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ECOLITASTER

ECOLITASTER. Making E. Coli tasting flavors Valencia iGEM 2006. BioCircuit. Vanillin. PBP. PBP. CRP. P. OmpR. cI. GFP. RFP. AND. Block diagram (Simulink). Mechanism. Our sensor is a mutated PBP that docks a vanillin molecule and perform an allosteric motion like in Looger et al.

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ECOLITASTER

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  1. ECOLITASTER Making E. Coli tasting flavors Valencia iGEM 2006

  2. BioCircuit Vanillin PBP PBP CRP P OmpR cI GFP RFP AND

  3. Block diagram (Simulink)

  4. Mechanism • Our sensor is a mutated PBP that docks a vanillin molecule and perform an allosteric motion like in Looger et al. • This motion is propagated to the kinase domain resulting in autophosphorylation of the EnvZ domain and phosphate transfer to OmpR (OmpR-P). • OmpR-P regulates our three ompC-based promoters. • 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. • Thus the AND promoter is activated and GFP is expressed at intermediate concentrations of OmpR-P. • The cells’ color is: • At high vanillin concentration: red. • At intermediate: yellow. • At low: green. • No vanillin: no color.

  5. 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.

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

  7. 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).

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