http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Robert+Baldwin&year=&month=2014.igem.org - User contributions [en]2024-03-29T05:02:05ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experimentsTeam:EPF Lausanne/Results/IFP experiments2014-10-18T01:07:35Z<p>Robert Baldwin: </p>
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<h2> <b><u>Characterisation of the spatiotemporal dynamics of the CpxR - split IFP 1.4 stress sensor </u> </b> </h2<br />
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<h3><b>Experiment 1: </b>Demonstration of CpxR's dimerization & Elucidation of its dimerization orientation [BBa_K1486056] </h3><br />
<p><br />
<u>Introduction</u> <br /><br />
CpxR is the relay protein in the stress resonsive CpxAR two component regulatory system. It has been shown by split beta galactosidase assay that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that <i>E. coli</i> CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in <i>E. coli</i>.</p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to determine if and how CpxR dimerised in vivo in <i>E. coli</i>. This experiment intended to get a first idea of the real-time temporal dynamics of the activation of CpxR (the cytoplasmic relay protein of the CpxA-R pathway) by KCl stress via CpxA (the periplasmic sensor protein of the CpxA-R pathway). This experiment is a first of its kind.<br />
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<p><br />
<u>Methods</u> <br /><br />
To evaluate if and how CpxR dimerized under KCl stress, we built by gibson assembly four constructs with the various possible orientations that the split IFP1.4 fragments could have with CpxR: IFP[1] and IFP[2] on the N-terminus of CpxR, IFP[1] on the N-terminus of CpxR and IFP[2] on the C-terminus of CpxR, and finally IFP[1] and IFP[2] on the N-terminus of CpxR. The split IFP fragments were provided by the Michnick Lab, and the CpxR coding region was amplified by PCR from extracted <i>E. coli</i> genome (Bacterial Genomic Miniprep Kit from Sigma Aldrich). The protocol for stressing the cells and reading the fluorescence can be downloaded <a href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
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<p><br />
<u>Results</u> <br /><br />
As seen in the graph bellow, induction of the signal was done at minute 24 (marked via a vertically spoted line). The construct with IFP fragments on the C-termina responded immediately to stress. In a fact we observed a 3 fold signal increase in 2 minutes. All other constructs we observed a low baseline signal non responsive to KCl stress. It is to be noted that the C-termina constructs always had higher signal levels than the other constructs. This leads us to believe that the PBS used to resuspend our cultures led to small levels of stress (the PBS we use does not contain KCl but traces of NaCl). The 30-fold signal increase from the baseline allows us to assert that our constructs responds to KCl stress.<br />
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<img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" alt="Construct Comparison" class="img-responsive"><br />
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<u>Discussion</u> <br /><br />
We successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus. This finding suggests that CpxR binds via its C-termina. This leads us to hypothesise that the CpxR dimerisation mechanisms is the same for other members of the highly conserved OmpR/PhoB subfamily. This hypothesis could allow the development of similar system that could the study other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.<br />
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<h3><b> Experiment 3: </b>Signal induction by various concentrations of KCl & signal shutdown by centrifugation </h3><br />
<p><br />
<u>Aim</u> <br /><br />
Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change.<br />
To do so, we read our signal for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal.<br />
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<p><br />
<u>Methods</u> <br /><br />
To evaluate if a modulation in KCl concentrations affected the intensity of the intensity of the fluorescent signal, and if a change in medium by centrifugation shutdown the signal; we read our signal on a plate reader for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal. The protocol for this experiment can be downloaded <a href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
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<p><br />
<u>Results</u> <br /><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis are possible for our system.<br />
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<img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" alt="GA1 Shutdown" class="img-responsive"><br />
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<a id="IFP_Exp4"></a><br />
<h3><b> Experiment 4: </b>Visualization of the the CpxR split IFP1.4 activation by KCl stress </h3><br />
<p><br />
<u>Aim</u> <br /><br />
Having shown that we were able to monitor the temporal dynamics of our construct, we wanted to see if we were able to analyze the spatial dynamics by microscopy.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To visualize the activation of our construct, we prepared cells as above for the previous plate-reader experiments, spread 10 µl on a glass slide added a coverslip and imaged them on a Zeiss Axioplan with a x100 objective and a APC (Cy5.5) filter. The pictures shown bellow were taken with a 5.1(s) integration time.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the pictures bellow, we were able to distinguish specific patterns within bacteria. We observed two phenotypes within our population: elongated and normal cells. The difference in these phenotypes was noticed in previous experiments and is most certainly due to the CpxR overexpression as we observed this also in non-stressed conditions. In the first phenotype (elongated) we were able to distinguish several bands that seem fairly uniformly distributed. In the second phenotype (normal) we observed a single band in the center of the bacteria. These observations led us to believe that CpxR might be involved in the division process of <i>E. coli</i> as it seems coherent for cells to slow down division upon stress. After looking into the literature, similar bands were visualizable in <i>E. coli</i> for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how <i>E. coli</i> monitor division under various environments.<br />
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<h4> References </h4><br />
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<a id="ref1"></a>1: S.K. Hatzios, S. Ringgaard, B. M. Davis, M. K. Waldor (2012, August 15). Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition. <i>Plos One</i>.<br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experimentsTeam:EPF Lausanne/Results/IFP experiments2014-10-18T01:06:25Z<p>Robert Baldwin: </p>
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<h2> <b><u>Characterisation of the spatiotemporal dynamics of the CpxR - split IFP 1.4 stress sensor </u> </b> </h2<br />
</div></div><br />
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<a id="IFP_Exp2"></a><br />
<h3><b>Experiment 1: </b>Demonstration of CpxR's dimerization & Elucidation of its dimerization orientation [BBa_K1486056] </h3><br />
<p><br />
<u>Introduction</u> <br /><br />
CpxR is the relay protein in the stress resonsive CpxAR two component regulatory system. It has been shown by split beta galactosidase assay that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that <i>E. coli</i> CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in <i>E. coli</i>.</p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to determine if and how CpxR dimerised in vivo in <i>E. coli</i>. This experiment intended to get a first idea of the real-time temporal dynamics of the activation of CpxR (the cytoplasmic relay protein of the CpxA-R pathway) by KCl stress via CpxA (the periplasmic sensor protein of the CpxA-R pathway). This experiment is a first of its kind.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To evaluate if and how CpxR dimerized under KCl stress, we built by gibson assembly four constructs with the various possible orientations that the split IFP1.4 fragments could have with CpxR: IFP[1] and IFP[2] on the N-terminus of CpxR, IFP[1] on the N-terminus of CpxR and IFP[2] on the C-terminus of CpxR, and finally IFP[1] and IFP[2] on the N-terminus of CpxR. The split IFP fragments were provided by the Michnick Lab, and the CpxR coding region was amplified by PCR from extracted <i>E. coli</i> genome (Bacterial Genomic Miniprep Kit from Sigma Aldrich). The protocol for stressing the cells and reading the fluorescence can be downloaded <a href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the graph bellow, induction of the signal was done at minute 24 (marked via a vertically spoted line). The construct with IFP fragments on the C-termina responded immediately to stress. In a fact we observed a 3 fold signal increase in 2 minutes. All other constructs we observed a low baseline signal non responsive to KCl stress. It is to be noted that the C-termina constructs always had higher signal levels than the other constructs. This leads us to believe that the PBS used to resuspend our cultures led to small levels of stress (the PBS we use does not contain KCl but traces of NaCl). The 30-fold signal increase from the baseline allows us to assert that our constructs responds to KCl stress.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" alt="Construct Comparison" class="img-responsive"><br />
</div><br />
<p><br />
<u>Discussion</u> <br /><br />
We successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus. This finding suggests that CpxR binds via its C-termina. This leads us to hypothesise that the CpxR dimerisation mechanisms is the same for other members of the highly conserved OmpR/PhoB subfamily. This hypothesis could allow the development of similar system that could the study other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.<br />
</p><br />
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<a id="IFP_Exp3"></a><br />
<h3><b> Experiment 3: </b>Signal induction by various concentrations of KCl & signal shutdown by centrifugation </h3><br />
<p><br />
<u>Aim</u> <br /><br />
Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change.<br />
To do so, we read our signal for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To evaluate if a modulation in KCl concentrations affected the intensity of the intensity of the fluorescent signal, and if a change in medium by centrifugation shutdown the signal; we read our signal on a plate reader for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal. The protocol for this experiment can be downloaded <a href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis are possible for our system.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" alt="GA1 Shutdown" class="img-responsive"><br />
</div><br />
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<a id="IFP_Exp4"></a><br />
<h3><b> Experiment 4: </b>Visualization of the the CpxR split IFP1.4 activation by KCl stress </h3><br />
<p><br />
<u>Aim</u> <br /><br />
Having shown that we were able to monitor the temporal dynamics of our construct, we wanted to see if we were able to analyze the spatial dynamics by microscopy.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To visualize the activation of our construct, we prepared cells as above for the previous plate-reader experiments, spread 10 µl on a glass slide added a coverslip and imaged them on a Zeiss Axioplan with a x100 objective and a APC (Cy5.5) filter. The pictures shown bellow were taken with a 5.1(s) integration time.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the pictures bellow, we were able to distinguish specific patterns within bacteria. We observed two phenotypes within our population: elongated and normal cells. The difference in these phenotypes was noticed in previous experiments and is most certainly due to the CpxR overexpression as we observed this also in non-stressed conditions. In the first phenotype (elongated) we were able to distinguish several bands that seem fairly uniformly distributed. In the second phenotype (normal) we observed a single band in the center of the bacteria. These observations led us to believe that CpxR might be involved in the division process of <i>E. coli</i> as it seems coherent for cells to slow down division upon stress. After looking into the literature, similar bands were visualizable in <i>E. coli</i> for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how <i>E. coli</i> monitor division under various environments.<br />
</p><br />
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<h4> References </h4><br />
<p><br />
<a id="ref1"></a>1: S.K. Hatzios, S. Ringgaard, B. M. Davis, M. K. Waldor (2012, August 15). Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition. <i>Plos One</i>.<br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/ResultsTeam:EPF Lausanne/Results2014-10-18T01:04:38Z<p>Robert Baldwin: </p>
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<h1 class="cntr"> <b>RESULTS</b> </h1><br />
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<!--replaced the title : Characterisation of the spatiotemporal dynamics of the CpxR - split IFP 1.4 stress sensor --><br />
<h2 id="IFP_Exp"> <b> Characterisation of the spatiotemporal dynamics of the CpxR stress sensor </b> </h2<br />
</div><br />
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<h3 id="IFP_Exp2"><b>Demonstration of CpxR's dimerization & Elucidation of its dimerization orientation</b></h3><br />
<br />
<p><br />
Previous in vitro experiments (FRET) have shown that CpxR dimerizes. Currently little in vivo information about CpxR’s dimerization is available. To characterize our stress responsive bacteria, we had to confirm that CpxR dimerized in vivo as well as elucidate CpxR’s dimerization orientation. <br />
</p><br />
<br /><br />
<p><br />
We synthesized four constructs with combinations of the split IFP1.4 fragments fused to the C or N terminal of CpxR. <br />
As seen in the graph bellow, induction of IFP1.4 signal by 50 mM KCl was done at t=24min. We clearly see that the construct with IFP fragments on the C-terminal immediately responded to stress by emitting fluorescence. In fact we observe a 3 fold signal increase in 2 minutes. On the other hand, the three other orientations were non-responsive to KCl stress.</p><br />
<br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" data-lightbox="chips" data-title="Construct Comparison"><img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" width="80%"/></a><br /><br />
</div><br />
<p><br />
This amazing 30-fold signal increase in little time from the baseline allows us to assert that only the strain synthesizing the IFP fragments on the C-terminals of CpxR responded to KCl stress. For a further analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
</p><br />
<br />
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<br />
<br />
<!--<br />
<p><br />
<u>Introduction</u> <br /><br />
CpxR is the relay protein in the stress responsive CpxAR two component regulatory system. It has been shown by split beta galactosidase assay that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that <i>E. coli</i> CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in <i>E. coli</i>.</p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to determine if and how CpxR dimerised in vivo in <i>E. coli</i>. This experiment intended to get a first idea of the real-time temporal dynamics of the activation of CpxR (the cytoplasmic relay protein of the CpxA-R pathway) by KCl stress via CpxA (the periplasmic sensor protein of the CpxA-R pathway). This experiment is a first of its kind.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To evaluate if and how CpxR dimerized under KCl stress, we built by gibson assembly four constructs with the various possible orientations that the split IFP1.4 fragments could have with CpxR: IFP[1] and IFP[2] on the N-terminus of CpxR, IFP[1] on the N-terminus of CpxR and IFP[2] on the C-terminus of CpxR, and finally IFP[1] and IFP[2] on the N-terminus of CpxR. The split IFP fragments were provided by the Michnick Lab, and the CpxR coding region was amplified by PCR from extracted <i>E. coli</i> genome (Bacterial Genomic Miniprep Kit from Sigma Aldrich). The protocol for stressing the cells and reading the fluorescence can be downloaded <a target="blank" href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the graph bellow, induction of the signal was done at minute 24 (marked via a vertically spoted line). The construct with IFP fragments on the C-termina responded immediately to stress. In a fact we observed a 3 fold signal increase in 2 minutes. All other constructs we observed a low baseline signal non responsive to KCl stress. It is to be noted that the C-termina constructs always had higher signal levels than the other constructs. This leads us to believe that the PBS used to resuspend our cultures led to small levels of stress (the PBS we use does not contain KCl but traces of NaCl). The 30-fold signal increase from the baseline allows us to assert that our constructs responds to KCl stress.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" alt="Construct Comparison" class="img-responsive"><br />
</div><br />
<p><br />
<u>Discussion</u> <br /><br />
We successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus. This finding suggests that CpxR binds via its C-termina. This leads us to hypothesise that the CpxR dimerisation mechanisms is the same for other members of the highly conserved OmpR/PhoB subfamily. This hypothesis could allow the development of similar system that could the study other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.<br />
</p><br />
<br /><br /><br />
--><br />
<br />
<br />
<br />
<br /><br /><br />
<br />
<br />
<h3 id="IFP_Exp3"> <b>Signal induction by various concentrations of KCl & signal shutdown by centrifugation</b></h3><br />
<br />
<p><br />
Having confirmed that KCl was a good inducer for our signal, we tested different concentration of KCl to modulate the signal and better characterize our biobrick. We also aimed to shutdown the signal by centrifugation and medium change. The signal was measured on a plate reader 20 minutes, before the addition of KCl. After 2 hours of measurement we centrifuged the cells for ten minutes and replaced the medium with PBS to be able to get a shutdown of the signal.</p><br />
<br />
<div class="cntr"><br />
<a target="_blank" href="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" data-lightbox="chips" data-title="The split-IFP are linked to cpxR on the C-terminal. During the experiment it was possible to shut down the signal"><img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" width="80%"/></a><br /><br />
</div><br />
<br />
<br />
<p><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to drastically shut down the signal, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis is possible for our system. For a thorough analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
</p><br />
<br />
<br />
<!--<br />
<p><br />
<u>Aim</u> <br /><br />
Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change.<br />
To do so, we read our signal for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To evaluate if a modulation in KCl concentrations affected the intensity of the intensity of the fluorescent signal, and if a change in medium by centrifugation shutdown the signal; we read our signal on a plate reader for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal. The protocol for this experiment can be downloaded <a target="_blank" href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis are possible for our system.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" alt="GA1 Shutdown" class="img-responsive"><br />
</div><br />
<br /><br /><br/><br/><br />
--><br />
<br />
<br /><br /><br />
<br />
<br />
<h3 id="IFP_Exp4"><b>Visualization of the the CpxR split IFP1.4 activation by KCl stress </b></h3><br />
<br />
<br />
<p><br />
<br />
<br />
Having shown that we were able to monitor the temporal dynamics of CpxR activation, we wanted to see if we could analyze CpxR’s spatial dynamics by microscopy. 10μl of our cells, previously stressed with 80 mM KCl were spread on a glass slide and imaged with a x100 objective and a APC (Cy5.5) filter.<br />
<br />
<br />
</p><br />
<br />
<br />
<p><br />
<br />
<br />
We noticed various characteristics of the cells from the picture bellow. First, IFP signal was much more present in stressed bacteria rather than in non-stressed bacteria. Secondly, we distinguished two specific phenotypes within bacteria: elongated and normal cells. We noticed that this difference was due to CpxR overexpression as we saw this phenomenon also in non-stressed conditions.<br /><br /><br />
<br />
<br />
In elongated cells, we were able to distinguish several bright bands of IFP signal that seem fairly uniformly distributed. In the normal phenotype we distinguished a single band in the centre of the bacteria. These observations led us to believe that CpxR might be involved in the division process of E. coli as it seems coherent for cells to slow down division upon stress. <br /><br /><br />
<br />
<br />
After looking into the literature, similar bands were visualizable in E. coli for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how E. coli monitor division under various environments. For a thorough analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
<br />
<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" data-lightbox="results" data-title="Results"><img src="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" alt="results" width="45%" class="pull-left"></a><br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/EPFL_2014_03_10_2014_Experiment-24.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/d/de/EPFL_2014_03_10_2014_Experiment-34.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/fd/EPFL_2014_03_10_2014_Experiment-35.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/0/08/EPFL_2014_03_10_2014_Experiment-37.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/a/a7/EPFL_2014_03_10_2014_Experiment-38.jpg" data-lightbox="results" data-title="Results"></a> <br />
<a href="https://static.igem.org/mediawiki/2014/2/2e/EPFL_2014_03_10_2014_Picture3.jpg" data-lightbox="results" data-title="Results"><br />
<a href="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" data-lightbox="results" data-title="Results"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" alt="results" width="45%" class="pull-right"></a><br />
</div><br />
<br />
<br />
<br />
<!--<br />
<br />
<p><br />
<u>Aim</u> <br /><br />
Having shown that we were able to monitor the temporal dynamics of our construct, we wanted to see if we were able to analyze the spatial dynamics by microscopy.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To visualize the activation of our construct, we prepared cells as above for the previous plate-reader experiments, spread 10 µl on a glass slide added a coverslip and imaged them on a Zeiss Axioplan with a x100 objective and a APC (Cy5.5) filter. The pictures shown bellow were taken with a 5.1(s) integration time.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the pictures bellow, we were able to distinguish specific patterns within bacteria. We observed two phenotypes within our population: elongated and normal cells. The difference in these phenotypes was noticed in previous experiments and is most certainly due to the CpxR overexpression as we observed this also in non-stressed conditions. In the first phenotype (elongated) we were able to distinguish several bands that seem fairly uniformly distributed. In the second phenotype (normal) we observed a single band in the center of the bacteria. These observations led us to believe that CpxR might be involved in the division process of <i>E. coli</i> as it seems coherent for cells to slow down division upon stress. After looking into the literature, similar bands were visualizable in <i>E. coli</i> for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how <i>E. coli</i> monitor division under various environments.<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"><img src="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" alt="results" width="45%" class="pull-left"></a><br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/EPFL_2014_03_10_2014_Experiment-24.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/d/de/EPFL_2014_03_10_2014_Experiment-34.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/fd/EPFL_2014_03_10_2014_Experiment-35.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/0/08/EPFL_2014_03_10_2014_Experiment-37.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/a/a7/EPFL_2014_03_10_2014_Experiment-38.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a> <br />
<a href="https://static.igem.org/mediawiki/2014/2/2e/EPFL_2014_03_10_2014_Picture3.jpg" data-lightbox="results" data-title="IFP signal in stressed cells"><br />
<a href="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" data-lightbox="results" data-title="IFP signal in non stressed cells"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" alt="results" width="45%" class="pull-right"></a><br />
</div><br />
<br />
--><br />
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<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
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<br />
<br/><br/><br/><br />
<!--<br />
<br />
<h3 id=“CpxR-promoter"><b>RFP under CpxR promoter to investigate time delay between transcription factor activation and protein synthesis</b></h3><br />
<br />
<p>To mesure the time it takes a cell to react to a stress input and if our modified CpxR still has its function as a transcription factor, we wanted to investigate the RFP expression of stressed cells that we co-transformed our CpxR-split-IPF construct with the <a target="_blank" href="http://parts.igem.org/Part:BBa_K339007">BBa_K339007</a>. Unfortunately we realised that the regulatory sequence of the RFP, the CpxR responsive promoter, was missing. So we constructed the biobrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486048">BBa_K1486048</a> which contains the regulatory sequence. After a few experiments we realised that KCl stress, which we have found to be a good signal inducer, did not work, even in cells expressing native CpxR. So we developed the biobricks <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486049">BBa_K1486049</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486050">BBa_K1486050</a>, which encode for RFP regulated by the CpxR responsive promoter, how it is found in the genome. In the figure below you see that neither of the three constructs was stress induced.</p><br />
