cellular biosensor having vanillin as an input and the expression of two reporters as output. To perform this, we constructed two devices: a sensor (based on a receptor and a two-component signal transductor) and an actuator (which received the intermedi- ate signal and expressed the reporters). We used a phos- phorylation mechanism from EnvZ as transmembranal pathway using OmpR-P as a standardised mediator (Fig. 1). To avoid interference with the natural pathways and have a better control of the kinase/phosphatase domain, we chose an EnvZ - E. coli strain as a chassis. The receptor domain of the sensor was inspired on the work by the group of Dr Hellinga, in which a sensor of