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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

ECOLITASTER

Making E. Coli tasting flavors

Valencia iGEM 2006


Biocircuit
BioCircuit

Vanillin

PBP

PBP

CRP

P

OmpR

cI

GFP

RFP

AND



Mechanism
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.


List of parts
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
Transfer Function

Color gradient

Y (red) = RFP

Y (green) = GFP

U = OMPR-P


Conclusions
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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