<br />
<div class="cntr img-border"><br />
<a href="https://static.igem.org/mediawiki/2014/9/91/BBa_K1486049_BBa_K1486048.png" data-lightbox="cpxr" data-title="CpxR promoted RFP under stress"><br />
<img src="https://static.igem.org/mediawiki/2014/9/91/BBa_K1486049_BBa_K1486048.png" alt="CpxR promoted RFP under stress" class="img-responsive" /></a><br />
</div><br />
<br />
<br/><br/><br/><br />
--><br />
<br />
<br />
<h3 id="IFP_Exp5"><b>Activation of CpxR - split IFP1.4 on microfluidic chip by chamber crushing </b></h3><br />
<br />
<p><br />
Knowing that we were able to able to visualize CpxR-IFP activation under a microscope, we proceeded to trying to activate the pathway by mechanical stress on a microfluidic chip - the ultimate barrier to building a functional BioPad !<br /><br /><br />
<br />
To induce this stress, we turned on the buttons of our SmashColi microfluidic chip at a pressure of 25 psi. We imaged chambers before stress and after stress (10 min after button activation). A drastic increase in signal was detected !<br />
<br />
<div class="cntr"><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/d/dc/Button_IFP_non_stressed_2_Cy5.jpg" data-lightbox="results_button" data-title="results_button"><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Button_IFP_non_stressed_2_Cy5.jpg" alt="results" width="45%" class="pull-left"> <br />
</a><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/7/77/Button_IFP_non_stressed_2.jpg" data-lightbox="results_button" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/9/99/Button_IFP_stressed_2.jpg" data-lightbox="results_button" data-title="Results"></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/f/f8/Button_IFP_stressed_2_Cy5.jpg" data-lightbox="results_button" data-title="results_button"><img src="https://static.igem.org/mediawiki/2014/f/f8/Button_IFP_stressed_2_Cy5.jpg" alt="results" width="45%" class="pull-right"> <br />
</a><br />
<br />
</div><br />
<br />
<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
<br />
<h3 id="IFP_Exp1"> <b> Characterisation of the pBAD promoter and folding ability of GFP fused to CpxR </b></h3><br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg" data-lightbox="img1"><img src="<br />
https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg" width="70%"></a><br />
</div> <br /><br /><br />
<br />
<p>This construct aimed to evaluate the expression of our construct and the characteristics of the arabinose promoter in <i>E. coli</i> by fusing a superfolder GFP protein to CpxR. The sfGFP was chosen because of its higher intensity compared to GFP. </p><br />
<p>Not knowing if CpxR would react the same way if sfGFP were attached to the N or C terminus, 2 biobricks were built, one with each of the orientations: <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486002">BBa_K1486002 (N terminus)</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486005">BBa_K1486005 (C terminus)</a>.<br />
</p><br />
<p><br />
We tested both constructs CpxR-sfGFP and sfGFP-CpxR on a plate reader to characterize the pBAD promoter, measuring the sfGFP signal in function of the arabinose concentration. We also measured the signal in a microfluidic chip. You can see in the graph below that the signal is increasing as the concentration of the arabinose increases. The construct with sfGFP at the C terminal lead to a higher GFP signal. Interestingly both curves increases in a very similar manner.<br />
<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/a/ac/Nterm_and_Cterm_log.png" data-lightbox="hellopony"><img src="<br />
https://static.igem.org/mediawiki/2014/a/ac/Nterm_and_Cterm_log.png" width="50%"></a><br />
</div><br />
<br/><br/><br />
<hr/><br />
<br />
<h2 id="Characterisation_of_the_split_luciferase"> <b>Characterisation of the split luciferase </b> </h2><br />
<br />
<h3 id="Characterisation_of_the_split_luciferase"><b>Split luciferase complementation assay using CheY and CheZ chemotaxis proteins </b> </h3><br />
<br />
<p><br />
CheY and CheZ are two proteins involved in the bacterial chemotaxis pathway. It has been shown by split luciferase complementation assay that these two proteins are not interacting in presence of chemoattractant, but start to interact in absence of chemoattractant or presence of chemorepellent. Based on the work of Waldor<sup><a href="#ref1">1</a></sup> Laboratory, we wanted to redo the experiment to test our own splits, with firefly (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486055">BBa_K1486055</a>) and renilla (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486054">BBa_K1486054</a>) luciferases.<br />
</p><br />
<br />
<p>As shown in the graphs (fig.1A and 1B), we didn't observe a high signal for our assay. However, the signal being higher than the blanks, it is an encouraging sign that the splits luciferase can be used for other experiments of this kind. A possible explanation for these results is that we didn't completely get rid of the arabinose, which acts as a chemoattractant. Moreover, CheY and CheZ being endogenously expressed in bacteria, there could be interferences with our fusion proteins and weakening of our signal. This should be tested again with CheY/CheZ knock out strains.<br /><br />
</p><br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Renilla"><img src="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Firefly"><img src="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
</div><br /><br />
<br /><br />
<p>We also could determine which of the luciferases would best suit our following experiments. As shown in fig. 2, for the same concentration of substrate, we see that firefly luciferase has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.</p><br /><br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" data-lightbox="chips" data-title="Bioluminescence assay"><img src="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" width="80%"/></a><br /><br />
</div><br />
</p><br />
</div><br />
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<!--<br />
<div class="cntr"><br />
<h2 id="Characterisation_of_the_split_luciferase"> <b><u>Characterisation of the split luciferase </u> </b> </h2><br />
</div><br />
<br /><br /><br />
<br />
<h3 id="Characterisation_of_the_split_luciferase"><b>Experiment 1: </b>CheY/CheZ fused to split Firefly/Renilla luciferase, and full Firefly/Renilla luciferase characterisation </h3><br />
<p><u>Introduction</u> <br /><br />
CheY and CheZ are two proteins involved in the bacterial chemotaxis pathway. It has been shown by split luciferase complementation assay that these two proteins are not interacting in presence of chemoattractant, but start to interact (CheZ being the phosphatase of CheY) in absence of chemoattractant or presence of chemorepellent. Based on the work of Waldor<sup><a href="#ref1">1</a></sup> Laboratory, we wanted to redo and adapt the experiment to test our own splits.<br /> <br /></p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to test the efficiency of split Renilla luciferase and split Firefly luciferase. We wanted to study the speed of the signal and the amount of substrate needed to have a performant response. <br /> <br /><br />
</p><br />
<p><br />
<u>Method</u> <br /><br />
To proceed to this complementation assay, we built two constructs, one to test split Renilla Luciferase and the other for split Firefly Luciferase The CheY was fused to the N-terminal part of each split, while the CheZ was fused to the C-terminal part. We used the full luciferases (Renilla : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486022"> BBa_K1486022 </a> and Firefly : <a target="_blank" href="http://parts.igem.org/Part:BBa_K325108"> BBa_K325108 </a> from Cambridge 2010 team) as positive controls and the non-fused splits (Renilla : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486021"> BBa_K1486021 </a> and Firefly : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486018"> BBa_K1486018 </a>) as negative controls.<br /> <br /><br />
The bioluminescence assay was performed as described <a target="_blank" href="https://static.igem.org/mediawiki/2014/6/6d/Protocol_-_Bioluminescence_assay.pdf">here</a>. <br /><br />
The constructs were designed and assembled as described <a target="_blank" href="https://static.igem.org/mediawiki/2014/3/3b/Constructs_design_CheYCheZ.pdf">here</a>.<br /><br /> <br /><br />
</p><br />
<p><u>Results</u> <br /><br />
As shown in the graphs below (fig.1A and 1B), we couldn't really observe a high signal for our assay. However, the signal being higher than the blanks, it is an encouraging sign that the splits luciferase can be used for other experiments of this kind. A possible explanation for these results is that arabinose being a chemoattractant, we might need to do more wash steps with PBS to get rid of the arabinose before taking the measurements. Moreover, CheY and CheZ being endogenously expressed in bacteria, the edogenous proteins could interfere with our fusion proteins and weaken our signal. This complementation assay should be tested with CheY/CheZ knock out strains, as it was done in Waldor Laboratory.<br /><br />
</p><br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Renilla"><img src="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Firefly"><img src="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
</div><br /><br />
<br /><br />
We also could determine which of the luciferases would best suit our following experiments. As shown in fig. 2, for the same concentration of substrate, we see that firefly luciferase has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.<br /><br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
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--><br />
<br />
<br />
<br />
<br />
<h2 id="Yeast_experiments"> <b>The PBS2-HOG1 split-GFP & split Renilla Luciferase stress response<br />
</b> </h2><br />
<br />
<br/><br/><br />
<br />
<h3 id="Yeast_exp1"><b>Confirmation of successful transformation via the Renilla Luciferase tag </b></h3><br />
<br/><br />
<p><br />
<br />
Since we had never transformed <i>S. cerevisiae</i>, we first needed to confirm our protein tag transformations as a positive control for subsequent experiments. We cultured the two strains we got from the transformation, the HOG1-rLuc (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486033">BBa_K1486033</a> ) and PBS2-rLuc (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486027">BBa_K1486027</a>), put them in a 96 well plate and tested their luminescence using a plate reader.</p><br />
<p><br />
<br />
The test was performed using two different concentrations of the substrate, coelenterazine-h(benzyl coelenterazine): 1μM and 5μM so that we could determine the optimal concentration we would use for the next assays. The plate started to be read directly after addition of the substrate in the dark and measures were taken every 56 seconds thereafter. Three wells for each sample were measured and below are shown the graphs of their average luminescence plotted over time. We observed a signal-to-noise ratio of 4 at 1μM of substrate and 10 at 5μM for the PBS2-rLuc strain. The HOG1-rLuc strain seemed problematic and died within the plate. Unable to verify the correct transformation, we reproduced the strain but were unable to confirm its validity before the wiki deadline.</p><br />
<br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/0e/Pbslucprettycomparaison_.png" data-lightbox="chips" data-title=""><img src="https://static.igem.org/mediawiki/2014/0/0e/Pbslucprettycomparaison_.png" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<p><br />
There is a clear luminescence generated by the cells, providing us with confirmation that our designs are correct and we determined which concentration of substrate to use to have a decent signal-to-background ratio.<br />
</p><br />
<br />
<br />
<br />
<h3 id="Yeast_exp2"><b>The split-sfGFP strain stress-response</b></h3><br />
<p><br />
<br />
<br />
Having confirmed that our design for yeast transformations was correct via the previous experiment, we performed stress-response tests upon our PBS2-HOG1-splitGFP (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486029">BBa_K1486029</a>) and (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486035">BBa_K1486035</a>) strain to determine whether the pathway is reactive to stress as theorized. A co-transformation was performed using linear fragments produced in the same way as for the first experiment to obtain the strain. The N-terminal split of the superfolder GFP was tagged onto PBS2 and the C-terminal onto HOG1. The strain was then tested along with non-transformed cells in a 96 well plate with various stresses (e.g. Acetic Acid, Ethanol, Glucose...). The cells were centrifuged and resuspended in PBS before loading into the plate.<br />
</p><br />
<br />
<br />
<div class="cntr"><br />
<br />
<div class="cntr"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/1/1d/Splitgfpgraph.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<p><br />
The most reactive of stresses turned out to be Acetic Acid 3.6%(shown above) followed by Ethanol 10%. Other stresses tested did not seem to show conclusive results and we were unable to determine more reactants before the wiki freeze deadline. To further assess the fluorescence 10μl of our cells were spread on a glass slide and imaged with a x63 objective and a Green LP filter<br />
(Excitation BP 450-490, Dichroïc FT 510, Emission LP 515). Below is shown a comparison of before and after 3.6% Acetic Acid stress in the microscopy image merged with the fluorescence. <br />
</p><br />
<br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/3b/Splitgfpyeast.jpg" data-lightbox="caca" data-title="Image of our split sfGFP expressing cells before and after the 3.6% acetic acid stress (x63 objective and a Green LP filter)"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3b/Splitgfpyeast.jpg" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<br />
<div class="cntr"><br />
<p><br />
For a more quantitative measure, the fluorescent cells to total cells ratio was calculated and illustrated below.<br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Splitgfpcount.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<br />
<p><br />
<br />
<br />
From the data shown above, we can see that fluorescence increases within cells in our split sfGFP strain in response to stress while it does not in non-transformed cells. We can thus conclude that the orientation of the split sfGFP constructs added to PBS2 and HOG1 allows proper re-assembly upon interaction of the two proteins. This result shows promise for the actual application of the project as a Biopad using yeast cells. <b>It is the first time a split gets succesfully implemented in the HOG1 pathway by direct attachment to proteins of the kinase cascade</b>. Detection of protein–protein interactions within the pathway were previously studied <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/yea.320080602/abstract"> using vesicle targeting</a>.<br />
<br />
<p/><br />
<br />
<br />
<br />
<br />
<br />
<h3 id="Yeast_exp3"><b>The split-Luciferase strain stress-response</b></h3><br />
<p><br />
After determining that the pathway is reactive to certain stresses and that the split-GFP strain increases its fluorescence, we performed the same test in the obtained split-luciferase (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486030">BBa_K1486030</a>) and (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486036">BBa_K1486036</a>) strain. Unfortunately, due to the difficult nature of detecting an instantaneous luminescence signal and the unstable nature of coelenterazine-Luciferase reaction in different conditions(inhibition due to various molecules such as glucose), our attempts in detecting it on the plate reader failed. Further tests are planned, including an experiment in the microfluidic chips, to directly measure the effect of mechanical pressure, but will not be done before the wiki freeze. <br />
</p><br />
<br />
<br />
<a id="Microfluidics_experiments"></a><br />
<br />
<br />
<br />
<h2 id="Micro_exp1"><b>Microfluidic achievements</b></h2><br />
<table class="table table-striped valign-middle table-center"><br />
<thead><br />
<tr><br />
<th></th><br />
<th>MITOMI</th><br />
<th>MITOMI modified</th><br />
<th>SmashColi</th><br />
<th>BioPad</th><br />
<th>FilterColi</th><br />
<th>CleanColi</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>Full chip</td><br />
<td class="cntr"><a target="_blank" href="https://static.igem.org/mediawiki/2014/a/ab/Mitomi11.png" data-lightbox="chips" data-title="MITOMI"><img src="https://static.igem.org/mediawiki/2014/a/ab/Mitomi11.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/6/64/Mitomimodif1.png" data-lightbox="chips" data-title="MITOMI Modified"><img src="https://static.igem.org/mediawiki/2014/6/64/Mitomimodif1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/1/15/Smash1.png" data-lightbox="chips" data-title="SmashColi"><img src="https://static.igem.org/mediawiki/2014/1/15/Smash1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/d/db/Biopad1.png" data-lightbox="chips" data-title="BioPad"><img src="https://static.igem.org/mediawiki/2014/d/db/Biopad1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/d/db/Filter1.png" data-lightbox="chips" data-title="FilterColi"><img src="https://static.igem.org/mediawiki/2014/d/db/Filter1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/b/b0/Screen_Shot_2014-10-16_at_22.15.39.png" data-lightbox="chips" data-title="CleanColi"><img src="https://static.igem.org/mediawiki/2014/b/b0/Screen_Shot_2014-10-16_at_22.15.39.png" width="70%"/></a><br /></td><br />
</tr><br />
<tr><br />
<td>Unit Cell</td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/0/0f/MitomiUnit1.png" data-lightbox="chips" data-title="MITOMI Unit"><img src="https://static.igem.org/mediawiki/2014/0/0f/MitomiUnit1.png" width="50%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/7/78/MitomimodifUnit.png" data-lightbox="chips" data-title="MITOMI Modified Unit"><img src="https://static.igem.org/mediawiki/2014/7/78/MitomimodifUnit.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/3/3a/Smahsunit1.png" data-lightbox="chips" data-title="SmashColi Unit"><img src="https://static.igem.org/mediawiki/2014/3/3a/Smahsunit1.png" width="30%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/6/60/Biopadunit1.png" data-lightbox="chips" data-title="BioPad Unit"><img src="https://static.igem.org/mediawiki/2014/6/60/Biopadunit1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/9/9e/FilterUnit.png" data-lightbox="chips" data-title="FilterColi Unit"><img src="https://static.igem.org/mediawiki/2014/9/9e/FilterUnit.png" width="70%"/></a><br /></td><br />
<td><span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Designed</td><br />
<td><span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
</tr><br />
<tr><br />
<td>Mold fabrication</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Fabrication of the chip</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Application</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br/> <p>To start our microfluidic experiments, we used the <a target="_blank" href="http://link.springer.com/protocol/10.1007%2F978-1-61779-292-2_6">MITOMI chip</a> that was designed in the laboratory of Pr. Maerkl.</p><br/><br />
<br/><br />
<br />
<br />
<br />
<br />
<br />
<h3 id="Micro_exp2"><b>Culturing <i>E. coli</i> with constitutive GFP on chip</b></h3><br />
<p>We loaded <i>E. coli</i>, which contained constitutive GFP, in the chip. By using LabVIEW, a protocol was launched overnight to ensure the growth of the cells (the protocol can be found <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#growth">here</a>).<br />
<br /></p><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/e/e4/Growth_small.gif" data-lightbox="pipi"><img src="<br />
https://static.igem.org/mediawiki/2014/e/e4/Growth_small.gif" width="50%"></a><br />
</div><br />
</p><br />
<p>The next morning, a scan of the chip was done to see the intensity of the GFP in the chip.<br /></p><br />
<div class=""><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/f/fe/Gfp.png" data-lightbox="prout"><img src="<br />
https://static.igem.org/mediawiki/2014/f/fe/Gfp.png" width="50%" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<br /><br />
<br />
<br />
<br />
<h3 id="Micro_exp3"><b>CpxR linked with GFP on the N terminal, induced by arabinose in <i>E. coli</i></b></h3><br />
<p>The experiment that was done on wetbench to show that CpxR linked with GFP was expressed with an arabinose promoter was replicated on a MITOMI chip.<br />
LB medium with arabinose was flowed in the upper half whereas LB medium without arabinose was flowed in the lower half. We scanned every hour for 5h (to know how it was done click <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#date-08-21-2014">here</a>).</p><br />
<div class="row"><br />
<div class="col col-md-6"><a href="https://static.igem.org/mediawiki/2014/4/4e/Truc2.png" data-lightbox="chips" data-title="Scan of the microfluidic chip at t = 0min. No signal is detected."><img src="https://static.igem.org/mediawiki/2014/4/4e/Truc2.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 1.</strong> Scan of the microfluidic chip at t = 0min. No signal is detected<br />
</div></div><br />
<div class="col col-md-6"><br />
<a href="https://static.igem.org/mediawiki/2014/3/32/Truc3.png" data-lightbox="chips" data-title="Scan of the microfluidic chip at t = 300min."><img src="https://static.igem.org/mediawiki/2014/3/32/Truc3.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 2.</strong> Scan of the microfluidic chip at t = 300min.</div></div><br />
</div><br />
<p>We analysed the scans and obtained the following results.</p><br />
<p><a href="https://static.igem.org/mediawiki/2014/4/4c/Gfp_ara.png" data-lightbox="chips" data-title="Figure 3. Evolution of CpxR-GFP fluorescence over time"><img src="https://static.igem.org/mediawiki/2014/4/4c/Gfp_ara.png" alt="" class="img-responsive" /></a></p><br />
<br />
<div class="cntr"> <br />
<strong>Figure 3.&nbsp;</strong> Evolution of CpxR-GFP fluorescence over time<br />
</div> <br />
<br />
<br/><br />
<br />
<br />
<br />
<h3 id="Micro_exp4"><b>On chip infrared detection</b></h3><br />
<p>As we focused part of our work on the IFP1.4, we needed confirmation of this signal detection in our microfluidic chips. Thus the aim of this experiment was to prove this fluorescence detection capability. Bacteria were loaded in the smash-coli chip. The first batch was KCl stressed and the second batch was unstressed. We then simply scanned the chip and analysed the results using ImageJ. For more details, please visit our <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#date-10-03-2014">notebook</a>. </p><br />
<div class="row"><br />
<div class="col col-md-6"><a href="https://static.igem.org/mediawiki/2014/c/cf/No_KCl_red.png" data-lightbox="chips" data-title="Figure 1. Cy5 scan of a chamber containing non-stressed CpxR-IFP bacteria"><img src="https://static.igem.org/mediawiki/2014/c/cf/No_KCl_red.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 1.</strong> Cy5 scan of a chamber containing non-stressed CpxR-IFP bacteria.<br />
</div></div><br />
<div class="col col-md-6"><br />
<a href="https://static.igem.org/mediawiki/2014/c/c2/KCL_red.png" data-lightbox="chips" data-title="Figure 2. Cy5 scan of a chamber containing KCl-stressed CpxR-IFP bacteria"><img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_red.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 2.</strong> Cy5 scan of a chamber containing KCl-stressed CpxR-IFP bacteria.</div></div><br />
</div><br />
<p>We analysed the scans and obtained the following results.</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/2/24/KCl_stack.PNG" alt="" class="img-responsive" /></p><br />
<strong>Figure 3.&nbsp;</strong> Histogram of KCl stressed cells and non-stressed cells.<br />
<br />
<br/><br/><br />
<br />
<hr><br />
<br />
<h3>BioPad Detector: Detection of sfGFP</h3><br />
<br />
<p> To get a first idea of how our BioPad was able to detect signal, we attempted to detect sfGFP signal emission by exciting our sfGFP with a 470 nm emitting LED and acquiring the images through our device. A sample of the video taken can be seen here bellow: </p><br />
<br />
<div class = "cntr"><br />
<video width="500" height="300" controls><br />
<source src="https://static.igem.org/mediawiki/2014/2/2f/Gfptracking_EPFL.mp4" type="video/mp4"><br />
</video><br />
</div><br />
<br />
<p><br />
To learn more about how the detector works check out our <a href="https://2014.igem.org/Team:EPF_Lausanne/Hardware"> Hardware page!</a><br />
</p><br />
<br />
<!--<h4> References </h4><br />
<p><br />
<a id="ref1"></a>1: S.K. Hatzios, S. Ringgaard, B. M. Davis, M. K. Waldor (2012, August 15). Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition. <i>Plos One</i>.<br />
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<li><a href="#IFP_Exp1">AraC/PBad Promoter & GFP fused to CpxR</a></li><br />
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<li><a href="#IFP_Exp4"></b>Microscopy of KCl stress </a></li><br />
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<li><a href="#Yeast_exp3">The split-Luciferase strain stress response</a></li><br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/OverviewTeam:EPF Lausanne/Overview2014-10-18T00:59:58Z<p>Robert Baldwin: </p>
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<h1 class="cntr">Overview</h1><br />
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<h2 class="section-heading" id="title_intro">Introduction</h2><br />
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Biological responses can be quick and precise! This is the message that our team wants to convey! With this in mind, our team brought forward a novel idea: <br />
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Combining Protein Complementation techniques with biosensors and microfluidics, allows fast spatiotemporal analysis of bacterial/yeast responses to stimuli.<br />
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Touch_bacteria_EPFL_Ted.png" alt="touch bacteria" width="190" class="pull-left img-left img-border" /><br />
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Biological responses can be quick! This is the message that the 2014 EPFL iGEM team want to convey. <br />
