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.
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.
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.
Tunable Cpx Activation
We further investigate CpxR dimerization by applying different levels of stress. Known stress factor KCl is added at a range of concentration, as to determine the necessary amount of stress to generate a fluorescent signal. This helps us in finding the amount of antigen Mantis would need. The protocol for this experiment is the same as before and can be found here.
The results in Figure 4 show that the intensity of the fluorescent signal is heavily dependent on the activation level, but each level of activator shows signal within two hours! This shows that the system is sensitive to small amounts of stress, which can imply that only low antigen concentrations could be detected.
From Figure 4 we can conclude that Cpx activation and the subsequently generated fluorescent signal is strongly linked to the addition of stress factor KCl. The low signal generated when no KCl is added shows that the system is specific, and fluorescence clearly increases when more stress is added to the system.References
- 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.
- 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.
