Difference between revisions of "Team:Wageningen UR/Composite Part"
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| + | <h4>Best Composite Part</h4> | ||
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| + | <a href="#introduction">Introduction</a> | ||
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| + | <a href="#results">Results</a> | ||
| + | </li> | ||
| + | <li> | ||
| + | <a href="#conclusions">Conclusions</a> | ||
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| − | + | <li><a href="https://2017.igem.org/Team:Wageningen_UR/Wet-lab">Wet-lab</a></li> | |
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| − | + | <section id="introduction"><br> | |
<section class="MI intro"> | <section class="MI intro"> | ||
<div class="Title"> | <div class="Title"> | ||
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| + | <h2>Introduction</h2> | ||
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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 | + | <b>Figure 1:</b> eYFPn and eYFPc are fused (seperately) to CpxR. This way BiFC is used to visualize the CpxR dimerization step [1]. |
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
<p> | <p> | ||
| − | <a href="http://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]. To optimize experimental results, <a href="https://2017.igem.org/Team:Wageningen_UR/Results/SpecificVisualization">wet-lab | + | <a href="http://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]. To optimize experimental results, <a href="https://2017.igem.org/Team:Wageningen_UR/Results/SpecificVisualization">wet-lab experiments</a> and <a href="https://2017.igem.org/Team:Wageningen_UR/Model/Cpx_Kinetics">computer models</a> were used. |
</p><p> | </p><p> | ||
| − | 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 | + | 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 phosphoryl group is transferred to response regulator CpxR. Phosphorylated CpxR can homodimerize and natively functions as a transcriptional regulator (Figure 1). More background information in the Cpx pathway can be found <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Cpx">here</a>. |
</p><p> | </p><p> | ||
| − | We can directly visualize Cpx pathway using BiFC. <a href="http://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 | + | We can directly visualize the Cpx pathway using BiFC. <a href="http://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 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 a 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 high copy number plasmid <a href="http://parts.igem.org/Part:pSB1C3">pSB1C3</a> via Golden Gate Assembly. |
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<div class="figure-center-caption"> | <div class="figure-center-caption"> | ||
| − | <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. | + | <b>Figure 2:</b> Schematic of translational unit <a href="http://parts.igem.org/Part:BBa_K2387032"> BBa_K2387032</a>. CpxR-eYFPn and CpxR-eYFPc fusions under control of the inducible araC/pBAD promoter. |
</div> | </div> | ||
| + | </section> | ||
| + | </section> | ||
| − | + | <section id="results"><br> | |
<section class="results"> | <section class="results"> | ||
<h2>Results</h2> | <h2>Results</h2> | ||
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<h3>Inducible Protein Expression<h3> | <h3>Inducible Protein Expression<h3> | ||
| − | <p>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 <i>E. coli</i> 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 [4]. | + | <p>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 <i>E. coli</i> 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]. View the full protocol |
<a href="https://static.igem.org/mediawiki/2017/d/d4/Cpx_Dimerization.pdf">here</a>. | <a href="https://static.igem.org/mediawiki/2017/d/d4/Cpx_Dimerization.pdf">here</a>. | ||
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<p> | <p> | ||
| − | 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 | + | 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 <a href="https://2017.igem.org/Team:Wageningen_UR/Model/IntegrationCpx#Initial_modeling"> computer model</a>! |
</p><p> | </p><p> | ||
We can thus conclude that the fluorescent signal of <a href="http://parts.igem.org/Part:BBa_K2387032"> BBa_K2387032</a> can be tuned through addition of L-arabinose. | We can thus conclude that the fluorescent signal of <a href="http://parts.igem.org/Part:BBa_K2387032"> BBa_K2387032</a> can be tuned through addition of L-arabinose. | ||
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<p> | <p> | ||