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<br /><br /><i>The BioPad project is intended to be a solid proof-of-concept of the combination of biosensors with protein complementation techniques to achieve fast spatiotemporal analysis of bacterial or yeast response to mechanical stimuli.</i> <br />
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<p class="lead"><br />
Our team explored this hypothesis by engineering two stress related pathways in <i>E. coli</i> and <i>S. cerevisiae</i> with in mind the development of a BioPad: a biological touchscreen consisting of a microfluidic chip, touch responsive bacteria/yeast, and a signal detector. Learn more about <a href="#howitworks">how the BioPad works !</a> </p><br />
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The pathway engineered in <i>E. coli</i>, the Cpx Pathway, is a two-component (CpxA and CpxR) regulatory system responsive to envelope stress. It controls the expression of "survival" genes whose products act in the periplasm to maintain membrane integrity. CpxA intermembrane kinase activates and autophosphorylates when sensing misfolded protein (due to stress for example). CpxA phosphorylates CpxR which homodimerizes, acting as a transcription factor. A full description of the pathway is available <a href="https://2014.igem.org/Team:EPF_Lausanne/Envelope_stress_responsive_bacteria">Envelope Stress Responsive Bacteria page !</a></p><br />
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<p class="lead"><br />
The pathway engineered in <i>E. coli</i>, the Cpx Pathway, is a two-component regulatory system responsive to envelope stress. A full description of the pathway is available <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Envelope_stress_responsive_bacteria#cpx">here.</a><br />
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In <i>S. cerevisiae</i> we modified the HOG Pathway - a MAPKK pathway responsive to osmotic stress. For more information concerning the HOG Pathway click <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Yeast">here.</a><br />
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<a href="https://static.igem.org/mediawiki/2014/f/fd/CpxR-HOG.jpg" data-lightbox="cpxr" data-title="EPFL microfluidic chips"><br />
<img src="https://static.igem.org/mediawiki/2014/f/fd/CpxR-HOG.jpg" alt="touch response" class="img-responsive" />Hello</a><br />
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<a href="https://static.igem.org/mediawiki/2014/d/d8/EPFLmicrofluidics.JPG" data-lightbox="image-1" data-title="EPFL microfluidic chips"><img src="https://static.igem.org/mediawiki/2014/d/d8/EPFLmicrofluidics.JPG" width="200" class="img-border"></a><br />
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<p class="lead"><br />
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Our project also includes an extensive <b>microfluidics section</b>. Our self designed chips helped us improve precision, safety, and quantification methods used throughout the project. To learn more about the microfluidic components of our project check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Microfluidics">this link.</a></p><br />
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<div class="pull-right img-right"><br />
<img src="http://www.raspberrypi.org/wp-content/uploads/2011/07/RaspiModelB.png" alt="first" width="200" class="img-border"><br />
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<p class="lead"><br />
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Last but not least, we designed a <b>novel signal detector</b> ! To make signal detection more practical we developed an automatised cheap tracking system made of a mini-computer (Raspberry Pi) and a mini-HD camera. More details concerning this the BioPad detector can be found <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Hardware">here.</a><br />
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<h2 id="CpxPathway">The Cpx Pathway in <i> E.Coli </i></h2><br />
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The natural function of the Cpx two component regulatory system in bacteria is to control the expression of ‘survival’ genes whose products act in the periplasm to maintain membrane integrity. This ensures continued bacterial growth even in environments with harmful extracytoplasmic stresses. The Cpx two component regulatory system belongs to the class I histidine kinases and includes three main proteins: CpxA, CpxR, and CpxP. For more detailed information about the Cpx pathway check out our <a href="https://2014.igem.org/Team:EPF_Lausanne/Envelope_stress_responsive_bacteria">Envelope Stress Responsive Bacteria page !</a></p><br />
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<h2 class="section-heading" id="howitworks">How the BioPad works - <i>E. coli</i></h2><br />
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<a href="https://static.igem.org/mediawiki/2014/b/b7/Puce_petit.jpg" data-lightbox="image-0" data-title="A potential BioPad"><br />
<img src="https://static.igem.org/mediawiki/2014/b/b7/Puce_petit.jpg" alt="Potential biopad" height="200" class="img-border" /></a><br />
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<p class="lead"><br />
Our BioPad is a self-designed PDMS microfluidic chip, made of thousands of compartments representing “pixels”. Each 300μm x 300μm x 50μm compartment contains a few layers of <i>E. coli</i>. When the surface of the chip is touched, a deformation on the chip - and thus of the compartments – leads to cellular membrane shear stress and protein aggregation/misfolding in the periplasm.<br/> <br/></p><br />
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The histidine kinase sensor CpxA auto-phosphorylates and transfers its phosphate to its corresponding relay protein, CpxR, resulting in its dimerization. We engineered the pathway, by fusing split reporter protein fragments to the CpxR (IFP1.4). This allows the two fragments to remain inactive until physical interaction of CpxR (stimulated by envelope stress) leads to the proper folding of IFP1.4 and reconstitution of its fluorescent properties. As the reconstitution of the split fragments of IFP1.4 are reversible, the system can be shutdown upon stress removal (CpxA changes conformation to become a phosphatase and induces CpxR’s dissociation).<br/> <br/> <br />
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The BioPad also includes a signal detector. The BioPad Detector is composed of an inexpensive credit card-sized single-board computer called Raspberry Pi, a highly sensitive digital camera with appropriate light filters, and a light-emitting source. It identifies and processes the position of the light/fluorescence emitted by the BioPad. The information about the position of the light relative to chip is then used to control the associated electronic device. <br/> <br />
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Our self-designed PDMS microfluidic chip, the BioPad, is made of hundreds of compartments representing "pixels." Each 30µm x 30µm x 3µm compartment contains a few layers of <i>E. coli</i>. When the surface of the chip is touched, a deformation of the chip - and thus of the compartments - leads to cellular membrane shear stress and protein aggregation/misfolding in the periplasm. <br />
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The aggregated/misfolded proteins are then sensed by the histidine kinase CpxA sensor, which auto-phosphorylates and transfers its phosphate to its corresponding relay protein, CpxR. Upon phosphorylation, CpxR homo-dimerizes. <br />
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Our engineered bacteria contain CpxR proteins fused to split fluorescent protein fragments (split IFP1.4) via a 10-amino acid, 2x GGGGS flexible linker. This allows us to detect CpxR dimerization, synonymous periplasmic stress and touch. Moreover, the split protein fragments are reversible. Therefore, when stress is removed, CpxA changes conformation and dephosphorylates CpxR allowing it to dissociate. The signal is shutdown and darkness returns The BioPad Detector (composed of an inexpensive CMOS called Raspberry Pi, a highly sensitive digital camera with appropriate light filters, and a light emitting source) identifies and processes the position of the light/fluorescence emitted by the BioPad. This information about the position of the light relative to chip is then used to control the associated electronic device.<br />
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<a href="https://static.igem.org/mediawiki/2014/1/15/Screen_Shot_2014-10-12_at_3.29.30_PM.png" data-lightbox="image-0" data-title="Fluorescence"><br />
<img src="https://static.igem.org/mediawiki/2014/1/15/Screen_Shot_2014-10-12_at_3.29.30_PM.png" alt="touch bacteria" height="200" class="img-border" /></a><br />
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<h2 id="thehogpathway">The HOG Pathway</h2><br />
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<p class="lead">The HOG (High Osmolarity Glycerol) pathway is a MAPK (Mitogen activated protein kinase) pathway which yeast cells use to coordinate intracellular activities to optimise survival and proliferation in not only hyper-osmotic stress but also heat shock, nitrogen stress and oxidative stress. It is represented below.</p><br />
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<img src="https://static.igem.org/mediawiki/2014/6/6d/Hog_pathway_copy.jpg" width="750" alt="HOG_pathway_description"><br />
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<p> The pathway includes five main proteins:</p><br />
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<li><p class="lead">Sho1/Sln1 – Membrane proteins which are classed as STREs (STress Response Elements) which sense the stress and initiate the pathway</p></li><br />
<li><p class="lead">Ste11 – The MAPKKK which phosphorylates PBS2</p></li><br />
<li><p class="lead">PBS2 – The MAPKK which phosphorylates HOG1</p></li><br />
<li><p class="lead">HOG1 – The MAPK which localizes to the nucleus upon phosphorylation and induces target gene transcription</p></li><br />
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<h2 id="howweengineered">Engineering the HOG pathway in <i>S. cerevisiae</i></h2><br />
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<p class="lead"><br />
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The <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Yeast">HOG pathway</a> controls the response to osmotic stress in yeast cells.<br />
Like bacteria, our modified <i>S. cerevisiae</i> cells can be loaded into the PDMS microfluidic chips we have designed. Thus cells are confined in small compartments made of a rather soft material. When the surface of the chip is touched, it leads to a deformation of the chip and its chambers. Since the HOG pathway is reactive to turgor pressure, the mechanical pressure applied activates it. Upon induction of the pathway, which is a classical MAP kinase pathway, PBS2 – a MAPKK – is phosphorylated and binds HOG1 – a MAPK – and in turn phosphorylates it.<br />
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<a href="https://static.igem.org/mediawiki/2014/7/7b/Schema_split.png" data-lightbox="img1" data-title="Schema of Hog1/Pbs2 split sfGFP and split rLuc"><br />
<img src="https://static.igem.org/mediawiki/2014/7/7b/Schema_split.png" width="55%" alt="" class="pull-left img-left img-border"></a><br />
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<p class="lead"><br />
Therefore, we have fused these two kinases to split fluorescent and luminescent proteins, via a 13-amino acid flexible linker, by homologous recombination. This allows us to detect the phosphorylation of Hog1 by Pbs2 in response to osmotic pressure or touch. We have used split sfGFP and split Renilla luciferase tags on the C-terminals of both proteins.<br />
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The split sfGFP is irreversible and was made to show the interaction between our two Pbs2 and Hog1 while we use the reversible split luciferase tags to assess the activation and inactivation of the pathway. In fact, when stress is removed, the signal should decline. The <b>BioTouch Detector </b> is programmed to identify and process the light position and can transmit the information to a computer.<br />
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<h2 class="section-heading" id="thebiopad">The BioPad Detector</h2><br />
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The signals induced by the BioTouch Chip are then processed by our self designed detection system: the BioTouch Detector. The BioTouch Detector is mainly made of a cheap computer (Raspberry Pi), a highly sensitive digital camera with appropriate light filters, and a light emitting source. The BioTouch Detector locates signals from various sources (infrared fluorescence, green fluorescence and luminescence), processes them and sends back the relative positions of the signals with respect to the BioTouch Pad. Thanks to this position, we are able to extract information such as giving a computer operating system that the position represents the position of the mouse on a screen, that the well at the given position is a suitable antibiotic candidate, or that a gene of interest has been activated. We therefore effectively control a computer or any other electronic device through a living interface: the BioTouch Pad.<br />
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<li><a href="#howweengineered">Engineering the HOG pathway in <i>S. cerevisiae</i></a> </li><br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/ResultsTeam:EPF Lausanne/Results2014-10-18T00:52:31Z<p>Robert Baldwin: </p>
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<!-- RESULTS --><br />
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<h1 class="cntr"> <b>RESULTS</b> </h1><br />
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<a id="IFP_Exp"></a><br />
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<h2> <b> Characterisation of the spatiotemporal dynamics of the CpxR stress sensor </b> </h2<br />
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<h3><b>Demonstration of CpxR's dimerization & Elucidation of its dimerization orientation</b></h3><br />
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Previous in vitro experiments (FRET) have shown that CpxR dimerizes. Currently little in vivo information about CpxR’s dimerization is available. To characterize our stress responsive bacteria, we had to confirm that CpxR dimerized in vivo as well as elucidate CpxR’s dimerization orientation. <br />
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We synthesized four constructs with combinations of the split IFP1.4 fragments fused to the C or N terminal of CpxR. <br />
As seen in the graph bellow, induction of IFP1.4 signal by 50 mM KCl was done at t=24min. We clearly see that the construct with IFP fragments on the C-terminal immediately responded to stress by emitting fluorescence. In fact we observe a 3 fold signal increase in 2 minutes. On the other hand, the three other orientations were non-responsive to KCl stress.</p><br />
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<a href="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" data-lightbox="chips" data-title="Construct Comparison"><img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" width="80%"/></a><br /><br />
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<p><br />
This amazing 30-fold signal increase in little time from the baseline allows us to assert that only the strain synthesizing the IFP fragments on the C-terminals of CpxR responded to KCl stress. For a further analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
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<p><br />
<u>Introduction</u> <br /><br />
CpxR is the relay protein in the stress responsive CpxAR two component regulatory system. It has been shown by split beta galactosidase assay that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that <i>E. coli</i> CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in <i>E. coli</i>.</p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to determine if and how CpxR dimerised in vivo in <i>E. coli</i>. This experiment intended to get a first idea of the real-time temporal dynamics of the activation of CpxR (the cytoplasmic relay protein of the CpxA-R pathway) by KCl stress via CpxA (the periplasmic sensor protein of the CpxA-R pathway). This experiment is a first of its kind.<br />
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<p><br />
<u>Methods</u> <br /><br />
To evaluate if and how CpxR dimerized under KCl stress, we built by gibson assembly four constructs with the various possible orientations that the split IFP1.4 fragments could have with CpxR: IFP[1] and IFP[2] on the N-terminus of CpxR, IFP[1] on the N-terminus of CpxR and IFP[2] on the C-terminus of CpxR, and finally IFP[1] and IFP[2] on the N-terminus of CpxR. The split IFP fragments were provided by the Michnick Lab, and the CpxR coding region was amplified by PCR from extracted <i>E. coli</i> genome (Bacterial Genomic Miniprep Kit from Sigma Aldrich). The protocol for stressing the cells and reading the fluorescence can be downloaded <a target="blank" href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the graph bellow, induction of the signal was done at minute 24 (marked via a vertically spoted line). The construct with IFP fragments on the C-termina responded immediately to stress. In a fact we observed a 3 fold signal increase in 2 minutes. All other constructs we observed a low baseline signal non responsive to KCl stress. It is to be noted that the C-termina constructs always had higher signal levels than the other constructs. This leads us to believe that the PBS used to resuspend our cultures led to small levels of stress (the PBS we use does not contain KCl but traces of NaCl). The 30-fold signal increase from the baseline allows us to assert that our constructs responds to KCl stress.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" alt="Construct Comparison" class="img-responsive"><br />
</div><br />
<p><br />
<u>Discussion</u> <br /><br />
We successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus. This finding suggests that CpxR binds via its C-termina. This leads us to hypothesise that the CpxR dimerisation mechanisms is the same for other members of the highly conserved OmpR/PhoB subfamily. This hypothesis could allow the development of similar system that could the study other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.<br />
</p><br />
<br /><br /><br />
--><br />
<br />
<br />
<br />
<br /><br /><br />
<br />
<a id="IFP_Exp3"></a><br />
<h3><b>Signal induction by various concentrations of KCl & signal shutdown by centrifugation</b></h3><br />
<br />
<p><br />
Having confirmed that KCl was a good inducer for our signal, we tested different concentration of KCl to modulate the signal and better characterize our biobrick. We also aimed to shutdown the signal by centrifugation and medium change. The signal was measured on a plate reader 20 minutes, before the addition of KCl. After 2 hours of measurement we centrifuged the cells for ten minutes and replaced the medium with PBS to be able to get a shutdown of the signal.</p><br />
<br />
<div class="cntr"><br />
<a target="_blank" href="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" data-lightbox="chips" data-title="The split-IFP are linked to cpxR on the C-terminal. During the experiment it was possible to shut down the signal"><img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" width="80%"/></a><br /><br />
</div><br />
<br />
<br />
<p><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to drastically shut down the signal, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis is possible for our system. For a thorough analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
</p><br />
<br />
<br />
<!--<br />
<p><br />
<u>Aim</u> <br /><br />
Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change.<br />
To do so, we read our signal for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To evaluate if a modulation in KCl concentrations affected the intensity of the intensity of the fluorescent signal, and if a change in medium by centrifugation shutdown the signal; we read our signal on a plate reader for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal. The protocol for this experiment can be downloaded <a target="_blank" href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis are possible for our system.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" alt="GA1 Shutdown" class="img-responsive"><br />
</div><br />
<br /><br /><br/><br/><br />
--><br />
<br />
<br /><br /><br />
<br />
<a id="IFP_Exp4"></a><br />
<h3><b>Visualization of the the CpxR split IFP1.4 activation by KCl stress </b></h3><br />
<br />
<br />
<p><br />
<br />
<br />
Having shown that we were able to monitor the temporal dynamics of CpxR activation, we wanted to see if we could analyze CpxR’s spatial dynamics by microscopy. 10μl of our cells, previously stressed with 80 mM KCl were spread on a glass slide and imaged with a x100 objective and a APC (Cy5.5) filter.<br />
<br />
<br />
</p><br />
<br />
<br />
<p><br />
<br />
<br />
We noticed various characteristics of the cells from the picture bellow. First, IFP signal was much more present in stressed bacteria rather than in non-stressed bacteria. Secondly, we distinguished two specific phenotypes within bacteria: elongated and normal cells. We noticed that this difference was due to CpxR overexpression as we saw this phenomenon also in non-stressed conditions.<br /><br /><br />
<br />
<br />
In elongated cells, we were able to distinguish several bright bands of IFP signal that seem fairly uniformly distributed. In the normal phenotype we distinguished a single band in the centre of the bacteria. These observations led us to believe that CpxR might be involved in the division process of E. coli as it seems coherent for cells to slow down division upon stress. <br /><br /><br />
<br />
<br />
After looking into the literature, similar bands were visualizable in E. coli for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how E. coli monitor division under various environments. For a thorough analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
<br />
<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" data-lightbox="results" data-title="Results"><img src="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" alt="results" width="45%" class="pull-left"></a><br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/EPFL_2014_03_10_2014_Experiment-24.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/d/de/EPFL_2014_03_10_2014_Experiment-34.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/fd/EPFL_2014_03_10_2014_Experiment-35.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/0/08/EPFL_2014_03_10_2014_Experiment-37.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/a/a7/EPFL_2014_03_10_2014_Experiment-38.jpg" data-lightbox="results" data-title="Results"></a> <br />
<a href="https://static.igem.org/mediawiki/2014/2/2e/EPFL_2014_03_10_2014_Picture3.jpg" data-lightbox="results" data-title="Results"><br />
<a href="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" data-lightbox="results" data-title="Results"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" alt="results" width="45%" class="pull-right"></a><br />
</div><br />
<br />
<br />
<br />
<!--<br />
<br />
<p><br />
<u>Aim</u> <br /><br />
Having shown that we were able to monitor the temporal dynamics of our construct, we wanted to see if we were able to analyze the spatial dynamics by microscopy.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To visualize the activation of our construct, we prepared cells as above for the previous plate-reader experiments, spread 10 µl on a glass slide added a coverslip and imaged them on a Zeiss Axioplan with a x100 objective and a APC (Cy5.5) filter. The pictures shown bellow were taken with a 5.1(s) integration time.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the pictures bellow, we were able to distinguish specific patterns within bacteria. We observed two phenotypes within our population: elongated and normal cells. The difference in these phenotypes was noticed in previous experiments and is most certainly due to the CpxR overexpression as we observed this also in non-stressed conditions. In the first phenotype (elongated) we were able to distinguish several bands that seem fairly uniformly distributed. In the second phenotype (normal) we observed a single band in the center of the bacteria. These observations led us to believe that CpxR might be involved in the division process of <i>E. coli</i> as it seems coherent for cells to slow down division upon stress. After looking into the literature, similar bands were visualizable in <i>E. coli</i> for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how <i>E. coli</i> monitor division under various environments.<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"><img src="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" alt="results" width="45%" class="pull-left"></a><br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/EPFL_2014_03_10_2014_Experiment-24.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/d/de/EPFL_2014_03_10_2014_Experiment-34.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/fd/EPFL_2014_03_10_2014_Experiment-35.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/0/08/EPFL_2014_03_10_2014_Experiment-37.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/a/a7/EPFL_2014_03_10_2014_Experiment-38.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a> <br />
<a href="https://static.igem.org/mediawiki/2014/2/2e/EPFL_2014_03_10_2014_Picture3.jpg" data-lightbox="results" data-title="IFP signal in stressed cells"><br />
<a href="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" data-lightbox="results" data-title="IFP signal in non stressed cells"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" alt="results" width="45%" class="pull-right"></a><br />
</div><br />
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--><br />
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<!--<br />
<a id=“CpxR-promoter"></a><br />
<h3><b>RFP under CpxR promoter to investigate time delay between transcription factor activation and protein synthesis</b></h3><br />
<br />
