Difference between revisions of "Team:Wageningen UR/Composite Part"

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                             <!--Introduction-->
 
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                                             <b>Figure 4:</b> eYFPn and eYFPc are fused (seperately) to CpxR. This way BiFC is used to visualize the CpxR dimerization step.
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                                             <b>Figure 1:</b> eYFPn and eYFPc are fused (seperately) to CpxR. This way BiFC is used to visualize the CpxR dimerization step.
 
                                             [1].
 
                                             [1].
 
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Here we show how we brought individual lab projects together and how we implement them in our device! We performed experiments in which we show that our cells are still viable after drying (which means that they can safely be shipped and still work properly), and that we can measure fluorescence in blood serum! Furthermore we combine the <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Cpx"> "Signal Transduction"</a> and <a href="https://2017.igem.org/Team:Wageningen_UR/Results/SpecificVisualization"> "Specific Visualization"</a> modules to directly measure antigens by coupling the affinity molecule, Cpx signal transduction and Bimolecular Fluorescence Complementation (BiFC) specific visualization. <mark> *Pending* Finally, we use the Mantis device to measure fluorescence emitted by our bacteria!</mark>
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<a href="parts.igem.org/Part:BBa_K2387032"> BBa_K2387032</a> 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].
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The Cpx signal transduction system is a native system of <i>E. coli</i> 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 <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Cpx">here</a>.
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We can directly visualize Cpx pathway using BiFC. <a href="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="http://parts.igem.org/Part:BBa_K1486000">CpxR (BBa_K1486000)</a>. We put these fusions under control of the inducible <a href="http://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="http://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="http://parts.igem.org/Part:pSB1C3">pSB1C3</a> via Golden Gate Assembly.
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                                            <b>Figure 2:</b> Schematic of translational unit <a href="parts.igem.org/Part:BBa_K2387032"> BBa_K2387032</a>. CpxR-eYFPn and CpxR-eYFPc fusions under control of the inducible araC/pBAD promoter.
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                             <li>T. Kerppola, “Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells,” <i>Annu. Rev. Biophys.</i>, vol. 37, pp. 465–87, 2008.</li>
 
                             <li>T. Kerppola, “Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells,” <i>Annu. Rev. Biophys.</i>, vol. 37, pp. 465–87, 2008.</li>
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<li>T. L. Raivio and T. J. Silhavy, “The sigmaE and Cpx regulatory pathways: Overlapping but distinct envelope stress responses,” <i>Curr. Opin. Microbiol.</i>, vol. 2, no. 2, pp. 159–165, 1999.</li>
 
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Revision as of 12:33, 30 October 2017

Best Composite Part

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].

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.

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.