Team:Wageningen UR/Composite Part

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BBa_K2387032 is created as a means to detect activation of the Cpx pathway of E. coli. This is done using a method called Bimolecular Fluorescence Complementation (BiFC) [1].

The Cpx signal transduction system is a native system of E. coli and it is used to sense environmental stress [2]. Upon sensing of stress, regulon CpxP titrates away from transmembrane signal transducer CpxA. CpxA then autophosphorylates and this phosphogroup is transferred to response regulator CpxR. Phosphorylated CpxR can homodimerize and natively functions as a transcriptional regulator. More background information in the Cpx pathway can be found here.

We can directly visualize Cpx pathway using BiFC. eYFP (BBa_E0030) was cleaved between amino acids 154 and 155 and we fused these N- and C-termini of to the C-terminus of CpxR (BBa_K1486000). We put these fusions under control of the inducible pBAD/araC promoter (BBa_BI0500) to enable controlled protein expression, and strong ribosome binding site (RBS) BBa_B0034 was placed upstream of the created fusions. This transcriptional unit (Figure 2) was constructed and placed in hgih copy number plasmid pSB1C3 via Golden Gate Assembly.

Figure 2: Schematic of translational unit BBa_K2387032. CpxR-eYFPn and CpxR-eYFPc fusions under control of the inducible araC/pBAD promoter.

References

  1. T. Kerppola, “Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells,” Annu. Rev. Biophys., vol. 37, pp. 465–87, 2008.
  2. T. L. Raivio and T. J. Silhavy, “The sigmaE and Cpx regulatory pathways: Overlapping but distinct envelope stress responses,” Curr. Opin. Microbiol., vol. 2, no. 2, pp. 159–165, 1999.