<p>To mesure the time it takes a cell to react to a stress input and if our modified CpxR still has its function as a transcription factor, we wanted to investigate the RFP expression of stressed cells that we co-transformed our CpxR-split-IPF construct with the <a target="_blank" href="http://parts.igem.org/Part:BBa_K339007">BBa_K339007</a>. Unfortunately we realised that the regulatory sequence of the RFP, the CpxR responsive promoter, was missing. So we constructed the biobrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486048">BBa_K1486048</a> which contains the regulatory sequence. After a few experiments we realised that KCl stress, which we have found to be a good signal inducer, did not work, even in cells expressing native CpxR. So we developed the biobricks <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486049">BBa_K1486049</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486050">BBa_K1486050</a>, which encode for RFP regulated by the CpxR responsive promoter, how it is found in the genome. In the figure below you see that neither of the three constructs was stress induced.</p><br />
<br />
<div class="cntr img-border"><br />
<a href="https://static.igem.org/mediawiki/2014/9/91/BBa_K1486049_BBa_K1486048.png" data-lightbox="cpxr" data-title="CpxR promoted RFP under stress"><br />
<img src="https://static.igem.org/mediawiki/2014/9/91/BBa_K1486049_BBa_K1486048.png" alt="CpxR promoted RFP under stress" class="img-responsive" /></a><br />
</div><br />
<br />
<br/><br/><br/><br />
--><br />
<br />
<a id="IFP_Exp5"></a><br />
<h3><b>Activation of CpxR - split IFP1.4 on microfluidic chip by chamber crushing </b></h3><br />
<br />
<p><br />
Knowing that we were able to able to visualize CpxR-IFP activation under a microscope, we proceeded to trying to activate the pathway by mechanical stress on a microfluidic chip - the ultimate barrier to building a functional BioPad !<br /><br /><br />
<br />
To induce this stress, we turned on the buttons of our SmashColi microfluidic chip at a pressure of 25 psi. We imaged chambers before stress and after stress (10 min after button activation). A drastic increase in signal was detected !<br />
<br />
<div class="cntr"><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/d/dc/Button_IFP_non_stressed_2_Cy5.jpg" data-lightbox="results_button" data-title="results_button"><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Button_IFP_non_stressed_2_Cy5.jpg" alt="results" width="45%" class="pull-left"> <br />
</a><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/7/77/Button_IFP_non_stressed_2.jpg" data-lightbox="results_button" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/9/99/Button_IFP_stressed_2.jpg" data-lightbox="results_button" data-title="Results"></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/f/f8/Button_IFP_stressed_2_Cy5.jpg" data-lightbox="results_button" data-title="results_button"><img src="https://static.igem.org/mediawiki/2014/f/f8/Button_IFP_stressed_2_Cy5.jpg" alt="results" width="45%" class="pull-right"> <br />
</a><br />
<br />
</div><br />
<br />
<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
<a id="IFP_Exp1"></a><br />
<h3> <b> Characterisation of the pBAD promoter and folding ability of GFP fused to CpxR </b></h3><br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg" data-lightbox="img1"><img src="<br />
https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg" width="70%"></a><br />
</div> <br /><br /><br />
<br />
<p>This construct aimed to evaluate the expression of our construct and the characteristics of the arabinose promoter in <i>E. coli</i> by fusing a superfolder GFP protein to CpxR. The sfGFP was chosen because of its higher intensity compared to GFP. </p><br />
<p>Not knowing if CpxR would react the same way if sfGFP were attached to the N or C terminus, 2 biobricks were built, one with each of the orientations: <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486002">BBa_K1486002 (N terminus)</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486005">BBa_K1486005 (C terminus)</a>.<br />
</p><br />
<p><br />
We tested both constructs CpxR-sfGFP and sfGFP-CpxR on a plate reader to characterize the pBAD promoter, measuring the sfGFP signal in function of the arabinose concentration. We also measured the signal in a microfluidic chip. You can see in the graph below that the signal is increasing as the concentration of the arabinose increases. The construct with sfGFP at the C terminal lead to a higher GFP signal. Interestingly both curves increases in a very similar manner.<br />
<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/a/ac/Nterm_and_Cterm_log.png" data-lightbox="hellopony"><img src="<br />
https://static.igem.org/mediawiki/2014/a/ac/Nterm_and_Cterm_log.png" width="50%"></a><br />
</div><br />
<br/><br/><br />
<hr/><br />
<br />
<h2 id="Characterisation_of_the_split_luciferase"> <b>Characterisation of the split luciferase </b> </h2><br />
<br />
<h3 id="Characterisation_of_the_split_luciferase"><b>Split luciferase complementation assay using CheY and CheZ chemotaxis proteins </b> </h3><br />
<br />
<p><br />
CheY and CheZ are two proteins involved in the bacterial chemotaxis pathway. It has been shown by split luciferase complementation assay that these two proteins are not interacting in presence of chemoattractant, but start to interact in absence of chemoattractant or presence of chemorepellent. Based on the work of Waldor<sup><a href="#ref1">1</a></sup> Laboratory, we wanted to redo the experiment to test our own splits, with firefly (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486055">BBa_K1486055</a>) and renilla (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486054">BBa_K1486054</a>) luciferases.<br />
</p><br />
<br />
<p>As shown in the graphs (fig.1A and 1B), we didn't observe a high signal for our assay. However, the signal being higher than the blanks, it is an encouraging sign that the splits luciferase can be used for other experiments of this kind. A possible explanation for these results is that we didn't completely get rid of the arabinose, which acts as a chemoattractant. Moreover, CheY and CheZ being endogenously expressed in bacteria, there could be interferences with our fusion proteins and weakening of our signal. This should be tested again with CheY/CheZ knock out strains.<br /><br />
</p><br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Renilla"><img src="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Firefly"><img src="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
</div><br /><br />
<br /><br />
<p>We also could determine which of the luciferases would best suit our following experiments. As shown in fig. 2, for the same concentration of substrate, we see that firefly luciferase has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.</p><br /><br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" data-lightbox="chips" data-title="Bioluminescence assay"><img src="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" width="80%"/></a><br /><br />
</div><br />
</p><br />
</div><br />
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<!--<br />
<div class="cntr"><br />
<h2 id="Characterisation_of_the_split_luciferase"> <b><u>Characterisation of the split luciferase </u> </b> </h2><br />
</div><br />
<br /><br /><br />
<br />
<h3 id="Characterisation_of_the_split_luciferase"><b>Experiment 1: </b>CheY/CheZ fused to split Firefly/Renilla luciferase, and full Firefly/Renilla luciferase characterisation </h3><br />
<p><u>Introduction</u> <br /><br />
CheY and CheZ are two proteins involved in the bacterial chemotaxis pathway. It has been shown by split luciferase complementation assay that these two proteins are not interacting in presence of chemoattractant, but start to interact (CheZ being the phosphatase of CheY) in absence of chemoattractant or presence of chemorepellent. Based on the work of Waldor<sup><a href="#ref1">1</a></sup> Laboratory, we wanted to redo and adapt the experiment to test our own splits.<br /> <br /></p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to test the efficiency of split Renilla luciferase and split Firefly luciferase. We wanted to study the speed of the signal and the amount of substrate needed to have a performant response. <br /> <br /><br />
</p><br />
<p><br />
<u>Method</u> <br /><br />
To proceed to this complementation assay, we built two constructs, one to test split Renilla Luciferase and the other for split Firefly Luciferase The CheY was fused to the N-terminal part of each split, while the CheZ was fused to the C-terminal part. We used the full luciferases (Renilla : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486022"> BBa_K1486022 </a> and Firefly : <a target="_blank" href="http://parts.igem.org/Part:BBa_K325108"> BBa_K325108 </a> from Cambridge 2010 team) as positive controls and the non-fused splits (Renilla : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486021"> BBa_K1486021 </a> and Firefly : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486018"> BBa_K1486018 </a>) as negative controls.<br /> <br /><br />
The bioluminescence assay was performed as described <a target="_blank" href="https://static.igem.org/mediawiki/2014/6/6d/Protocol_-_Bioluminescence_assay.pdf">here</a>. <br /><br />
The constructs were designed and assembled as described <a target="_blank" href="https://static.igem.org/mediawiki/2014/3/3b/Constructs_design_CheYCheZ.pdf">here</a>.<br /><br /> <br /><br />
</p><br />
<p><u>Results</u> <br /><br />
As shown in the graphs below (fig.1A and 1B), we couldn't really observe a high signal for our assay. However, the signal being higher than the blanks, it is an encouraging sign that the splits luciferase can be used for other experiments of this kind. A possible explanation for these results is that arabinose being a chemoattractant, we might need to do more wash steps with PBS to get rid of the arabinose before taking the measurements. Moreover, CheY and CheZ being endogenously expressed in bacteria, the edogenous proteins could interfere with our fusion proteins and weaken our signal. This complementation assay should be tested with CheY/CheZ knock out strains, as it was done in Waldor Laboratory.<br /><br />
</p><br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Renilla"><img src="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Firefly"><img src="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
</div><br /><br />
<br /><br />
We also could determine which of the luciferases would best suit our following experiments. As shown in fig. 2, for the same concentration of substrate, we see that firefly luciferase has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.<br /><br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<br /><br />
--><br />
<br />
<br />
<a id="Yeast_experiments"></a><br />
<br />
<h2> <b>The PBS2-HOG1 split-GFP & split Renilla Luciferase stress response<br />
</b> </h2><br />
<br />
<br/><br/><br />
<a id="Yeast_exp1"></a><br />
<h3><b>Confirmation of successful transformation via the Renilla Luciferase tag </b></h3><br />
<br/><br />
<p><br />
<br />
Since we had never transformed <i>S. cerevisiae</i>, we first needed to confirm our protein tag transformations as a positive control for subsequent experiments. We cultured the two strains we got from the transformation, the HOG1-rLuc (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486033">BBa_K1486033</a> ) and PBS2-rLuc (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486027">BBa_K1486027</a>), put them in a 96 well plate and tested their luminescence using a plate reader.</p><br />
<p><br />
<br />
The test was performed using two different concentrations of the substrate, coelenterazine-h(benzyl coelenterazine): 1μM and 5μM so that we could determine the optimal concentration we would use for the next assays. The plate started to be read directly after addition of the substrate in the dark and measures were taken every 56 seconds thereafter. Three wells for each sample were measured and below are shown the graphs of their average luminescence plotted over time. We observed a signal-to-noise ratio of 4 at 1μM of substrate and 10 at 5μM for the PBS2-rLuc strain. The HOG1-rLuc strain seemed problematic and died within the plate. Unable to verify the correct transformation, we reproduced the strain but were unable to confirm its validity before the wiki deadline.</p><br />
<br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/0e/Pbslucprettycomparaison_.png" data-lightbox="chips" data-title=""><img src="https://static.igem.org/mediawiki/2014/0/0e/Pbslucprettycomparaison_.png" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<p><br />
There is a clear luminescence generated by the cells, providing us with confirmation that our designs are correct and we determined which concentration of substrate to use to have a decent signal-to-background ratio.<br />
</p><br />
<br />
<br />
<a id="Yeast_exp2"></a><br />
<h3><b>The split-sfGFP strain stress-response</b></h3><br />
<p><br />
<br />
<br />
Having confirmed that our design for yeast transformations was correct via the previous experiment, we performed stress-response tests upon our PBS2-HOG1-splitGFP (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486029">BBa_K1486029</a>) and (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486035">BBa_K1486035</a>) strain to determine whether the pathway is reactive to stress as theorized. A co-transformation was performed using linear fragments produced in the same way as for the first experiment to obtain the strain. The N-terminal split of the superfolder GFP was tagged onto PBS2 and the C-terminal onto HOG1. The strain was then tested along with non-transformed cells in a 96 well plate with various stresses (e.g. Acetic Acid, Ethanol, Glucose...). The cells were centrifuged and resuspended in PBS before loading into the plate.<br />
</p><br />
<br />
<br />
<div class="cntr"><br />
<br />
<div class="cntr"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/1/1d/Splitgfpgraph.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<p><br />
The most reactive of stresses turned out to be Acetic Acid 3.6%(shown above) followed by Ethanol 10%. Other stresses tested did not seem to show conclusive results and we were unable to determine more reactants before the wiki freeze deadline. To further assess the fluorescence 10μl of our cells were spread on a glass slide and imaged with a x63 objective and a Green LP filter<br />
(Excitation BP 450-490, Dichroïc FT 510, Emission LP 515). Below is shown a comparison of before and after 3.6% Acetic Acid stress in the microscopy image merged with the fluorescence. <br />
</p><br />
<br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/3b/Splitgfpyeast.jpg" data-lightbox="caca" data-title="Image of our split sfGFP expressing cells before and after the 3.6% acetic acid stress (x63 objective and a Green LP filter)"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3b/Splitgfpyeast.jpg" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<br />
<div class="cntr"><br />
<p><br />
For a more quantitative measure, the fluorescent cells to total cells ratio was calculated and illustrated below.<br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Splitgfpcount.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<br />
<p><br />
<br />
<br />
From the data shown above, we can see that fluorescence increases within cells in our split sfGFP strain in response to stress while it does not in non-transformed cells. We can thus conclude that the orientation of the split sfGFP constructs added to PBS2 and HOG1 allows proper re-assembly upon interaction of the two proteins. This result shows promise for the actual application of the project as a Biopad using yeast cells. <b>It is the first time a split gets succesfully implemented in the HOG1 pathway by direct attachment to proteins of the kinase cascade</b>. Detection of protein–protein interactions within the pathway were previously studied <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/yea.320080602/abstract"> using vesicle targeting</a>.<br />
<br />
<p/><br />
<br />
<br />
<br />
<br />
<a id="Yeast_exp3"></a><br />
<h3><b>The split-Luciferase strain stress-response</b></h3><br />
<p><br />
After determining that the pathway is reactive to certain stresses and that the split-GFP strain increases its fluorescence, we performed the same test in the obtained split-luciferase (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486030">BBa_K1486030</a>) and (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486036">BBa_K1486036</a>) strain. Unfortunately, due to the difficult nature of detecting an instantaneous luminescence signal and the unstable nature of coelenterazine-Luciferase reaction in different conditions(inhibition due to various molecules such as glucose), our attempts in detecting it on the plate reader failed. Further tests are planned, including an experiment in the microfluidic chips, to directly measure the effect of mechanical pressure, but will not be done before the wiki freeze. <br />
</p><br />
<br />
<br />
<a id="Microfluidics_experiments"></a><br />
<br />
<br />
<br />
<a id="Micro_exp1"></a><br />
<h2><b>Microfluidic achievements</b></h2><br />
<table class="table table-striped valign-middle table-center"><br />
<thead><br />
<tr><br />
<th></th><br />
<th>MITOMI</th><br />
<th>MITOMI modified</th><br />
<th>SmashColi</th><br />
<th>BioPad</th><br />
<th>FilterColi</th><br />
<th>CleanColi</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>Full chip</td><br />
<td class="cntr"><a target="_blank" href="https://static.igem.org/mediawiki/2014/a/ab/Mitomi11.png" data-lightbox="chips" data-title="MITOMI"><img src="https://static.igem.org/mediawiki/2014/a/ab/Mitomi11.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/6/64/Mitomimodif1.png" data-lightbox="chips" data-title="MITOMI Modified"><img src="https://static.igem.org/mediawiki/2014/6/64/Mitomimodif1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/1/15/Smash1.png" data-lightbox="chips" data-title="SmashColi"><img src="https://static.igem.org/mediawiki/2014/1/15/Smash1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/d/db/Biopad1.png" data-lightbox="chips" data-title="BioPad"><img src="https://static.igem.org/mediawiki/2014/d/db/Biopad1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/d/db/Filter1.png" data-lightbox="chips" data-title="FilterColi"><img src="https://static.igem.org/mediawiki/2014/d/db/Filter1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/b/b0/Screen_Shot_2014-10-16_at_22.15.39.png" data-lightbox="chips" data-title="CleanColi"><img src="https://static.igem.org/mediawiki/2014/b/b0/Screen_Shot_2014-10-16_at_22.15.39.png" width="70%"/></a><br /></td><br />
</tr><br />
<tr><br />
<td>Unit Cell</td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/0/0f/MitomiUnit1.png" data-lightbox="chips" data-title="MITOMI Unit"><img src="https://static.igem.org/mediawiki/2014/0/0f/MitomiUnit1.png" width="50%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/7/78/MitomimodifUnit.png" data-lightbox="chips" data-title="MITOMI Modified Unit"><img src="https://static.igem.org/mediawiki/2014/7/78/MitomimodifUnit.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/3/3a/Smahsunit1.png" data-lightbox="chips" data-title="SmashColi Unit"><img src="https://static.igem.org/mediawiki/2014/3/3a/Smahsunit1.png" width="30%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/6/60/Biopadunit1.png" data-lightbox="chips" data-title="BioPad Unit"><img src="https://static.igem.org/mediawiki/2014/6/60/Biopadunit1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/9/9e/FilterUnit.png" data-lightbox="chips" data-title="FilterColi Unit"><img src="https://static.igem.org/mediawiki/2014/9/9e/FilterUnit.png" width="70%"/></a><br /></td><br />
<td><span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Designed</td><br />
<td><span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
</tr><br />
<tr><br />
<td>Mold fabrication</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Fabrication of the chip</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Application</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br/> <p>To start our microfluidic experiments, we used the <a target="_blank" href="http://link.springer.com/protocol/10.1007%2F978-1-61779-292-2_6">MITOMI chip</a> that was designed in the laboratory of Pr. Maerkl.</p><br/><br />
<br/><br />
<br />
<br />
<br />
<br />
<a id="Micro_exp2"></a><br />
<h3><b>Culturing <i>E. coli</i> with constitutive GFP on chip</b></h3><br />
<p>We loaded <i>E. coli</i>, which contained constitutive GFP, in the chip. By using LabVIEW, a protocol was launched overnight to ensure the growth of the cells (the protocol can be found <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#growth">here</a>).<br />
<br /></p><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/e/e4/Growth_small.gif" data-lightbox="pipi"><img src="<br />
https://static.igem.org/mediawiki/2014/e/e4/Growth_small.gif" width="50%"></a><br />
</div><br />
</p><br />
<p>The next morning, a scan of the chip was done to see the intensity of the GFP in the chip.<br /></p><br />
<div class=""><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/f/fe/Gfp.png" data-lightbox="prout"><img src="<br />
https://static.igem.org/mediawiki/2014/f/fe/Gfp.png" width="50%" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<br /><br />
<br />
<a id="Micro_exp3"></a><br />
<br />
<h3><b>CpxR linked with GFP on the N terminal, induced by arabinose in <i>E. coli</i></b></h3><br />
<p>The experiment that was done on wetbench to show that CpxR linked with GFP was expressed with an arabinose promoter was replicated on a MITOMI chip.<br />
LB medium with arabinose was flowed in the upper half whereas LB medium without arabinose was flowed in the lower half. We scanned every hour for 5h (to know how it was done click <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#date-08-21-2014">here</a>).</p><br />
<div class="row"><br />
<div class="col col-md-6"><a href="https://static.igem.org/mediawiki/2014/4/4e/Truc2.png" data-lightbox="chips" data-title="Scan of the microfluidic chip at t = 0min. No signal is detected."><img src="https://static.igem.org/mediawiki/2014/4/4e/Truc2.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 1.</strong> Scan of the microfluidic chip at t = 0min. No signal is detected<br />
</div></div><br />
<div class="col col-md-6"><br />
<a href="https://static.igem.org/mediawiki/2014/3/32/Truc3.png" data-lightbox="chips" data-title="Scan of the microfluidic chip at t = 300min."><img src="https://static.igem.org/mediawiki/2014/3/32/Truc3.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 2.</strong> Scan of the microfluidic chip at t = 300min.</div></div><br />
</div><br />
<p>We analysed the scans and obtained the following results.</p><br />
<p><a href="https://static.igem.org/mediawiki/2014/4/4c/Gfp_ara.png" data-lightbox="chips" data-title="Figure 3. Evolution of CpxR-GFP fluorescence over time"><img src="https://static.igem.org/mediawiki/2014/4/4c/Gfp_ara.png" alt="" class="img-responsive" /></a></p><br />
<br />
<div class="cntr"> <br />
<strong>Figure 3.&nbsp;</strong> Evolution of CpxR-GFP fluorescence over time<br />
</div> <br />
<br />
<br/><br />
<br />
<a id="Micro_exp4"></a><br />
<br />
<h3><b>On chip infrared detection</b></h3><br />
<p>As we focused part of our work on the IFP1.4, we needed confirmation of this signal detection in our microfluidic chips. Thus the aim of this experiment was to prove this fluorescence detection capability. Bacteria were loaded in the smash-coli chip. The first batch was KCl stressed and the second batch was unstressed. We then simply scanned the chip and analysed the results using ImageJ. For more details, please visit our <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#date-10-03-2014">notebook</a>. </p><br />
<div class="row"><br />
<div class="col col-md-6"><a href="https://static.igem.org/mediawiki/2014/c/cf/No_KCl_red.png" data-lightbox="chips" data-title="Figure 1. Cy5 scan of a chamber containing non-stressed CpxR-IFP bacteria"><img src="https://static.igem.org/mediawiki/2014/c/cf/No_KCl_red.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 1.</strong> Cy5 scan of a chamber containing non-stressed CpxR-IFP bacteria.<br />
</div></div><br />
<div class="col col-md-6"><br />
<a href="https://static.igem.org/mediawiki/2014/c/c2/KCL_red.png" data-lightbox="chips" data-title="Figure 2. Cy5 scan of a chamber containing KCl-stressed CpxR-IFP bacteria"><img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_red.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 2.</strong> Cy5 scan of a chamber containing KCl-stressed CpxR-IFP bacteria.</div></div><br />
</div><br />
<p>We analysed the scans and obtained the following results.</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/2/24/KCl_stack.PNG" alt="" class="img-responsive" /></p><br />
<strong>Figure 3.&nbsp;</strong> Histogram of KCl stressed cells and non-stressed cells.<br />
<br />
<br/><br/><br />
<br />
<hr><br />
<br />
<h3>BioPad Detector: Detection of sfGFP</h3><br />
<br />
<p> To get a first idea of how our BioPad was able to detect signal, we attempted to detect sfGFP signal emission by exciting our sfGFP with a 470 nm emitting LED and acquiring the images through our device. A sample of the video taken can be seen here bellow: </p><br />
<br />
<div class = "cntr"><br />
<video width="500" height="300" controls><br />
<source src="https://static.igem.org/mediawiki/2014/2/2f/Gfptracking_EPFL.mp4" type="video/mp4"><br />
</video><br />
</div><br />
<br />
<p><br />
To learn more about how the detector works check out our <a href="https://2014.igem.org/Team:EPF_Lausanne/Hardware"> Hardware page!</a><br />
</p><br />
<br />
<!--<h4> References </h4><br />
<p><br />
<a id="ref1"></a>1: S.K. Hatzios, S. Ringgaard, B. M. Davis, M. K. Waldor (2012, August 15). Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition. <i>Plos One</i>.<br />
</p>--><br />
</div><br />
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<ul class="nav sidenav box" data-spy="affix" data-offset-top="200" data-offset-bottom="600"><br />
<br />
<li class="active"><a href="#IFP_Exp">Characterization of spatiotemporal dynamics of the CpxR stress sensor</a><br />
<ul class="nav"><br />
<li><a href="#IFP_Exp1">AraC/PBad Promoter & GFP fused to CpxR</a></li><br />
<li><a href="#IFP_Exp2">CpxR dimerization & Orientation Elucidation</a></li><br />
<li><a href="#IFP_Exp3">Titration of KCl & signal shutdown </a></li><br />
<li><a href="#IFP_Exp4"></b>Microscopy of KCl stress </a></li><br />
<li><a href="#IFP_Exp5"></b>Mechanical stress on microfluidic chip</a></li><br />
<br />
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</li><br />
<br />
<li><a href="#Characterisation_of_the_split_luciferase">Luciferases & split luciferases</a><br />
<ul class="nav"><br />
<li><a href="#Characterisation_of_the_split_luciferase">CheY/CheZ - split Firefly/Renilla luciferase & full Firefly/Renilla luciferase</a></li><br />