| − | We further investigate CpxR dimerization by applying different levels of stress. Known stress factor KCl is added at a range of | + | We further investigate CpxR dimerization by applying different levels of stress. Known stress factor KCl is added at a range of concentrations, 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 |
<a href="https://static.igem.org/mediawiki/2017/d/d4/Cpx_Dimerization.pdf">here</a>. | <a href="https://static.igem.org/mediawiki/2017/d/d4/Cpx_Dimerization.pdf">here</a>. | ||
</p> | </p> | ||
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<p> | <p> | ||
| − | 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 | + | 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 a wide range of antigen concentrations could be detected. |
</p><p> | </p><p> | ||
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. | 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. | ||
</p> | </p> | ||
| + | |||
| + | </section> | ||
| + | </section> | ||
| + | |||
| + | <section id="conclusions"><br> | ||
| + | <section class="conclusions"> | ||
<h2>Conclusions</h2> | <h2>Conclusions</h2> | ||
<p> | <p> | ||
| − | <a href="http://parts.igem.org/Part:BBa_K2387032"> BBa_K2387032</a> is proven to be a useful tool in directly visualizing Cpx activation. A fluorescent signal is | + | <a href="http://parts.igem.org/Part:BBa_K2387032"> BBa_K2387032</a> is proven to be a useful tool in directly visualizing Cpx activation. A fluorescent signal is specifically generated within a matter of hours. We also show that this signal can be tuned through addition of a range of L-arabinose concentrations. Furthermore, we show that Cpx activation is dependent on the addition of stress factor KCl, and this can be measured in a wide range of concentrations. We furthermore show that <a href="http://parts.igem.org/Part:BBa_K2387032"> BBa_K2387032</a> can also be implemented in <a href="https://2017.igem.org/Team:Wageningen_UR/Results/SpecificVisualization#optimalactivation"><i>E. coli</i> K12ΔCpxR</a>. |
</p><p> | </p><p> | ||
This BioBrick can be readily used in any whole cell biosensor which makes use of the Cpx pathway. This pathway can be adapted to measure a wide range of factors, such as antigens, salt concentrations or pH change [5]. | This BioBrick can be readily used in any whole cell biosensor which makes use of the Cpx pathway. This pathway can be adapted to measure a wide range of factors, such as antigens, salt concentrations or pH change [5]. | ||
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</div> | </div> | ||
</section> | </section> | ||
| + | </section> | ||
<!-- References --> | <!-- References --> | ||
Latest revision as of 22:30, 1 November 2017
Best Composite Part
Introduction
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 experiments 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 phosphoryl group is transferred to response regulator CpxR. Phosphorylated CpxR can homodimerize and natively functions as a transcriptional regulator (Figure 1). More background information in the Cpx pathway can be found here.
We can directly visualize the Cpx pathway using BiFC. eYFP (BBa_E0030) was cleaved between amino acids 154 and 155 and we fused these N- and C-termini 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 a strong ribosome binding site (RBS) (BBa_B0034) was placed upstream of the created fusions. This transcriptional unit (Figure 2) was constructed and placed in high 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]. View 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 concentrations, 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 a wide range of 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.
Conclusions
BBa_K2387032 is proven to be a useful tool in directly visualizing Cpx activation. A fluorescent signal is specifically generated within a matter of hours. We also show that this signal can be tuned through addition of a range of L-arabinose concentrations. Furthermore, we show that Cpx activation is dependent on the addition of stress factor KCl, and this can be measured in a wide range of concentrations. We furthermore show that BBa_K2387032 can also be implemented in E. coli K12ΔCpxR.
This BioBrick can be readily used in any whole cell biosensor which makes use of the Cpx pathway. This pathway can be adapted to measure a wide range of factors, such as antigens, salt concentrations or pH change [5].
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.
- Fleischer, R., Heermann, R., Jung, K., & Hunke, S. (2007). Purification, reconstitution, and characterization of the CpxRAP envelope stress system of Escherichia coli. Journal of Biological Chemistry, 282(12), 8583–8593.
- 2014 iGEM EFPL Results Page
- P. N. Danese and T. J. Silhavy, “CpxP, a stress-combative member of the Cpx regulon,” J. Bacteriol., vol. 180, no. 4, pp. 831–839, 1998.
