Difference between revisions of "Part:BBa K2387032"

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BBa_K2387032 is created as a means to detect activation of the Cpx pathway of <i>E. coli</i>. This is done using a method called Bimolecular Fluorescence Complementation (BiFC) [1]. To optimize experimental results, wet-lab experience and computer models were used.
 
BBa_K2387032 is created as a means to detect activation of the Cpx pathway of <i>E. coli</i>. This is done using a method called Bimolecular Fluorescence Complementation (BiFC) [1]. To optimize experimental results, wet-lab experience and computer models were used.
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<a href="https://parts.igem.org/Part:BBa_E0030"> eYFP (BBa_E0030)</a> was cleaved between amino acids 154 and 155 and we fused these N- and C-termini of  to the C-terminus of <a href="https://parts.igem.org/Part:BBa_K1486000">CpxR (BBa_K1486000)</a>. We put these fusions under control of the inducible <a href="https://parts.igem.org/Part:BBa_I0500">pBAD/araC promoter (BBa_BI0500)</a> to enable controlled protein expression, and strong ribosome binding site (RBS) <a href="https://parts.igem.org/Part:BBa_B0034"> BBa_B0034</a> was placed upstream of the created fusions. This transcriptional unit (Figure 2) was constructed and placed in hgih copy number plasmid <a href="https://parts.igem.org/Part:pSB1C3">pSB1C3</a> via Golden Gate Assembly.

Revision as of 19:44, 30 October 2017


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.

<a href="https://parts.igem.org/Part:BBa_E0030"> eYFP (BBa_E0030)</a> was cleaved between amino acids 154 and 155 and we fused these N- and C-termini of to the C-terminus of <a href="https://parts.igem.org/Part:BBa_K1486000">CpxR (BBa_K1486000)</a>. We put these fusions under control of the inducible <a href="https://parts.igem.org/Part:BBa_I0500">pBAD/araC promoter (BBa_BI0500)</a> to enable controlled protein expression, and strong ribosome binding site (RBS) <a href="https://parts.igem.org/Part:BBa_B0034"> BBa_B0034</a> was placed upstream of the created fusions. This transcriptional unit (Figure 2) was constructed and placed in hgih copy number plasmid <a href="https://parts.igem.org/Part:pSB1C3">pSB1C3</a> via Golden Gate Assembly.