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</li><br />
<li><a href="#Yeast_experiments">Yeast</a><br />
<ul class="nav"><br />
<li><a href="#Yeast_exp1">Confirmation of successful transformation via the Renilla Luciferase tag</a></li><br />
<li><a href="#Yeast_exp2">The split-GFP strain stress-response </a></li><br />
<li><a href="#Yeast_exp3">The split-Luciferase strain stress response</a></li><br />
</ul> <br />
</li><br />
<br />
</li><br />
<li><a href="#Microfluidics_experiments">Microfluidics</a><br />
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<li><a href="#Micro_exp1">Chips</a></li><br />
<li><a href="#Micro_exp2">Culturing <i>E.coli</i> </a></li><br />
<li><a href="#Micro_exp3">sfGFP-CpxR fusion under pBAD</a></li><br />
<li><a href="#Micro_exp4">On chip infrared detection</a></li><br />
</ul> <br />
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<!-- END ABSTRACT --><br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/ResultsTeam:EPF Lausanne/Results2014-10-18T00:51:00Z<p>Robert Baldwin: </p>
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<!-- RESULTS --><br />
<div class="align-left"><br />
<h1 class="cntr"> <b>RESULTS</b> </h1><br />
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<a id="IFP_Exp"></a><br />
<!--replaced the title : Characterisation of the spatiotemporal dynamics of the CpxR - split IFP 1.4 stress sensor --><br />
<h2> <b> Characterisation of the spatiotemporal dynamics of the CpxR stress sensor </b> </h2<br />
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<a id="IFP_Exp2"></a><br />
<h3><b>Demonstration of CpxR's dimerization & Elucidation of its dimerization orientation</b></h3><br />
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<p><br />
Previous in vitro experiments (FRET) have shown that CpxR dimerizes. Currently little in vivo information about CpxR’s dimerization is available. To characterize our stress responsive bacteria, we had to confirm that CpxR dimerized in vivo as well as elucidate CpxR’s dimerization orientation. <br />
</p><br />
<br /><br />
<p><br />
We synthesized four constructs with combinations of the split IFP1.4 fragments fused to the C or N terminal of CpxR. <br />
As seen in the graph bellow, induction of IFP1.4 signal by 50 mM KCl was done at t=24min. We clearly see that the construct with IFP fragments on the C-terminal immediately responded to stress by emitting fluorescence. In fact we observe a 3 fold signal increase in 2 minutes. On the other hand, the three other orientations were non-responsive to KCl stress.</p><br />
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<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" data-lightbox="chips" data-title="Construct Comparison"><img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" width="80%"/></a><br /><br />
</div><br />
<p><br />
This amazing 30-fold signal increase in little time from the baseline allows us to assert that only the strain synthesizing the IFP fragments on the C-terminals of CpxR responded to KCl stress. For a further analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
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<!--<br />
<p><br />
<u>Introduction</u> <br /><br />
CpxR is the relay protein in the stress responsive CpxAR two component regulatory system. It has been shown by split beta galactosidase assay that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that <i>E. coli</i> CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in <i>E. coli</i>.</p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to determine if and how CpxR dimerised in vivo in <i>E. coli</i>. This experiment intended to get a first idea of the real-time temporal dynamics of the activation of CpxR (the cytoplasmic relay protein of the CpxA-R pathway) by KCl stress via CpxA (the periplasmic sensor protein of the CpxA-R pathway). This experiment is a first of its kind.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To evaluate if and how CpxR dimerized under KCl stress, we built by gibson assembly four constructs with the various possible orientations that the split IFP1.4 fragments could have with CpxR: IFP[1] and IFP[2] on the N-terminus of CpxR, IFP[1] on the N-terminus of CpxR and IFP[2] on the C-terminus of CpxR, and finally IFP[1] and IFP[2] on the N-terminus of CpxR. The split IFP fragments were provided by the Michnick Lab, and the CpxR coding region was amplified by PCR from extracted <i>E. coli</i> genome (Bacterial Genomic Miniprep Kit from Sigma Aldrich). The protocol for stressing the cells and reading the fluorescence can be downloaded <a target="blank" href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the graph bellow, induction of the signal was done at minute 24 (marked via a vertically spoted line). The construct with IFP fragments on the C-termina responded immediately to stress. In a fact we observed a 3 fold signal increase in 2 minutes. All other constructs we observed a low baseline signal non responsive to KCl stress. It is to be noted that the C-termina constructs always had higher signal levels than the other constructs. This leads us to believe that the PBS used to resuspend our cultures led to small levels of stress (the PBS we use does not contain KCl but traces of NaCl). The 30-fold signal increase from the baseline allows us to assert that our constructs responds to KCl stress.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" alt="Construct Comparison" class="img-responsive"><br />
</div><br />
<p><br />
<u>Discussion</u> <br /><br />
We successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus. This finding suggests that CpxR binds via its C-termina. This leads us to hypothesise that the CpxR dimerisation mechanisms is the same for other members of the highly conserved OmpR/PhoB subfamily. This hypothesis could allow the development of similar system that could the study other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.<br />
</p><br />
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<a id="IFP_Exp3"></a><br />
<h3><b>Signal induction by various concentrations of KCl & signal shutdown by centrifugation</b></h3><br />
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<p><br />
Having confirmed that KCl was a good inducer for our signal, we tested different concentration of KCl to modulate the signal and better characterize our biobrick. We also aimed to shutdown the signal by centrifugation and medium change. The signal was measured on a plate reader 20 minutes, before the addition of KCl. After 2 hours of measurement we centrifuged the cells for ten minutes and replaced the medium with PBS to be able to get a shutdown of the signal.</p><br />
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<div class="cntr"><br />
<a target="_blank" href="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" data-lightbox="chips" data-title="The split-IFP are linked to cpxR on the C-terminal. During the experiment it was possible to shut down the signal"><img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" width="80%"/></a><br /><br />
</div><br />
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<p><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to drastically shut down the signal, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis is possible for our system. For a thorough analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
</p><br />
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<!--<br />
<p><br />
<u>Aim</u> <br /><br />
Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change.<br />
To do so, we read our signal for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To evaluate if a modulation in KCl concentrations affected the intensity of the intensity of the fluorescent signal, and if a change in medium by centrifugation shutdown the signal; we read our signal on a plate reader for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal. The protocol for this experiment can be downloaded <a target="_blank" href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis are possible for our system.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" alt="GA1 Shutdown" class="img-responsive"><br />
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<a id="IFP_Exp4"></a><br />
<h3><b>Visualization of the the CpxR split IFP1.4 activation by KCl stress </b></h3><br />
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<p><br />
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Having shown that we were able to monitor the temporal dynamics of CpxR activation, we wanted to see if we could analyze CpxR’s spatial dynamics by microscopy. 10μl of our cells, previously stressed with 80 mM KCl were spread on a glass slide and imaged with a x100 objective and a APC (Cy5.5) filter.<br />
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</p><br />
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<p><br />
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We noticed various characteristics of the cells from the picture bellow. First, IFP signal was much more present in stressed bacteria rather than in non-stressed bacteria. Secondly, we distinguished two specific phenotypes within bacteria: elongated and normal cells. We noticed that this difference was due to CpxR overexpression as we saw this phenomenon also in non-stressed conditions.<br /><br /><br />
<br />
<br />
In elongated cells, we were able to distinguish several bright bands of IFP signal that seem fairly uniformly distributed. In the normal phenotype we distinguished a single band in the centre of the bacteria. These observations led us to believe that CpxR might be involved in the division process of E. coli as it seems coherent for cells to slow down division upon stress. <br /><br /><br />
<br />
<br />
After looking into the literature, similar bands were visualizable in E. coli for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how E. coli monitor division under various environments. For a thorough analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
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</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" data-lightbox="results" data-title="Results"><img src="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" alt="results" width="45%" class="pull-left"></a><br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/EPFL_2014_03_10_2014_Experiment-24.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/d/de/EPFL_2014_03_10_2014_Experiment-34.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/fd/EPFL_2014_03_10_2014_Experiment-35.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/0/08/EPFL_2014_03_10_2014_Experiment-37.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/a/a7/EPFL_2014_03_10_2014_Experiment-38.jpg" data-lightbox="results" data-title="Results"></a> <br />
<a href="https://static.igem.org/mediawiki/2014/2/2e/EPFL_2014_03_10_2014_Picture3.jpg" data-lightbox="results" data-title="Results"><br />
<a href="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" data-lightbox="results" data-title="Results"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" alt="results" width="45%" class="pull-right"></a><br />
</div><br />
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<!--<br />
<br />
<p><br />
<u>Aim</u> <br /><br />
Having shown that we were able to monitor the temporal dynamics of our construct, we wanted to see if we were able to analyze the spatial dynamics by microscopy.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To visualize the activation of our construct, we prepared cells as above for the previous plate-reader experiments, spread 10 µl on a glass slide added a coverslip and imaged them on a Zeiss Axioplan with a x100 objective and a APC (Cy5.5) filter. The pictures shown bellow were taken with a 5.1(s) integration time.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the pictures bellow, we were able to distinguish specific patterns within bacteria. We observed two phenotypes within our population: elongated and normal cells. The difference in these phenotypes was noticed in previous experiments and is most certainly due to the CpxR overexpression as we observed this also in non-stressed conditions. In the first phenotype (elongated) we were able to distinguish several bands that seem fairly uniformly distributed. In the second phenotype (normal) we observed a single band in the center of the bacteria. These observations led us to believe that CpxR might be involved in the division process of <i>E. coli</i> as it seems coherent for cells to slow down division upon stress. After looking into the literature, similar bands were visualizable in <i>E. coli</i> for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how <i>E. coli</i> monitor division under various environments.<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"><img src="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" alt="results" width="45%" class="pull-left"></a><br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/EPFL_2014_03_10_2014_Experiment-24.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/d/de/EPFL_2014_03_10_2014_Experiment-34.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/fd/EPFL_2014_03_10_2014_Experiment-35.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/0/08/EPFL_2014_03_10_2014_Experiment-37.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/a/a7/EPFL_2014_03_10_2014_Experiment-38.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a> <br />
<a href="https://static.igem.org/mediawiki/2014/2/2e/EPFL_2014_03_10_2014_Picture3.jpg" data-lightbox="results" data-title="IFP signal in stressed cells"><br />
<a href="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" data-lightbox="results" data-title="IFP signal in non stressed cells"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" alt="results" width="45%" class="pull-right"></a><br />
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<!--<br />
<a id=“CpxR-promoter"></a><br />
<h3><b>RFP under CpxR promoter to investigate time delay between transcription factor activation and protein synthesis</b></h3><br />
<br />
<p>To mesure the time it takes a cell to react to a stress input and if our modified CpxR still has its function as a transcription factor, we wanted to investigate the RFP expression of stressed cells that we co-transformed our CpxR-split-IPF construct with the <a target="_blank" href="http://parts.igem.org/Part:BBa_K339007">BBa_K339007</a>. Unfortunately we realised that the regulatory sequence of the RFP, the CpxR responsive promoter, was missing. So we constructed the biobrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486048">BBa_K1486048</a> which contains the regulatory sequence. After a few experiments we realised that KCl stress, which we have found to be a good signal inducer, did not work, even in cells expressing native CpxR. So we developed the biobricks <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486049">BBa_K1486049</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486050">BBa_K1486050</a>, which encode for RFP regulated by the CpxR responsive promoter, how it is found in the genome. In the figure below you see that neither of the three constructs was stress induced.</p><br />
<br />
<div class="cntr img-border"><br />
<a href="https://static.igem.org/mediawiki/2014/9/91/BBa_K1486049_BBa_K1486048.png" data-lightbox="cpxr" data-title="CpxR promoted RFP under stress"><br />
<img src="https://static.igem.org/mediawiki/2014/9/91/BBa_K1486049_BBa_K1486048.png" alt="CpxR promoted RFP under stress" class="img-responsive" /></a><br />
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<a id="IFP_Exp5"></a><br />
<h3><b>Activation of CpxR - split IFP1.4 on microfluidic chip by chamber crushing </b></h3><br />
<br />
<p><br />
Knowing that we were able to able to visualize CpxR-IFP activation under a microscope, we proceeded to trying to activate the pathway by mechanical stress on a microfluidic chip - the ultimate barrier to building a functional BioPad !<br /><br /><br />
<br />
To induce this stress, we turned on the buttons of our SmashColi microfluidic chip at a pressure of 25 psi. We imaged chambers before stress and after stress (10 min after button activation). A drastic increase in signal was detected !<br />
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<div class="cntr"><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/d/dc/Button_IFP_non_stressed_2_Cy5.jpg" data-lightbox="results_button" data-title="results_button"><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Button_IFP_non_stressed_2_Cy5.jpg" alt="results" width="45%" class="pull-left"> <br />
</a><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/7/77/Button_IFP_non_stressed_2.jpg" data-lightbox="results_button" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/9/99/Button_IFP_stressed_2.jpg" data-lightbox="results_button" data-title="Results"></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/f/f8/Button_IFP_stressed_2_Cy5.jpg" data-lightbox="results_button" data-title="results_button"><img src="https://static.igem.org/mediawiki/2014/f/f8/Button_IFP_stressed_2_Cy5.jpg" alt="results" width="45%" class="pull-right"> <br />
</a><br />
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</div><br />
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<a id="IFP_Exp1"></a><br />
<h3> <b> Characterisation of the pBAD promoter and folding ability of GFP fused to CpxR </b></h3><br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg" data-lightbox="img1"><img src="<br />
https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg" width="70%"></a><br />
</div> <br /><br /><br />
<br />
<p>This construct aimed to evaluate the expression of our construct and the characteristics of the arabinose promoter in <i>E. coli</i> by fusing a superfolder GFP protein to CpxR. The sfGFP was chosen because of its higher intensity compared to GFP. </p><br />
<p>Not knowing if CpxR would react the same way if sfGFP were attached to the N or C terminus, 2 biobricks were built, one with each of the orientations: <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486002">BBa_K1486002 (N terminus)</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486005">BBa_K1486005 (C terminus)</a>.<br />
</p><br />
<p><br />
We tested both constructs CpxR-sfGFP and sfGFP-CpxR on a plate reader to characterize the pBAD promoter, measuring the sfGFP signal in function of the arabinose concentration. We also measured the signal in a microfluidic chip. You can see in the graph below that the signal is increasing as the concentration of the arabinose increases. The construct with sfGFP at the C terminal lead to a higher GFP signal. Interestingly both curves increases in a very similar manner.<br />
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</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/a/ac/Nterm_and_Cterm_log.png" data-lightbox="hellopony"><img src="<br />
https://static.igem.org/mediawiki/2014/a/ac/Nterm_and_Cterm_log.png" width="50%"></a><br />
</div><br />
<br/><br/><br />
<hr/><br />
<br />
<h2 id="Characterisation_of_the_split_luciferase"> <b>Characterisation of the split luciferase </b> </h2><br />
<br />
<h3 id="Characterisation_of_the_split_luciferase"><b>Split luciferase complementation assay using CheY and CheZ chemotaxis proteins </b> </h3><br />
<br />
<p><br />
CheY and CheZ are two proteins involved in the bacterial chemotaxis pathway. It has been shown by split luciferase complementation assay that these two proteins are not interacting in presence of chemoattractant, but start to interact in absence of chemoattractant or presence of chemorepellent. Based on the work of Waldor<sup><a href="#ref1">1</a></sup> Laboratory, we wanted to redo the experiment to test our own splits, with firefly (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486055">BBa_K1486055</a>) and renilla (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486054">BBa_K1486054</a>) luciferases.<br />
</p><br />
<br />
<p>As shown in the graphs (fig.1A and 1B), we didn't observe a high signal for our assay. However, the signal being higher than the blanks, it is an encouraging sign that the splits luciferase can be used for other experiments of this kind. A possible explanation for these results is that we didn't completely get rid of the arabinose, which acts as a chemoattractant. Moreover, CheY and CheZ being endogenously expressed in bacteria, there could be interferences with our fusion proteins and weakening of our signal. This should be tested again with CheY/CheZ knock out strains.<br /><br />
</p><br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Renilla"><img src="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Firefly"><img src="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
</div><br /><br />
<br /><br />
<p>We also could determine which of the luciferases would best suit our following experiments. As shown in fig. 2, for the same concentration of substrate, we see that firefly luciferase has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.</p><br /><br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" data-lightbox="chips" data-title="Bioluminescence assay"><img src="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" width="80%"/></a><br /><br />
</div><br />
</p><br />
</div><br />
<br /><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!--<br />
<div class="cntr"><br />
<h2 id="Characterisation_of_the_split_luciferase"> <b><u>Characterisation of the split luciferase </u> </b> </h2><br />
</div><br />
<br /><br /><br />
<br />
<h3 id="Characterisation_of_the_split_luciferase"><b>Experiment 1: </b>CheY/CheZ fused to split Firefly/Renilla luciferase, and full Firefly/Renilla luciferase characterisation </h3><br />
<p><u>Introduction</u> <br /><br />
CheY and CheZ are two proteins involved in the bacterial chemotaxis pathway. It has been shown by split luciferase complementation assay that these two proteins are not interacting in presence of chemoattractant, but start to interact (CheZ being the phosphatase of CheY) in absence of chemoattractant or presence of chemorepellent. Based on the work of Waldor<sup><a href="#ref1">1</a></sup> Laboratory, we wanted to redo and adapt the experiment to test our own splits.<br /> <br /></p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to test the efficiency of split Renilla luciferase and split Firefly luciferase. We wanted to study the speed of the signal and the amount of substrate needed to have a performant response. <br /> <br /><br />
</p><br />
<p><br />
<u>Method</u> <br /><br />
To proceed to this complementation assay, we built two constructs, one to test split Renilla Luciferase and the other for split Firefly Luciferase The CheY was fused to the N-terminal part of each split, while the CheZ was fused to the C-terminal part. We used the full luciferases (Renilla : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486022"> BBa_K1486022 </a> and Firefly : <a target="_blank" href="http://parts.igem.org/Part:BBa_K325108"> BBa_K325108 </a> from Cambridge 2010 team) as positive controls and the non-fused splits (Renilla : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486021"> BBa_K1486021 </a> and Firefly : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486018"> BBa_K1486018 </a>) as negative controls.<br /> <br /><br />
The bioluminescence assay was performed as described <a target="_blank" href="https://static.igem.org/mediawiki/2014/6/6d/Protocol_-_Bioluminescence_assay.pdf">here</a>. <br /><br />
The constructs were designed and assembled as described <a target="_blank" href="https://static.igem.org/mediawiki/2014/3/3b/Constructs_design_CheYCheZ.pdf">here</a>.<br /><br /> <br /><br />
</p><br />
<p><u>Results</u> <br /><br />
As shown in the graphs below (fig.1A and 1B), we couldn't really observe a high signal for our assay. However, the signal being higher than the blanks, it is an encouraging sign that the splits luciferase can be used for other experiments of this kind. A possible explanation for these results is that arabinose being a chemoattractant, we might need to do more wash steps with PBS to get rid of the arabinose before taking the measurements. Moreover, CheY and CheZ being endogenously expressed in bacteria, the edogenous proteins could interfere with our fusion proteins and weaken our signal. This complementation assay should be tested with CheY/CheZ knock out strains, as it was done in Waldor Laboratory.<br /><br />
</p><br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Renilla"><img src="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Firefly"><img src="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
</div><br /><br />
<br /><br />
We also could determine which of the luciferases would best suit our following experiments. As shown in fig. 2, for the same concentration of substrate, we see that firefly luciferase has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.<br /><br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<br /><br />
--><br />
<br />
<br />
<a id="Yeast_experiments"></a><br />
<br />
<h2> <b>The PBS2-HOG1 split-GFP & split Renilla Luciferase stress response<br />
</b> </h2><br />
<br />
<br/><br/><br />
<a id="Yeast_exp1"></a><br />
<h3><b>Confirmation of successful transformation via the Renilla Luciferase tag </b></h3><br />
<br/><br />
<p><br />
<br />
Since we had never transformed <i>S. cerevisiae</i>, we first needed to confirm our protein tag transformations as a positive control for subsequent experiments. We cultured the two strains we got from the transformation, the HOG1-rLuc (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486033">BBa_K1486033</a> ) and PBS2-rLuc (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486027">BBa_K1486027</a>), put them in a 96 well plate and tested their luminescence using a plate reader.</p><br />
<p><br />
<br />
The test was performed using two different concentrations of the substrate, coelenterazine-h(benzyl coelenterazine): 1μM and 5μM so that we could determine the optimal concentration we would use for the next assays. The plate started to be read directly after addition of the substrate in the dark and measures were taken every 56 seconds thereafter. Three wells for each sample were measured and below are shown the graphs of their average luminescence plotted over time. We observed a signal-to-noise ratio of 4 at 1μM of substrate and 10 at 5μM for the PBS2-rLuc strain. The HOG1-rLuc strain seemed problematic and died within the plate. Unable to verify the correct transformation, we reproduced the strain but were unable to confirm its validity before the wiki deadline.</p><br />
