ECOLITASTER Making E. Coli tasting flavors Valencia iGEM 2006

BioCircuit RFP GFP cI CRP Vanillin P OmpR PBP AND PBP

Block diagram (Simulink)

Mechanism 1)Our sensor is a mutated PBP that docks a vanillin molecule and perform an allosteric motion like in Looger et al. 2)This motion is propagated to the kinase domain resulting in autophosphorylation of the EnvZ domain and phosphate transfer to OmpR (OmpR-P). 3)OmpR-P regulates our three ompC-based promoters. 4)The threshold concentration of OmpR-P that activates the promoter of the CRP gene should be lower than the one repressing the promoter of the cI gene. Therefore, at intermediate concentrations of OmpR-P, CRP and cI are expressed. 5)Thus the AND promoter is activated and GFP is expressed at intermediate concentrations of OmpR-P. 6)The cells’ color is: 1)At high vanillin concentration: red. 2)At intermediate: yellow. 3)At low: green. 4)No vanillin: no color.

List of Parts A CDS corresponding to a fusion protein between a designed periplasmic binding protein (PBP) docking vanillin and a transmembrane histidine kinase (Looger et al. Nature 2003). –Computational design of PBP domain. Make corresponding BioBrick. A promoter with two regulators (activators) using AND logic (Joung et al. Science 1994). –Part already designed. Make BioBrick. Two promoters activated by OmpR-P (i.e. ompC promoter). –The first is already a part in the Registry. –Design a mutation of OmpR-P operator to reduce binding. Promoter repressed by OmpR-P. –Graft OmpR-P operator downstream of a promoter. Mutate operator to reduce binding if necessary. Make BioBrick. Four CDS: –Coding cI part from the Registry. –Coding CRP from Joung et al. Make BioBrick. –Two reporters: GFP & RFP, they should have similar expression levels. Change codon usage in CDS to adjust their expression levels. Make BioBrick.

Transfer Function Y (red) = RFP Y (green) = GFP U = OMPR-P Color gradient

Conclusions It is a simple circuit with only 4 genes. We use well known proteins: cI, CRP, GFP, RFP. It allows to design a cool “taste” sensor (e.g. a protein that senses vanillin). E. Coli that tastes! We use a standard two-component signal transduction system part and we could use other sensors (e.g. UT Austin’s light sensor or Hellinga’s TNT sensor) if ours fails. It is a robust system. It allows to add new parts (e.g. a promoter with AND logic) to the Registry and design new ones (e.g. a promoter repressed by OmpR-P).