Part:BBa_K2387032

CpxR-eYFPn[1-154] and CpxR-eYPFc[155-238] + araC/pBAD promoter

The [http://parts.igem.com/Part:BBa_K2387005 N-terminus of eYFP] and the[http://parts.igem.com/Part:BBa_K2387006 C-terminus of eYFP] are fused to Cpx response regulator [http://parts.igem.com/Part:BBa_K2387002 CpxR] in order to visualize the activation of the Cpx pathway. Upon activation of the Cpx pathway, CpxR gets phosphorylated by E. coli endogenous CpxA after which it can homodimerize. This protein-protein interaction can be visualized using BiFC, hence the fusion of eYFPn[1-154] and eYFPc[155-238]. The fusion is put under the control of the L-arabinose inducible araC/pBAD promoter. Strong RBS BBa_B0034 is used to regulate transcription.

CpxR-eYFPn and CpxR-eYFPc fusions were linked together using a [http://parts.igem.com/Part:BBa_K1486004 Flexible Linker] consisting of two times the amino acids GGGGS.

This part is used to visualize the activation of the Cpx pathway via Bimolecular Fluorescence Complementation by using the CpxR-CpxR interaction.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal XhoI site found at 1283
  Illegal XhoI site found at 2498
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 1694
  Illegal AgeI site found at 2909
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

Usage and Biology

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.

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 was constructed and placed in high copy number plasmid pSB1C3 via Golden Gate Assembly.

Results - L-arabinose inducibility

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. Check out the full protocol here.

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

Results - KCl inducibility

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.

Figure 4: CpxR dimerization visualized with L-arabinose concentration of 0.2% and different activator concentrations over time.

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.