<br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/0e/Pbslucprettycomparaison_.png" data-lightbox="chips" data-title=""><img src="https://static.igem.org/mediawiki/2014/0/0e/Pbslucprettycomparaison_.png" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<p><br />
There is a clear luminescence generated by the cells, providing us with confirmation that our designs are correct and we determined which concentration of substrate to use to have a decent signal-to-background ratio.<br />
</p><br />
<br />
<br />
<a id="Yeast_exp2"></a><br />
<h3><b>The split-sfGFP strain stress-response</b></h3><br />
<p><br />
<br />
<br />
Having confirmed that our design for yeast transformations was correct via the previous experiment, we performed stress-response tests upon our PBS2-HOG1-splitGFP (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486029">BBa_K1486029</a>) and (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486035">BBa_K1486035</a>) strain to determine whether the pathway is reactive to stress as theorized. A co-transformation was performed using linear fragments produced in the same way as for the first experiment to obtain the strain. The N-terminal split of the superfolder GFP was tagged onto PBS2 and the C-terminal onto HOG1. The strain was then tested along with non-transformed cells in a 96 well plate with various stresses (e.g. Acetic Acid, Ethanol, Glucose...). The cells were centrifuged and resuspended in PBS before loading into the plate.<br />
</p><br />
<br />
<br />
<div class="cntr"><br />
<br />
<div class="cntr"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/1/1d/Splitgfpgraph.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<p><br />
The most reactive of stresses turned out to be Acetic Acid 3.6%(shown above) followed by Ethanol 10%. Other stresses tested did not seem to show conclusive results and we were unable to determine more reactants before the wiki freeze deadline. To further assess the fluorescence 10μl of our cells were spread on a glass slide and imaged with a x63 objective and a Green LP filter<br />
(Excitation BP 450-490, Dichroïc FT 510, Emission LP 515). Below is shown a comparison of before and after 3.6% Acetic Acid stress in the microscopy image merged with the fluorescence. <br />
</p><br />
<br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/3b/Splitgfpyeast.jpg" data-lightbox="caca" data-title="Image of our split sfGFP expressing cells before and after the 3.6% acetic acid stress (x63 objective and a Green LP filter)"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3b/Splitgfpyeast.jpg" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<br />
<div class="cntr"><br />
<p><br />
For a more quantitative measure, the fluorescent cells to total cells ratio was calculated and illustrated below.<br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Splitgfpcount.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<br />
<p><br />
<br />
<br />
From the data shown above, we can see that fluorescence increases within cells in our split sfGFP strain in response to stress while it does not in non-transformed cells. We can thus conclude that the orientation of the split sfGFP constructs added to PBS2 and HOG1 allows proper re-assembly upon interaction of the two proteins. This result shows promise for the actual application of the project as a Biopad using yeast cells. <b>It is the first time a split gets succesfully implemented in the HOG1 pathway by direct attachment to proteins of the kinase cascade</b>. Detection of protein–protein interactions within the pathway were previously studied <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/yea.320080602/abstract"> using vesicle targeting</a>.<br />
<br />
<p/><br />
<br />
<br />
<br />
<br />
<a id="Yeast_exp3"></a><br />
<h3><b>The split-Luciferase strain stress-response</b></h3><br />
<p><br />
After determining that the pathway is reactive to certain stresses and that the split-GFP strain increases its fluorescence, we performed the same test in the obtained split-luciferase (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486030">BBa_K1486030</a>) and (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486036">BBa_K1486036</a>) strain. Unfortunately, due to the difficult nature of detecting an instantaneous luminescence signal and the unstable nature of coelenterazine-Luciferase reaction in different conditions(inhibition due to various molecules such as glucose), our attempts in detecting it on the plate reader failed. Further tests are planned, including an experiment in the microfluidic chips, to directly measure the effect of mechanical pressure, but will not be done before the wiki freeze. <br />
</p><br />
<br />
<br />
<a id="Microfluidics_experiments"></a><br />
<br />
<br />
<br />
<a id="Micro_exp1"></a><br />
<h2><b>Microfluidic achievements</b></h2><br />
<table class="table table-striped valign-middle table-center"><br />
<thead><br />
<tr><br />
<th></th><br />
<th>MITOMI</th><br />
<th>MITOMI modified</th><br />
<th>SmashColi</th><br />
<th>BioPad</th><br />
<th>FilterColi</th><br />
<th>CleanColi</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>Full chip</td><br />
<td class="cntr"><a target="_blank" href="https://static.igem.org/mediawiki/2014/a/ab/Mitomi11.png" data-lightbox="chips" data-title="MITOMI"><img src="https://static.igem.org/mediawiki/2014/a/ab/Mitomi11.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/6/64/Mitomimodif1.png" data-lightbox="chips" data-title="MITOMI Modified"><img src="https://static.igem.org/mediawiki/2014/6/64/Mitomimodif1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/1/15/Smash1.png" data-lightbox="chips" data-title="SmashColi"><img src="https://static.igem.org/mediawiki/2014/1/15/Smash1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/d/db/Biopad1.png" data-lightbox="chips" data-title="BioPad"><img src="https://static.igem.org/mediawiki/2014/d/db/Biopad1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/d/db/Filter1.png" data-lightbox="chips" data-title="FilterColi"><img src="https://static.igem.org/mediawiki/2014/d/db/Filter1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/b/b0/Screen_Shot_2014-10-16_at_22.15.39.png" data-lightbox="chips" data-title="CleanColi"><img src="https://static.igem.org/mediawiki/2014/b/b0/Screen_Shot_2014-10-16_at_22.15.39.png" width="70%"/></a><br /></td><br />
</tr><br />
<tr><br />
<td>Unit Cell</td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/0/0f/MitomiUnit1.png" data-lightbox="chips" data-title="MITOMI Unit"><img src="https://static.igem.org/mediawiki/2014/0/0f/MitomiUnit1.png" width="50%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/7/78/MitomimodifUnit.png" data-lightbox="chips" data-title="MITOMI Modified Unit"><img src="https://static.igem.org/mediawiki/2014/7/78/MitomimodifUnit.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/3/3a/Smahsunit1.png" data-lightbox="chips" data-title="SmashColi Unit"><img src="https://static.igem.org/mediawiki/2014/3/3a/Smahsunit1.png" width="30%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/6/60/Biopadunit1.png" data-lightbox="chips" data-title="BioPad Unit"><img src="https://static.igem.org/mediawiki/2014/6/60/Biopadunit1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/9/9e/FilterUnit.png" data-lightbox="chips" data-title="FilterColi Unit"><img src="https://static.igem.org/mediawiki/2014/9/9e/FilterUnit.png" width="70%"/></a><br /></td><br />
<td><span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Designed</td><br />
<td><span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
</tr><br />
<tr><br />
<td>Mold fabrication</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Fabrication of the chip</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Application</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br/> <p>To start our microfluidic experiments, we used the <a target="_blank" href="http://link.springer.com/protocol/10.1007%2F978-1-61779-292-2_6">MITOMI chip</a> that was designed in the laboratory of Pr. Maerkl.</p><br/><br />
<br/><br />
<br />
<br />
<br />
<br />
<a id="Micro_exp2"></a><br />
<h3><b>Culturing <i>E. coli</i> with constitutive GFP on chip</b></h3><br />
<p>We loaded <i>E. coli</i>, which contained constitutive GFP, in the chip. By using LabVIEW, a protocol was launched overnight to ensure the growth of the cells (the protocol can be found <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#growth">here</a>).<br />
<br /></p><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/e/e4/Growth_small.gif" data-lightbox="pipi"><img src="<br />
https://static.igem.org/mediawiki/2014/e/e4/Growth_small.gif" width="50%"></a><br />
</div><br />
</p><br />
<p>The next morning, a scan of the chip was done to see the intensity of the GFP in the chip.<br /></p><br />
<div class=""><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/f/fe/Gfp.png" data-lightbox="prout"><img src="<br />
https://static.igem.org/mediawiki/2014/f/fe/Gfp.png" width="50%" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<br /><br />
<br />
<a id="Micro_exp3"></a><br />
<br />
<h3><b>CpxR linked with GFP on the N terminal, induced by arabinose in <i>E. coli</i></b></h3><br />
<p>The experiment that was done on wetbench to show that CpxR linked with GFP was expressed with an arabinose promoter was replicated on a MITOMI chip.<br />
LB medium with arabinose was flowed in the upper half whereas LB medium without arabinose was flowed in the lower half. We scanned every hour for 5h (to know how it was done click <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#date-08-21-2014">here</a>).</p><br />
<div class="row"><br />
<div class="col col-md-6"><a href="https://static.igem.org/mediawiki/2014/4/4e/Truc2.png" data-lightbox="chips" data-title="Scan of the microfluidic chip at t = 0min. No signal is detected."><img src="https://static.igem.org/mediawiki/2014/4/4e/Truc2.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 1.</strong> Scan of the microfluidic chip at t = 0min. No signal is detected<br />
</div></div><br />
<div class="col col-md-6"><br />
<a href="https://static.igem.org/mediawiki/2014/3/32/Truc3.png" data-lightbox="chips" data-title="Scan of the microfluidic chip at t = 300min."><img src="https://static.igem.org/mediawiki/2014/3/32/Truc3.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 2.</strong> Scan of the microfluidic chip at t = 300min.</div></div><br />
</div><br />
<p>We analysed the scans and obtained the following results.</p><br />
<p><a href="https://static.igem.org/mediawiki/2014/4/4c/Gfp_ara.png" data-lightbox="chips" data-title="Figure 3. Evolution of CpxR-GFP fluorescence over time"><img src="https://static.igem.org/mediawiki/2014/4/4c/Gfp_ara.png" alt="" class="img-responsive" /></a></p><br />
<br />
<div class="cntr"> <br />
<strong>Figure 3.&nbsp;</strong> Evolution of CpxR-GFP fluorescence over time<br />
</div> <br />
<br />
<br/><br />
<br />
<a id="Micro_exp4"></a><br />
<br />
<h3><b>On chip infrared detection</b></h3><br />
<p>As we focused part of our work on the IFP1.4, we needed confirmation of this signal detection in our microfluidic chips. Thus the aim of this experiment was to prove this fluorescence detection capability. Bacteria were loaded in the smash-coli chip. The first batch was KCl stressed and the second batch was unstressed. We then simply scanned the chip and analysed the results using ImageJ. For more details, please visit our <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#date-10-03-2014">notebook</a>. </p><br />
<div class="row"><br />
<div class="col col-md-6"><a href="https://static.igem.org/mediawiki/2014/c/cf/No_KCl_red.png" data-lightbox="chips" data-title="Figure 1. Cy5 scan of a chamber containing non-stressed CpxR-IFP bacteria"><img src="https://static.igem.org/mediawiki/2014/c/cf/No_KCl_red.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 1.</strong> Cy5 scan of a chamber containing non-stressed CpxR-IFP bacteria.<br />
</div></div><br />
<div class="col col-md-6"><br />
<a href="https://static.igem.org/mediawiki/2014/c/c2/KCL_red.png" data-lightbox="chips" data-title="Figure 2. Cy5 scan of a chamber containing KCl-stressed CpxR-IFP bacteria"><img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_red.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 2.</strong> Cy5 scan of a chamber containing KCl-stressed CpxR-IFP bacteria.</div></div><br />
</div><br />
<p>We analysed the scans and obtained the following results.</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/2/24/KCl_stack.PNG" alt="" class="img-responsive" /></p><br />
<strong>Figure 3.&nbsp;</strong> Histogram of KCl stressed cells and non-stressed cells.<br />
<br />
<br/><br/><br />
<br />
<hr><br />
<br />
<h3>BioPad Detector: Detection of sfGFP</h3><br />
<br />
<p> To get a first idea of how our BioPad was able to detect signal, we attempted to detect sfGFP signal emission by exciting our sfGFP with a 470 nm emitting LED and acquiring the images through our device. A sample of the video taken can be seen here bellow: </p><br />
<br />
<video width="320" height="240" controls><br />
<source src="https://static.igem.org/mediawiki/2014/2/2f/Gfptracking_EPFL.mp4" type="video/mp4"><br />
</video><br />
<br />
<p><br />
To learn more about how the detector works check out our <a href="https://2014.igem.org/Team:EPF_Lausanne/Hardware"> Hardware page!</a><br />
</p><br />
<br />
<!--<h4> References </h4><br />
<p><br />
<a id="ref1"></a>1: S.K. Hatzios, S. Ringgaard, B. M. Davis, M. K. Waldor (2012, August 15). Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition. <i>Plos One</i>.<br />
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<li><a href="#IFP_Exp4"></b>Microscopy of KCl stress </a></li><br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/ResultsTeam:EPF Lausanne/Results2014-10-18T00:50:05Z<p>Robert Baldwin: </p>
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<!-- RESULTS --><br />
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<h1 class="cntr"> <b>RESULTS</b> </h1><br />
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<a id="IFP_Exp"></a><br />
<!--replaced the title : Characterisation of the spatiotemporal dynamics of the CpxR - split IFP 1.4 stress sensor --><br />
<h2> <b> Characterisation of the spatiotemporal dynamics of the CpxR stress sensor </b> </h2<br />
</div><br />
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<br /><br /><br />
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<a id="IFP_Exp2"></a><br />
<h3><b>Demonstration of CpxR's dimerization & Elucidation of its dimerization orientation</b></h3><br />
<br />
<p><br />
Previous in vitro experiments (FRET) have shown that CpxR dimerizes. Currently little in vivo information about CpxR’s dimerization is available. To characterize our stress responsive bacteria, we had to confirm that CpxR dimerized in vivo as well as elucidate CpxR’s dimerization orientation. <br />
</p><br />
<br /><br />
<p><br />
We synthesized four constructs with combinations of the split IFP1.4 fragments fused to the C or N terminal of CpxR. <br />
As seen in the graph bellow, induction of IFP1.4 signal by 50 mM KCl was done at t=24min. We clearly see that the construct with IFP fragments on the C-terminal immediately responded to stress by emitting fluorescence. In fact we observe a 3 fold signal increase in 2 minutes. On the other hand, the three other orientations were non-responsive to KCl stress.</p><br />
<br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" data-lightbox="chips" data-title="Construct Comparison"><img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" width="80%"/></a><br /><br />
</div><br />
<p><br />
This amazing 30-fold signal increase in little time from the baseline allows us to assert that only the strain synthesizing the IFP fragments on the C-terminals of CpxR responded to KCl stress. For a further analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
</p><br />
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<!--<br />
<p><br />
<u>Introduction</u> <br /><br />
CpxR is the relay protein in the stress responsive CpxAR two component regulatory system. It has been shown by split beta galactosidase assay that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that <i>E. coli</i> CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in <i>E. coli</i>.</p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to determine if and how CpxR dimerised in vivo in <i>E. coli</i>. This experiment intended to get a first idea of the real-time temporal dynamics of the activation of CpxR (the cytoplasmic relay protein of the CpxA-R pathway) by KCl stress via CpxA (the periplasmic sensor protein of the CpxA-R pathway). This experiment is a first of its kind.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To evaluate if and how CpxR dimerized under KCl stress, we built by gibson assembly four constructs with the various possible orientations that the split IFP1.4 fragments could have with CpxR: IFP[1] and IFP[2] on the N-terminus of CpxR, IFP[1] on the N-terminus of CpxR and IFP[2] on the C-terminus of CpxR, and finally IFP[1] and IFP[2] on the N-terminus of CpxR. The split IFP fragments were provided by the Michnick Lab, and the CpxR coding region was amplified by PCR from extracted <i>E. coli</i> genome (Bacterial Genomic Miniprep Kit from Sigma Aldrich). The protocol for stressing the cells and reading the fluorescence can be downloaded <a target="blank" href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the graph bellow, induction of the signal was done at minute 24 (marked via a vertically spoted line). The construct with IFP fragments on the C-termina responded immediately to stress. In a fact we observed a 3 fold signal increase in 2 minutes. All other constructs we observed a low baseline signal non responsive to KCl stress. It is to be noted that the C-termina constructs always had higher signal levels than the other constructs. This leads us to believe that the PBS used to resuspend our cultures led to small levels of stress (the PBS we use does not contain KCl but traces of NaCl). The 30-fold signal increase from the baseline allows us to assert that our constructs responds to KCl stress.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" alt="Construct Comparison" class="img-responsive"><br />
</div><br />
<p><br />
<u>Discussion</u> <br /><br />
We successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus. This finding suggests that CpxR binds via its C-termina. This leads us to hypothesise that the CpxR dimerisation mechanisms is the same for other members of the highly conserved OmpR/PhoB subfamily. This hypothesis could allow the development of similar system that could the study other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.<br />
</p><br />
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--><br />
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<a id="IFP_Exp3"></a><br />
<h3><b>Signal induction by various concentrations of KCl & signal shutdown by centrifugation</b></h3><br />
<br />
<p><br />
Having confirmed that KCl was a good inducer for our signal, we tested different concentration of KCl to modulate the signal and better characterize our biobrick. We also aimed to shutdown the signal by centrifugation and medium change. The signal was measured on a plate reader 20 minutes, before the addition of KCl. After 2 hours of measurement we centrifuged the cells for ten minutes and replaced the medium with PBS to be able to get a shutdown of the signal.</p><br />
<br />
<div class="cntr"><br />
<a target="_blank" href="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" data-lightbox="chips" data-title="The split-IFP are linked to cpxR on the C-terminal. During the experiment it was possible to shut down the signal"><img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" width="80%"/></a><br /><br />
</div><br />
<br />
<br />
<p><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to drastically shut down the signal, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis is possible for our system. For a thorough analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
</p><br />
<br />
<br />
<!--<br />
<p><br />
<u>Aim</u> <br /><br />
Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change.<br />
To do so, we read our signal for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To evaluate if a modulation in KCl concentrations affected the intensity of the intensity of the fluorescent signal, and if a change in medium by centrifugation shutdown the signal; we read our signal on a plate reader for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal. The protocol for this experiment can be downloaded <a target="_blank" href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis are possible for our system.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" alt="GA1 Shutdown" class="img-responsive"><br />
</div><br />
<br /><br /><br/><br/><br />
--><br />
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<a id="IFP_Exp4"></a><br />
<h3><b>Visualization of the the CpxR split IFP1.4 activation by KCl stress </b></h3><br />
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<br />
<p><br />
<br />
<br />
Having shown that we were able to monitor the temporal dynamics of CpxR activation, we wanted to see if we could analyze CpxR’s spatial dynamics by microscopy. 10μl of our cells, previously stressed with 80 mM KCl were spread on a glass slide and imaged with a x100 objective and a APC (Cy5.5) filter.<br />
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</p><br />
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<p><br />
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<br />
We noticed various characteristics of the cells from the picture bellow. First, IFP signal was much more present in stressed bacteria rather than in non-stressed bacteria. Secondly, we distinguished two specific phenotypes within bacteria: elongated and normal cells. We noticed that this difference was due to CpxR overexpression as we saw this phenomenon also in non-stressed conditions.<br /><br /><br />
<br />
<br />
In elongated cells, we were able to distinguish several bright bands of IFP signal that seem fairly uniformly distributed. In the normal phenotype we distinguished a single band in the centre of the bacteria. These observations led us to believe that CpxR might be involved in the division process of E. coli as it seems coherent for cells to slow down division upon stress. <br /><br /><br />
<br />
<br />
After looking into the literature, similar bands were visualizable in E. coli for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how E. coli monitor division under various environments. For a thorough analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
<br />
<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" data-lightbox="results" data-title="Results"><img src="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" alt="results" width="45%" class="pull-left"></a><br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/EPFL_2014_03_10_2014_Experiment-24.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/d/de/EPFL_2014_03_10_2014_Experiment-34.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/fd/EPFL_2014_03_10_2014_Experiment-35.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/0/08/EPFL_2014_03_10_2014_Experiment-37.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/a/a7/EPFL_2014_03_10_2014_Experiment-38.jpg" data-lightbox="results" data-title="Results"></a> <br />
<a href="https://static.igem.org/mediawiki/2014/2/2e/EPFL_2014_03_10_2014_Picture3.jpg" data-lightbox="results" data-title="Results"><br />
<a href="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" data-lightbox="results" data-title="Results"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" alt="results" width="45%" class="pull-right"></a><br />
</div><br />
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<br />
<br />
<!--<br />
<br />
<p><br />
<u>Aim</u> <br /><br />
Having shown that we were able to monitor the temporal dynamics of our construct, we wanted to see if we were able to analyze the spatial dynamics by microscopy.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To visualize the activation of our construct, we prepared cells as above for the previous plate-reader experiments, spread 10 µl on a glass slide added a coverslip and imaged them on a Zeiss Axioplan with a x100 objective and a APC (Cy5.5) filter. The pictures shown bellow were taken with a 5.1(s) integration time.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the pictures bellow, we were able to distinguish specific patterns within bacteria. We observed two phenotypes within our population: elongated and normal cells. The difference in these phenotypes was noticed in previous experiments and is most certainly due to the CpxR overexpression as we observed this also in non-stressed conditions. In the first phenotype (elongated) we were able to distinguish several bands that seem fairly uniformly distributed. In the second phenotype (normal) we observed a single band in the center of the bacteria. These observations led us to believe that CpxR might be involved in the division process of <i>E. coli</i> as it seems coherent for cells to slow down division upon stress. After looking into the literature, similar bands were visualizable in <i>E. coli</i> for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how <i>E. coli</i> monitor division under various environments.<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"><img src="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" alt="results" width="45%" class="pull-left"></a><br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/EPFL_2014_03_10_2014_Experiment-24.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/d/de/EPFL_2014_03_10_2014_Experiment-34.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/fd/EPFL_2014_03_10_2014_Experiment-35.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/0/08/EPFL_2014_03_10_2014_Experiment-37.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/a/a7/EPFL_2014_03_10_2014_Experiment-38.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a> <br />
<a href="https://static.igem.org/mediawiki/2014/2/2e/EPFL_2014_03_10_2014_Picture3.jpg" data-lightbox="results" data-title="IFP signal in stressed cells"><br />
<a href="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" data-lightbox="results" data-title="IFP signal in non stressed cells"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" alt="results" width="45%" class="pull-right"></a><br />
</div><br />
<br />
--><br />
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<br />
<br />
<br/><br/><br/><br />
<!--<br />
<a id=“CpxR-promoter"></a><br />
