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.

Cornell iGEM 2021 Literature Research

Cornell iGEM 2021 proposes an improve to this part, which detects activation of the Cpx pathway of E. coli. Our modification adds a methyltransferase gene to the plasmid backbone. Since this plasmid is typically native to E. coli, transforming Bacillus subtilis — a common alternative chassis — with this plasmid would subject it to the host’s Restriction-Modification system. Since Bacillus subtilis’ R-M system targets foreign unmethylated DNA, we can prevent the degradation of this plasmid by using E. coli as an intermediary to facilitate methylation. While E. coli already possesses basal methyl transferase activity, introducing this gene will dramatically increase the efficiency of this process; and in doing so, increase the efficiency of transforming vectors into alternate bacteria, such as Bacillus subtilis.

Sources
[1] Suzuki, T., & Yasui, K. (2011). Plasmid artificial modification: a novel method for efficient DNA transfer into bacteria. In Strain Engineering (pp. 309-326). Humana Press.