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]. To optimize experimental results, wet-lab experience and computer models were used.

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.

Results


Inducible Protein Expression

After confirming that the araC/pBAD promoter works and assembling the necessary constructs, we can start visualizing Cpx pathway activation with BiFC. In order to simplify the experiments we activate the Cpx pathway with a known stress factor, KCl [3]. We perform all experiments in E. coli K12. We grow the cells in saltless LB and induce protein expression with a range of 0.02 - 0.2% L-arabinose. CpxR dimerization and subsequent fluorescence is measured over time, and the system is activated at t=20 min with 75 mM KCl (indicated by the arrow) [4]. Check out the full protocol here.

Figure 3: CpxR dimerization visualized with different L-arabinose concentrations over time.

The results clearly show a rapid increase in fluorescence after activation of the Cpx pathway when we visualize CpxR-CpxR dimerization, and we see that the signal gets stronger when CpxR-eYFP is expressed at higher levels (Figure 3). It is clear that within two hours a strong fluorescent signal is detected! We also correctly predicted a positive CpxR dimerization result in our computer model!

We can thus conclude that the fluorescent signal of BBa_K2387032 can be tuned through addition of L-arabinose.

Cpx Activation

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.