<h3><b>RFP under CpxR promoter to investigate time delay between transcription factor activation and protein synthesis</b></h3><br />
<br />
<p>To mesure the time it takes a cell to react to a stress input and if our modified CpxR still has its function as a transcription factor, we wanted to investigate the RFP expression of stressed cells that we co-transformed our CpxR-split-IPF construct with the <a target="_blank" href="http://parts.igem.org/Part:BBa_K339007">BBa_K339007</a>. Unfortunately we realised that the regulatory sequence of the RFP, the CpxR responsive promoter, was missing. So we constructed the biobrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486048">BBa_K1486048</a> which contains the regulatory sequence. After a few experiments we realised that KCl stress, which we have found to be a good signal inducer, did not work, even in cells expressing native CpxR. So we developed the biobricks <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486049">BBa_K1486049</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486050">BBa_K1486050</a>, which encode for RFP regulated by the CpxR responsive promoter, how it is found in the genome. In the figure below you see that neither of the three constructs was stress induced.</p><br />
<br />
<div class="cntr img-border"><br />
<a href="https://static.igem.org/mediawiki/2014/9/91/BBa_K1486049_BBa_K1486048.png" data-lightbox="cpxr" data-title="CpxR promoted RFP under stress"><br />
<img src="https://static.igem.org/mediawiki/2014/9/91/BBa_K1486049_BBa_K1486048.png" alt="CpxR promoted RFP under stress" class="img-responsive" /></a><br />
</div><br />
<br />
<br/><br/><br/><br />
--><br />
<br />
<a id="IFP_Exp5"></a><br />
<h3><b>Activation of CpxR - split IFP1.4 on microfluidic chip by chamber crushing </b></h3><br />
<br />
<p><br />
Knowing that we were able to able to visualize CpxR-IFP activation under a microscope, we proceeded to trying to activate the pathway by mechanical stress on a microfluidic chip - the ultimate barrier to building a functional BioPad !<br /><br /><br />
<br />
To induce this stress, we turned on the buttons of our SmashColi microfluidic chip at a pressure of 25 psi. We imaged chambers before stress and after stress (10 min after button activation). A drastic increase in signal was detected !<br />
<br />
<div class="cntr"><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/d/dc/Button_IFP_non_stressed_2_Cy5.jpg" data-lightbox="results_button" data-title="results_button"><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Button_IFP_non_stressed_2_Cy5.jpg" alt="results" width="45%" class="pull-left"> <br />
</a><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/7/77/Button_IFP_non_stressed_2.jpg" data-lightbox="results_button" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/9/99/Button_IFP_stressed_2.jpg" data-lightbox="results_button" data-title="Results"></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/f/f8/Button_IFP_stressed_2_Cy5.jpg" data-lightbox="results_button" data-title="results_button"><img src="https://static.igem.org/mediawiki/2014/f/f8/Button_IFP_stressed_2_Cy5.jpg" alt="results" width="45%" class="pull-right"> <br />
</a><br />
<br />
</div><br />
<br />
<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
<a id="IFP_Exp1"></a><br />
<h3> <b> Characterisation of the pBAD promoter and folding ability of GFP fused to CpxR </b></h3><br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg" data-lightbox="img1"><img src="<br />
https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg" width="70%"></a><br />
</div> <br /><br /><br />
<br />
<p>This construct aimed to evaluate the expression of our construct and the characteristics of the arabinose promoter in <i>E. coli</i> by fusing a superfolder GFP protein to CpxR. The sfGFP was chosen because of its higher intensity compared to GFP. </p><br />
<p>Not knowing if CpxR would react the same way if sfGFP were attached to the N or C terminus, 2 biobricks were built, one with each of the orientations: <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486002">BBa_K1486002 (N terminus)</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486005">BBa_K1486005 (C terminus)</a>.<br />
</p><br />
<p><br />
We tested both constructs CpxR-sfGFP and sfGFP-CpxR on a plate reader to characterize the pBAD promoter, measuring the sfGFP signal in function of the arabinose concentration. We also measured the signal in a microfluidic chip. You can see in the graph below that the signal is increasing as the concentration of the arabinose increases. The construct with sfGFP at the C terminal lead to a higher GFP signal. Interestingly both curves increases in a very similar manner.<br />
<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/a/ac/Nterm_and_Cterm_log.png" data-lightbox="hellopony"><img src="<br />
https://static.igem.org/mediawiki/2014/a/ac/Nterm_and_Cterm_log.png" width="50%"></a><br />
</div><br />
<br/><br/><br />
<hr/><br />
<br />
<h2 id="Characterisation_of_the_split_luciferase"> <b>Characterisation of the split luciferase </b> </h2><br />
<br />
<h3 id="Characterisation_of_the_split_luciferase"><b>Split luciferase complementation assay using CheY and CheZ chemotaxis proteins </b> </h3><br />
<br />
<p><br />
CheY and CheZ are two proteins involved in the bacterial chemotaxis pathway. It has been shown by split luciferase complementation assay that these two proteins are not interacting in presence of chemoattractant, but start to interact in absence of chemoattractant or presence of chemorepellent. Based on the work of Waldor<sup><a href="#ref1">1</a></sup> Laboratory, we wanted to redo the experiment to test our own splits, with firefly (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486055">BBa_K1486055</a>) and renilla (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486054">BBa_K1486054</a>) luciferases.<br />
</p><br />
<br />
<p>As shown in the graphs (fig.1A and 1B), we didn't observe a high signal for our assay. However, the signal being higher than the blanks, it is an encouraging sign that the splits luciferase can be used for other experiments of this kind. A possible explanation for these results is that we didn't completely get rid of the arabinose, which acts as a chemoattractant. Moreover, CheY and CheZ being endogenously expressed in bacteria, there could be interferences with our fusion proteins and weakening of our signal. This should be tested again with CheY/CheZ knock out strains.<br /><br />
</p><br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Renilla"><img src="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Firefly"><img src="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
</div><br /><br />
<br /><br />
<p>We also could determine which of the luciferases would best suit our following experiments. As shown in fig. 2, for the same concentration of substrate, we see that firefly luciferase has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.</p><br /><br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" data-lightbox="chips" data-title="Bioluminescence assay"><img src="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" width="80%"/></a><br /><br />
</div><br />
</p><br />
</div><br />
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<!--<br />
<div class="cntr"><br />
<h2 id="Characterisation_of_the_split_luciferase"> <b><u>Characterisation of the split luciferase </u> </b> </h2><br />
</div><br />
<br /><br /><br />
<br />
<h3 id="Characterisation_of_the_split_luciferase"><b>Experiment 1: </b>CheY/CheZ fused to split Firefly/Renilla luciferase, and full Firefly/Renilla luciferase characterisation </h3><br />
<p><u>Introduction</u> <br /><br />
CheY and CheZ are two proteins involved in the bacterial chemotaxis pathway. It has been shown by split luciferase complementation assay that these two proteins are not interacting in presence of chemoattractant, but start to interact (CheZ being the phosphatase of CheY) in absence of chemoattractant or presence of chemorepellent. Based on the work of Waldor<sup><a href="#ref1">1</a></sup> Laboratory, we wanted to redo and adapt the experiment to test our own splits.<br /> <br /></p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to test the efficiency of split Renilla luciferase and split Firefly luciferase. We wanted to study the speed of the signal and the amount of substrate needed to have a performant response. <br /> <br /><br />
</p><br />
<p><br />
<u>Method</u> <br /><br />
To proceed to this complementation assay, we built two constructs, one to test split Renilla Luciferase and the other for split Firefly Luciferase The CheY was fused to the N-terminal part of each split, while the CheZ was fused to the C-terminal part. We used the full luciferases (Renilla : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486022"> BBa_K1486022 </a> and Firefly : <a target="_blank" href="http://parts.igem.org/Part:BBa_K325108"> BBa_K325108 </a> from Cambridge 2010 team) as positive controls and the non-fused splits (Renilla : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486021"> BBa_K1486021 </a> and Firefly : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486018"> BBa_K1486018 </a>) as negative controls.<br /> <br /><br />
The bioluminescence assay was performed as described <a target="_blank" href="https://static.igem.org/mediawiki/2014/6/6d/Protocol_-_Bioluminescence_assay.pdf">here</a>. <br /><br />
The constructs were designed and assembled as described <a target="_blank" href="https://static.igem.org/mediawiki/2014/3/3b/Constructs_design_CheYCheZ.pdf">here</a>.<br /><br /> <br /><br />
</p><br />
<p><u>Results</u> <br /><br />
As shown in the graphs below (fig.1A and 1B), we couldn't really observe a high signal for our assay. However, the signal being higher than the blanks, it is an encouraging sign that the splits luciferase can be used for other experiments of this kind. A possible explanation for these results is that arabinose being a chemoattractant, we might need to do more wash steps with PBS to get rid of the arabinose before taking the measurements. Moreover, CheY and CheZ being endogenously expressed in bacteria, the edogenous proteins could interfere with our fusion proteins and weaken our signal. This complementation assay should be tested with CheY/CheZ knock out strains, as it was done in Waldor Laboratory.<br /><br />
</p><br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Renilla"><img src="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Firefly"><img src="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
</div><br /><br />
<br /><br />
We also could determine which of the luciferases would best suit our following experiments. As shown in fig. 2, for the same concentration of substrate, we see that firefly luciferase has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.<br /><br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
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--><br />
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<br />
<a id="Yeast_experiments"></a><br />
<br />
<h2> <b>The PBS2-HOG1 split-GFP & split Renilla Luciferase stress response<br />
</b> </h2><br />
<br />
<br/><br/><br />
<a id="Yeast_exp1"></a><br />
<h3><b>Confirmation of successful transformation via the Renilla Luciferase tag </b></h3><br />
<br/><br />
<p><br />
<br />
Since we had never transformed <i>S. cerevisiae</i>, we first needed to confirm our protein tag transformations as a positive control for subsequent experiments. We cultured the two strains we got from the transformation, the HOG1-rLuc (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486033">BBa_K1486033</a> ) and PBS2-rLuc (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486027">BBa_K1486027</a>), put them in a 96 well plate and tested their luminescence using a plate reader.</p><br />
<p><br />
<br />
The test was performed using two different concentrations of the substrate, coelenterazine-h(benzyl coelenterazine): 1μM and 5μM so that we could determine the optimal concentration we would use for the next assays. The plate started to be read directly after addition of the substrate in the dark and measures were taken every 56 seconds thereafter. Three wells for each sample were measured and below are shown the graphs of their average luminescence plotted over time. We observed a signal-to-noise ratio of 4 at 1μM of substrate and 10 at 5μM for the PBS2-rLuc strain. The HOG1-rLuc strain seemed problematic and died within the plate. Unable to verify the correct transformation, we reproduced the strain but were unable to confirm its validity before the wiki deadline.</p><br />
<br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/0e/Pbslucprettycomparaison_.png" data-lightbox="chips" data-title=""><img src="https://static.igem.org/mediawiki/2014/0/0e/Pbslucprettycomparaison_.png" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<p><br />
There is a clear luminescence generated by the cells, providing us with confirmation that our designs are correct and we determined which concentration of substrate to use to have a decent signal-to-background ratio.<br />
</p><br />
<br />
<br />
<a id="Yeast_exp2"></a><br />
<h3><b>The split-sfGFP strain stress-response</b></h3><br />
<p><br />
<br />
<br />
Having confirmed that our design for yeast transformations was correct via the previous experiment, we performed stress-response tests upon our PBS2-HOG1-splitGFP (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486029">BBa_K1486029</a>) and (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486035">BBa_K1486035</a>) strain to determine whether the pathway is reactive to stress as theorized. A co-transformation was performed using linear fragments produced in the same way as for the first experiment to obtain the strain. The N-terminal split of the superfolder GFP was tagged onto PBS2 and the C-terminal onto HOG1. The strain was then tested along with non-transformed cells in a 96 well plate with various stresses (e.g. Acetic Acid, Ethanol, Glucose...). The cells were centrifuged and resuspended in PBS before loading into the plate.<br />
</p><br />
<br />
<br />
<div class="cntr"><br />
<br />
<div class="cntr"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/1/1d/Splitgfpgraph.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<p><br />
The most reactive of stresses turned out to be Acetic Acid 3.6%(shown above) followed by Ethanol 10%. Other stresses tested did not seem to show conclusive results and we were unable to determine more reactants before the wiki freeze deadline. To further assess the fluorescence 10μl of our cells were spread on a glass slide and imaged with a x63 objective and a Green LP filter<br />
(Excitation BP 450-490, Dichroïc FT 510, Emission LP 515). Below is shown a comparison of before and after 3.6% Acetic Acid stress in the microscopy image merged with the fluorescence. <br />
</p><br />
<br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/3b/Splitgfpyeast.jpg" data-lightbox="caca" data-title="Image of our split sfGFP expressing cells before and after the 3.6% acetic acid stress (x63 objective and a Green LP filter)"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3b/Splitgfpyeast.jpg" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<br />
<div class="cntr"><br />
<p><br />
For a more quantitative measure, the fluorescent cells to total cells ratio was calculated and illustrated below.<br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Splitgfpcount.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<br />
<p><br />
<br />
<br />
From the data shown above, we can see that fluorescence increases within cells in our split sfGFP strain in response to stress while it does not in non-transformed cells. We can thus conclude that the orientation of the split sfGFP constructs added to PBS2 and HOG1 allows proper re-assembly upon interaction of the two proteins. This result shows promise for the actual application of the project as a Biopad using yeast cells. <b>It is the first time a split gets succesfully implemented in the HOG1 pathway by direct attachment to proteins of the kinase cascade</b>. Detection of protein–protein interactions within the pathway were previously studied <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/yea.320080602/abstract"> using vesicle targeting</a>.<br />
<br />
<p/><br />
<br />
<br />
<br />
<br />
<a id="Yeast_exp3"></a><br />
<h3><b>The split-Luciferase strain stress-response</b></h3><br />
<p><br />
After determining that the pathway is reactive to certain stresses and that the split-GFP strain increases its fluorescence, we performed the same test in the obtained split-luciferase (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486030">BBa_K1486030</a>) and (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486036">BBa_K1486036</a>) strain. Unfortunately, due to the difficult nature of detecting an instantaneous luminescence signal and the unstable nature of coelenterazine-Luciferase reaction in different conditions(inhibition due to various molecules such as glucose), our attempts in detecting it on the plate reader failed. Further tests are planned, including an experiment in the microfluidic chips, to directly measure the effect of mechanical pressure, but will not be done before the wiki freeze. <br />
</p><br />
<br />
<br />
<a id="Microfluidics_experiments"></a><br />
<br />
<br />
<br />
<a id="Micro_exp1"></a><br />
<h2><b>Microfluidic achievements</b></h2><br />
<table class="table table-striped valign-middle table-center"><br />
<thead><br />
<tr><br />
<th></th><br />
<th>MITOMI</th><br />
<th>MITOMI modified</th><br />
<th>SmashColi</th><br />
<th>BioPad</th><br />
<th>FilterColi</th><br />
<th>CleanColi</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>Full chip</td><br />
<td class="cntr"><a target="_blank" href="https://static.igem.org/mediawiki/2014/a/ab/Mitomi11.png" data-lightbox="chips" data-title="MITOMI"><img src="https://static.igem.org/mediawiki/2014/a/ab/Mitomi11.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/6/64/Mitomimodif1.png" data-lightbox="chips" data-title="MITOMI Modified"><img src="https://static.igem.org/mediawiki/2014/6/64/Mitomimodif1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/1/15/Smash1.png" data-lightbox="chips" data-title="SmashColi"><img src="https://static.igem.org/mediawiki/2014/1/15/Smash1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/d/db/Biopad1.png" data-lightbox="chips" data-title="BioPad"><img src="https://static.igem.org/mediawiki/2014/d/db/Biopad1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/d/db/Filter1.png" data-lightbox="chips" data-title="FilterColi"><img src="https://static.igem.org/mediawiki/2014/d/db/Filter1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/b/b0/Screen_Shot_2014-10-16_at_22.15.39.png" data-lightbox="chips" data-title="CleanColi"><img src="https://static.igem.org/mediawiki/2014/b/b0/Screen_Shot_2014-10-16_at_22.15.39.png" width="70%"/></a><br /></td><br />
</tr><br />
<tr><br />
<td>Unit Cell</td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/0/0f/MitomiUnit1.png" data-lightbox="chips" data-title="MITOMI Unit"><img src="https://static.igem.org/mediawiki/2014/0/0f/MitomiUnit1.png" width="50%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/7/78/MitomimodifUnit.png" data-lightbox="chips" data-title="MITOMI Modified Unit"><img src="https://static.igem.org/mediawiki/2014/7/78/MitomimodifUnit.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/3/3a/Smahsunit1.png" data-lightbox="chips" data-title="SmashColi Unit"><img src="https://static.igem.org/mediawiki/2014/3/3a/Smahsunit1.png" width="30%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/6/60/Biopadunit1.png" data-lightbox="chips" data-title="BioPad Unit"><img src="https://static.igem.org/mediawiki/2014/6/60/Biopadunit1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/9/9e/FilterUnit.png" data-lightbox="chips" data-title="FilterColi Unit"><img src="https://static.igem.org/mediawiki/2014/9/9e/FilterUnit.png" width="70%"/></a><br /></td><br />
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<td>Designed</td><br />
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<td>Mold fabrication</td><br />
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<td>Fabrication of the chip</td><br />
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<td>Application</td><br />
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<br/> <p>To start our microfluidic experiments, we used the <a target="_blank" href="http://link.springer.com/protocol/10.1007%2F978-1-61779-292-2_6">MITOMI chip</a> that was designed in the laboratory of Pr. Maerkl.</p><br/><br />
<br/><br />
<br />
<br />
<br />
<br />
<a id="Micro_exp2"></a><br />
<h3><b>Culturing <i>E. coli</i> with constitutive GFP on chip</b></h3><br />
<p>We loaded <i>E. coli</i>, which contained constitutive GFP, in the chip. By using LabVIEW, a protocol was launched overnight to ensure the growth of the cells (the protocol can be found <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#growth">here</a>).<br />
<br /></p><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/e/e4/Growth_small.gif" data-lightbox="pipi"><img src="<br />
https://static.igem.org/mediawiki/2014/e/e4/Growth_small.gif" width="50%"></a><br />
</div><br />
</p><br />
<p>The next morning, a scan of the chip was done to see the intensity of the GFP in the chip.<br /></p><br />
<div class=""><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/f/fe/Gfp.png" data-lightbox="prout"><img src="<br />
https://static.igem.org/mediawiki/2014/f/fe/Gfp.png" width="50%" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<br /><br />
<br />
<a id="Micro_exp3"></a><br />
<br />
<h3><b>CpxR linked with GFP on the N terminal, induced by arabinose in <i>E. coli</i></b></h3><br />
<p>The experiment that was done on wetbench to show that CpxR linked with GFP was expressed with an arabinose promoter was replicated on a MITOMI chip.<br />
LB medium with arabinose was flowed in the upper half whereas LB medium without arabinose was flowed in the lower half. We scanned every hour for 5h (to know how it was done click <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#date-08-21-2014">here</a>).</p><br />
<div class="row"><br />
<div class="col col-md-6"><a href="https://static.igem.org/mediawiki/2014/4/4e/Truc2.png" data-lightbox="chips" data-title="Scan of the microfluidic chip at t = 0min. No signal is detected."><img src="https://static.igem.org/mediawiki/2014/4/4e/Truc2.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 1.</strong> Scan of the microfluidic chip at t = 0min. No signal is detected<br />
</div></div><br />
<div class="col col-md-6"><br />
<a href="https://static.igem.org/mediawiki/2014/3/32/Truc3.png" data-lightbox="chips" data-title="Scan of the microfluidic chip at t = 300min."><img src="https://static.igem.org/mediawiki/2014/3/32/Truc3.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 2.</strong> Scan of the microfluidic chip at t = 300min.</div></div><br />
</div><br />
<p>We analysed the scans and obtained the following results.</p><br />
<p><a href="https://static.igem.org/mediawiki/2014/4/4c/Gfp_ara.png" data-lightbox="chips" data-title="Figure 3. Evolution of CpxR-GFP fluorescence over time"><img src="https://static.igem.org/mediawiki/2014/4/4c/Gfp_ara.png" alt="" class="img-responsive" /></a></p><br />
<br />
<div class="cntr"> <br />
<strong>Figure 3.&nbsp;</strong> Evolution of CpxR-GFP fluorescence over time<br />
</div> <br />
<br />
<br/><br />
<br />
<a id="Micro_exp4"></a><br />
<br />
<h3><b>On chip infrared detection</b></h3><br />
<p>As we focused part of our work on the IFP1.4, we needed confirmation of this signal detection in our microfluidic chips. Thus the aim of this experiment was to prove this fluorescence detection capability. Bacteria were loaded in the smash-coli chip. The first batch was KCl stressed and the second batch was unstressed. We then simply scanned the chip and analysed the results using ImageJ. For more details, please visit our <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#date-10-03-2014">notebook</a>. </p><br />
<div class="row"><br />
<div class="col col-md-6"><a href="https://static.igem.org/mediawiki/2014/c/cf/No_KCl_red.png" data-lightbox="chips" data-title="Figure 1. Cy5 scan of a chamber containing non-stressed CpxR-IFP bacteria"><img src="https://static.igem.org/mediawiki/2014/c/cf/No_KCl_red.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 1.</strong> Cy5 scan of a chamber containing non-stressed CpxR-IFP bacteria.<br />
</div></div><br />
<div class="col col-md-6"><br />
<a href="https://static.igem.org/mediawiki/2014/c/c2/KCL_red.png" data-lightbox="chips" data-title="Figure 2. Cy5 scan of a chamber containing KCl-stressed CpxR-IFP bacteria"><img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_red.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 2.</strong> Cy5 scan of a chamber containing KCl-stressed CpxR-IFP bacteria.</div></div><br />
</div><br />
<p>We analysed the scans and obtained the following results.</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/2/24/KCl_stack.PNG" alt="" class="img-responsive" /></p><br />
<strong>Figure 3.&nbsp;</strong> Histogram of KCl stressed cells and non-stressed cells.<br />
<br />
<br/><br/><br />
<br />
<hr><br />
<br />
<h3>BioPad Detector: Detection of sfGFP</h3><br />
<br />
<p> To get a first idea of how our BioPad was able to detect signal, we attempted to detect sfGFP signal emission by exciting our sfGFP with a 470 nm emitting LED and acquiring the images through our device. A sample of the video taken can be seen here bellow: </p><br />
<br />
https://static.igem.org/mediawiki/2014/2/2f/Gfptracking_EPFL.mp4<br />
<br />
<p><br />
To learn more about how the detector works check out our <a href="https://2014.igem.org/Team:EPF_Lausanne/Hardware"> Hardware page!</a><br />
</p><br />
<br />
<!--<h4> References </h4><br />
<p><br />
<a id="ref1"></a>1: S.K. Hatzios, S. Ringgaard, B. M. Davis, M. K. Waldor (2012, August 15). Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition. <i>Plos One</i>.<br />
</p>--><br />
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<li><a href="#IFP_Exp1">AraC/PBad Promoter & GFP fused to CpxR</a></li><br />
<li><a href="#IFP_Exp2">CpxR dimerization & Orientation Elucidation</a></li><br />
<li><a href="#IFP_Exp3">Titration of KCl & signal shutdown </a></li><br />
<li><a href="#IFP_Exp4"></b>Microscopy of KCl stress </a></li><br />
<li><a href="#IFP_Exp5"></b>Mechanical stress on microfluidic chip</a></li><br />
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<li><a href="#Yeast_exp1">Confirmation of successful transformation via the Renilla Luciferase tag</a></li><br />
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<li><a href="#Yeast_exp3">The split-Luciferase strain stress response</a></li><br />
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<li><a href="#Micro_exp4">On chip infrared detection</a></li><br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/ResultsTeam:EPF Lausanne/Results2014-10-18T00:49:42Z<p>Robert Baldwin: </p>
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<h1 class="cntr"> <b>RESULTS</b> </h1><br />
<br /><br />
<br />
<br />
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<br />
<a id="IFP_Exp"></a><br />
<!--replaced the title : Characterisation of the spatiotemporal dynamics of the CpxR - split IFP 1.4 stress sensor --><br />
<h2> <b> Characterisation of the spatiotemporal dynamics of the CpxR stress sensor </b> </h2<br />
</div><br />
<br />
<br /><br /><br />
<br />
<br />
<br />
<a id="IFP_Exp2"></a><br />
<h3><b>Demonstration of CpxR's dimerization & Elucidation of its dimerization orientation</b></h3><br />
<br />
<p><br />
Previous in vitro experiments (FRET) have shown that CpxR dimerizes. Currently little in vivo information about CpxR’s dimerization is available. To characterize our stress responsive bacteria, we had to confirm that CpxR dimerized in vivo as well as elucidate CpxR’s dimerization orientation. <br />
</p><br />
<br /><br />
<p><br />
We synthesized four constructs with combinations of the split IFP1.4 fragments fused to the C or N terminal of CpxR. <br />
As seen in the graph bellow, induction of IFP1.4 signal by 50 mM KCl was done at t=24min. We clearly see that the construct with IFP fragments on the C-terminal immediately responded to stress by emitting fluorescence. In fact we observe a 3 fold signal increase in 2 minutes. On the other hand, the three other orientations were non-responsive to KCl stress.</p><br />
<br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" data-lightbox="chips" data-title="Construct Comparison"><img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" width="80%"/></a><br /><br />
</div><br />
<p><br />
This amazing 30-fold signal increase in little time from the baseline allows us to assert that only the strain synthesizing the IFP fragments on the C-terminals of CpxR responded to KCl stress. For a further analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
</p><br />
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<!--<br />
<p><br />
<u>Introduction</u> <br /><br />
CpxR is the relay protein in the stress responsive CpxAR two component regulatory system. It has been shown by split beta galactosidase assay that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that <i>E. coli</i> CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in <i>E. coli</i>.</p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to determine if and how CpxR dimerised in vivo in <i>E. coli</i>. This experiment intended to get a first idea of the real-time temporal dynamics of the activation of CpxR (the cytoplasmic relay protein of the CpxA-R pathway) by KCl stress via CpxA (the periplasmic sensor protein of the CpxA-R pathway). This experiment is a first of its kind.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To evaluate if and how CpxR dimerized under KCl stress, we built by gibson assembly four constructs with the various possible orientations that the split IFP1.4 fragments could have with CpxR: IFP[1] and IFP[2] on the N-terminus of CpxR, IFP[1] on the N-terminus of CpxR and IFP[2] on the C-terminus of CpxR, and finally IFP[1] and IFP[2] on the N-terminus of CpxR. The split IFP fragments were provided by the Michnick Lab, and the CpxR coding region was amplified by PCR from extracted <i>E. coli</i> genome (Bacterial Genomic Miniprep Kit from Sigma Aldrich). The protocol for stressing the cells and reading the fluorescence can be downloaded <a target="blank" href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the graph bellow, induction of the signal was done at minute 24 (marked via a vertically spoted line). The construct with IFP fragments on the C-termina responded immediately to stress. In a fact we observed a 3 fold signal increase in 2 minutes. All other constructs we observed a low baseline signal non responsive to KCl stress. It is to be noted that the C-termina constructs always had higher signal levels than the other constructs. This leads us to believe that the PBS used to resuspend our cultures led to small levels of stress (the PBS we use does not contain KCl but traces of NaCl). The 30-fold signal increase from the baseline allows us to assert that our constructs responds to KCl stress.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" alt="Construct Comparison" class="img-responsive"><br />
</div><br />
<p><br />
<u>Discussion</u> <br /><br />
We successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus. This finding suggests that CpxR binds via its C-termina. This leads us to hypothesise that the CpxR dimerisation mechanisms is the same for other members of the highly conserved OmpR/PhoB subfamily. This hypothesis could allow the development of similar system that could the study other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.<br />
</p><br />
<br /><br /><br />
--><br />
<br />
<br />
<br />
<br /><br /><br />
<br />
<a id="IFP_Exp3"></a><br />
<h3><b>Signal induction by various concentrations of KCl & signal shutdown by centrifugation</b></h3><br />
<br />
<p><br />
Having confirmed that KCl was a good inducer for our signal, we tested different concentration of KCl to modulate the signal and better characterize our biobrick. We also aimed to shutdown the signal by centrifugation and medium change. The signal was measured on a plate reader 20 minutes, before the addition of KCl. After 2 hours of measurement we centrifuged the cells for ten minutes and replaced the medium with PBS to be able to get a shutdown of the signal.</p><br />
<br />
<div class="cntr"><br />
<a target="_blank" href="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" data-lightbox="chips" data-title="The split-IFP are linked to cpxR on the C-terminal. During the experiment it was possible to shut down the signal"><img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" width="80%"/></a><br /><br />
</div><br />
<br />
<br />
<p><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to drastically shut down the signal, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis is possible for our system. For a thorough analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
</p><br />
<br />
<br />
<!--<br />
<p><br />
<u>Aim</u> <br /><br />
Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change.<br />
To do so, we read our signal for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To evaluate if a modulation in KCl concentrations affected the intensity of the intensity of the fluorescent signal, and if a change in medium by centrifugation shutdown the signal; we read our signal on a plate reader for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal. The protocol for this experiment can be downloaded <a target="_blank" href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis are possible for our system.<br />
</p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" alt="GA1 Shutdown" class="img-responsive"><br />
</div><br />
<br /><br /><br/><br/><br />
--><br />
<br />
<br /><br /><br />
<br />
<a id="IFP_Exp4"></a><br />
<h3><b>Visualization of the the CpxR split IFP1.4 activation by KCl stress </b></h3><br />
<br />
<br />
<p><br />
<br />
<br />
Having shown that we were able to monitor the temporal dynamics of CpxR activation, we wanted to see if we could analyze CpxR’s spatial dynamics by microscopy. 10μl of our cells, previously stressed with 80 mM KCl were spread on a glass slide and imaged with a x100 objective and a APC (Cy5.5) filter.<br />
<br />
<br />
</p><br />
<br />
<br />
<p><br />
<br />
<br />
We noticed various characteristics of the cells from the picture bellow. First, IFP signal was much more present in stressed bacteria rather than in non-stressed bacteria. Secondly, we distinguished two specific phenotypes within bacteria: elongated and normal cells. We noticed that this difference was due to CpxR overexpression as we saw this phenomenon also in non-stressed conditions.<br /><br /><br />
<br />
<br />
In elongated cells, we were able to distinguish several bright bands of IFP signal that seem fairly uniformly distributed. In the normal phenotype we distinguished a single band in the centre of the bacteria. These observations led us to believe that CpxR might be involved in the division process of E. coli as it seems coherent for cells to slow down division upon stress. <br /><br /><br />
<br />
<br />
After looking into the literature, similar bands were visualizable in E. coli for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how E. coli monitor division under various environments. For a thorough analysis of this experiment check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results/IFP_experiments">this link!</a><br />
<br />
<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" data-lightbox="results" data-title="Results"><img src="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" alt="results" width="45%" class="pull-left"></a><br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/EPFL_2014_03_10_2014_Experiment-24.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/d/de/EPFL_2014_03_10_2014_Experiment-34.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/fd/EPFL_2014_03_10_2014_Experiment-35.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/0/08/EPFL_2014_03_10_2014_Experiment-37.jpg" data-lightbox="results" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/a/a7/EPFL_2014_03_10_2014_Experiment-38.jpg" data-lightbox="results" data-title="Results"></a> <br />
<a href="https://static.igem.org/mediawiki/2014/2/2e/EPFL_2014_03_10_2014_Picture3.jpg" data-lightbox="results" data-title="Results"><br />
<a href="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" data-lightbox="results" data-title="Results"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" alt="results" width="45%" class="pull-right"></a><br />
</div><br />
<br />
<br />
<br />
<!--<br />
<br />
<p><br />
<u>Aim</u> <br /><br />
Having shown that we were able to monitor the temporal dynamics of our construct, we wanted to see if we were able to analyze the spatial dynamics by microscopy.<br />
</p><br />
<p><br />
<u>Methods</u> <br /><br />
To visualize the activation of our construct, we prepared cells as above for the previous plate-reader experiments, spread 10 µl on a glass slide added a coverslip and imaged them on a Zeiss Axioplan with a x100 objective and a APC (Cy5.5) filter. The pictures shown bellow were taken with a 5.1(s) integration time.<br />
</p><br />
<p><br />
<u>Results</u> <br /><br />
As seen in the pictures bellow, we were able to distinguish specific patterns within bacteria. We observed two phenotypes within our population: elongated and normal cells. The difference in these phenotypes was noticed in previous experiments and is most certainly due to the CpxR overexpression as we observed this also in non-stressed conditions. In the first phenotype (elongated) we were able to distinguish several bands that seem fairly uniformly distributed. In the second phenotype (normal) we observed a single band in the center of the bacteria. These observations led us to believe that CpxR might be involved in the division process of <i>E. coli</i> as it seems coherent for cells to slow down division upon stress. After looking into the literature, similar bands were visualizable in <i>E. coli</i> for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how <i>E. coli</i> monitor division under various environments.<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"><img src="https://static.igem.org/mediawiki/2014/0/07/EPFL_2014_03_10_2014_Experiment-46.jpg" alt="results" width="45%" class="pull-left"></a><br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/EPFL_2014_03_10_2014_Experiment-24.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/d/de/EPFL_2014_03_10_2014_Experiment-34.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/fd/EPFL_2014_03_10_2014_Experiment-35.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/0/08/EPFL_2014_03_10_2014_Experiment-37.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a><br />
<a href="https://static.igem.org/mediawiki/2014/a/a7/EPFL_2014_03_10_2014_Experiment-38.jpg" data-lightbox="Results" data-title="IFP signal in stressed cells"></a> <br />
<a href="https://static.igem.org/mediawiki/2014/2/2e/EPFL_2014_03_10_2014_Picture3.jpg" data-lightbox="results" data-title="IFP signal in stressed cells"><br />
<a href="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" data-lightbox="results" data-title="IFP signal in non stressed cells"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Neg_IFP_EPFL.jpg" alt="results" width="45%" class="pull-right"></a><br />
</div><br />
<br />
--><br />
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<br />
<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
<br />
<br />
<br/><br/><br/><br />
<!--<br />
<a id=“CpxR-promoter"></a><br />
<h3><b>RFP under CpxR promoter to investigate time delay between transcription factor activation and protein synthesis</b></h3><br />
<br />
<p>To mesure the time it takes a cell to react to a stress input and if our modified CpxR still has its function as a transcription factor, we wanted to investigate the RFP expression of stressed cells that we co-transformed our CpxR-split-IPF construct with the <a target="_blank" href="http://parts.igem.org/Part:BBa_K339007">BBa_K339007</a>. Unfortunately we realised that the regulatory sequence of the RFP, the CpxR responsive promoter, was missing. So we constructed the biobrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486048">BBa_K1486048</a> which contains the regulatory sequence. After a few experiments we realised that KCl stress, which we have found to be a good signal inducer, did not work, even in cells expressing native CpxR. So we developed the biobricks <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486049">BBa_K1486049</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486050">BBa_K1486050</a>, which encode for RFP regulated by the CpxR responsive promoter, how it is found in the genome. In the figure below you see that neither of the three constructs was stress induced.</p><br />
<br />
<div class="cntr img-border"><br />
<a href="https://static.igem.org/mediawiki/2014/9/91/BBa_K1486049_BBa_K1486048.png" data-lightbox="cpxr" data-title="CpxR promoted RFP under stress"><br />
<img src="https://static.igem.org/mediawiki/2014/9/91/BBa_K1486049_BBa_K1486048.png" alt="CpxR promoted RFP under stress" class="img-responsive" /></a><br />
</div><br />
<br />
<br/><br/><br/><br />
--><br />
<br />
<a id="IFP_Exp5"></a><br />
<h3><b>Activation of CpxR - split IFP1.4 on microfluidic chip by chamber crushing </b></h3><br />
<br />
<p><br />
Knowing that we were able to able to visualize CpxR-IFP activation under a microscope, we proceeded to trying to activate the pathway by mechanical stress on a microfluidic chip - the ultimate barrier to building a functional BioPad !<br /><br /><br />
<br />
To induce this stress, we turned on the buttons of our SmashColi microfluidic chip at a pressure of 25 psi. We imaged chambers before stress and after stress (10 min after button activation). A drastic increase in signal was detected !<br />
<br />
<div class="cntr"><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/d/dc/Button_IFP_non_stressed_2_Cy5.jpg" data-lightbox="results_button" data-title="results_button"><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Button_IFP_non_stressed_2_Cy5.jpg" alt="results" width="45%" class="pull-left"> <br />
</a><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/7/77/Button_IFP_non_stressed_2.jpg" data-lightbox="results_button" data-title="Results"></a><br />
<a href="https://static.igem.org/mediawiki/2014/9/99/Button_IFP_stressed_2.jpg" data-lightbox="results_button" data-title="Results"></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/f/f8/Button_IFP_stressed_2_Cy5.jpg" data-lightbox="results_button" data-title="results_button"><img src="https://static.igem.org/mediawiki/2014/f/f8/Button_IFP_stressed_2_Cy5.jpg" alt="results" width="45%" class="pull-right"> <br />
</a><br />
<br />
</div><br />
<br />
<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
<a id="IFP_Exp1"></a><br />
<h3> <b> Characterisation of the pBAD promoter and folding ability of GFP fused to CpxR </b></h3><br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg" data-lightbox="img1"><img src="<br />
https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg" width="70%"></a><br />
</div> <br /><br /><br />
<br />
<p>This construct aimed to evaluate the expression of our construct and the characteristics of the arabinose promoter in <i>E. coli</i> by fusing a superfolder GFP protein to CpxR. The sfGFP was chosen because of its higher intensity compared to GFP. </p><br />
<p>Not knowing if CpxR would react the same way if sfGFP were attached to the N or C terminus, 2 biobricks were built, one with each of the orientations: <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486002">BBa_K1486002 (N terminus)</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486005">BBa_K1486005 (C terminus)</a>.<br />
</p><br />
<p><br />
We tested both constructs CpxR-sfGFP and sfGFP-CpxR on a plate reader to characterize the pBAD promoter, measuring the sfGFP signal in function of the arabinose concentration. We also measured the signal in a microfluidic chip. You can see in the graph below that the signal is increasing as the concentration of the arabinose increases. The construct with sfGFP at the C terminal lead to a higher GFP signal. Interestingly both curves increases in a very similar manner.<br />
<br />
</p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/a/ac/Nterm_and_Cterm_log.png" data-lightbox="hellopony"><img src="<br />
https://static.igem.org/mediawiki/2014/a/ac/Nterm_and_Cterm_log.png" width="50%"></a><br />
</div><br />
<br/><br/><br />
<hr/><br />
<br />
<h2 id="Characterisation_of_the_split_luciferase"> <b>Characterisation of the split luciferase </b> </h2><br />
<br />
<h3 id="Characterisation_of_the_split_luciferase"><b>Split luciferase complementation assay using CheY and CheZ chemotaxis proteins </b> </h3><br />
<br />
<p><br />
CheY and CheZ are two proteins involved in the bacterial chemotaxis pathway. It has been shown by split luciferase complementation assay that these two proteins are not interacting in presence of chemoattractant, but start to interact in absence of chemoattractant or presence of chemorepellent. Based on the work of Waldor<sup><a href="#ref1">1</a></sup> Laboratory, we wanted to redo the experiment to test our own splits, with firefly (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486055">BBa_K1486055</a>) and renilla (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486054">BBa_K1486054</a>) luciferases.<br />
</p><br />
<br />
<p>As shown in the graphs (fig.1A and 1B), we didn't observe a high signal for our assay. However, the signal being higher than the blanks, it is an encouraging sign that the splits luciferase can be used for other experiments of this kind. A possible explanation for these results is that we didn't completely get rid of the arabinose, which acts as a chemoattractant. Moreover, CheY and CheZ being endogenously expressed in bacteria, there could be interferences with our fusion proteins and weakening of our signal. This should be tested again with CheY/CheZ knock out strains.<br /><br />
</p><br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Renilla"><img src="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Firefly"><img src="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
</div><br /><br />
<br /><br />
<p>We also could determine which of the luciferases would best suit our following experiments. As shown in fig. 2, for the same concentration of substrate, we see that firefly luciferase has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.</p><br /><br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" data-lightbox="chips" data-title="Bioluminescence assay"><img src="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" width="80%"/></a><br /><br />
</div><br />
</p><br />
</div><br />
<br /><br />
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<br />
<br />
<br />
<br />
<br />
<br />
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<br />
<!--<br />
<div class="cntr"><br />
<h2 id="Characterisation_of_the_split_luciferase"> <b><u>Characterisation of the split luciferase </u> </b> </h2><br />
</div><br />
<br /><br /><br />
<br />
<h3 id="Characterisation_of_the_split_luciferase"><b>Experiment 1: </b>CheY/CheZ fused to split Firefly/Renilla luciferase, and full Firefly/Renilla luciferase characterisation </h3><br />
<p><u>Introduction</u> <br /><br />
CheY and CheZ are two proteins involved in the bacterial chemotaxis pathway. It has been shown by split luciferase complementation assay that these two proteins are not interacting in presence of chemoattractant, but start to interact (CheZ being the phosphatase of CheY) in absence of chemoattractant or presence of chemorepellent. Based on the work of Waldor<sup><a href="#ref1">1</a></sup> Laboratory, we wanted to redo and adapt the experiment to test our own splits.<br /> <br /></p><br />
<p><br />
<u>Aim</u> <br /><br />
This experiment aimed to test the efficiency of split Renilla luciferase and split Firefly luciferase. We wanted to study the speed of the signal and the amount of substrate needed to have a performant response. <br /> <br /><br />
</p><br />
<p><br />
<u>Method</u> <br /><br />
To proceed to this complementation assay, we built two constructs, one to test split Renilla Luciferase and the other for split Firefly Luciferase The CheY was fused to the N-terminal part of each split, while the CheZ was fused to the C-terminal part. We used the full luciferases (Renilla : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486022"> BBa_K1486022 </a> and Firefly : <a target="_blank" href="http://parts.igem.org/Part:BBa_K325108"> BBa_K325108 </a> from Cambridge 2010 team) as positive controls and the non-fused splits (Renilla : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486021"> BBa_K1486021 </a> and Firefly : <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486018"> BBa_K1486018 </a>) as negative controls.<br /> <br /><br />
The bioluminescence assay was performed as described <a target="_blank" href="https://static.igem.org/mediawiki/2014/6/6d/Protocol_-_Bioluminescence_assay.pdf">here</a>. <br /><br />
The constructs were designed and assembled as described <a target="_blank" href="https://static.igem.org/mediawiki/2014/3/3b/Constructs_design_CheYCheZ.pdf">here</a>.<br /><br /> <br /><br />
</p><br />
<p><u>Results</u> <br /><br />
As shown in the graphs below (fig.1A and 1B), we couldn't really observe a high signal for our assay. However, the signal being higher than the blanks, it is an encouraging sign that the splits luciferase can be used for other experiments of this kind. A possible explanation for these results is that arabinose being a chemoattractant, we might need to do more wash steps with PBS to get rid of the arabinose before taking the measurements. Moreover, CheY and CheZ being endogenously expressed in bacteria, the edogenous proteins could interfere with our fusion proteins and weaken our signal. This complementation assay should be tested with CheY/CheZ knock out strains, as it was done in Waldor Laboratory.<br /><br />
</p><br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Renilla"><img src="https://static.igem.org/mediawiki/2014/3/30/Renilla-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
<a href="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" data-lightbox="cheYcheZ" data-title="Firefly"><img src="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" alt="cheYcheZ" width="45%"></a><br />
</div><br /><br />
<br /><br />
We also could determine which of the luciferases would best suit our following experiments. As shown in fig. 2, for the same concentration of substrate, we see that firefly luciferase has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.<br /><br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f7/Controls-CheYCheZexp.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<br /><br />
--><br />
<br />
<br />
<a id="Yeast_experiments"></a><br />
<br />
<h2> <b>The PBS2-HOG1 split-GFP & split Renilla Luciferase stress response<br />
</b> </h2><br />
<br />
<br/><br/><br />
<a id="Yeast_exp1"></a><br />
<h3><b>Confirmation of successful transformation via the Renilla Luciferase tag </b></h3><br />
<br/><br />
<p><br />
<br />
Since we had never transformed <i>S. cerevisiae</i>, we first needed to confirm our protein tag transformations as a positive control for subsequent experiments. We cultured the two strains we got from the transformation, the HOG1-rLuc (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486033">BBa_K1486033</a> ) and PBS2-rLuc (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486027">BBa_K1486027</a>), put them in a 96 well plate and tested their luminescence using a plate reader.</p><br />
<p><br />
<br />
The test was performed using two different concentrations of the substrate, coelenterazine-h(benzyl coelenterazine): 1μM and 5μM so that we could determine the optimal concentration we would use for the next assays. The plate started to be read directly after addition of the substrate in the dark and measures were taken every 56 seconds thereafter. Three wells for each sample were measured and below are shown the graphs of their average luminescence plotted over time. We observed a signal-to-noise ratio of 4 at 1μM of substrate and 10 at 5μM for the PBS2-rLuc strain. The HOG1-rLuc strain seemed problematic and died within the plate. Unable to verify the correct transformation, we reproduced the strain but were unable to confirm its validity before the wiki deadline.</p><br />
<br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/0e/Pbslucprettycomparaison_.png" data-lightbox="chips" data-title=""><img src="https://static.igem.org/mediawiki/2014/0/0e/Pbslucprettycomparaison_.png" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<p><br />
There is a clear luminescence generated by the cells, providing us with confirmation that our designs are correct and we determined which concentration of substrate to use to have a decent signal-to-background ratio.<br />
</p><br />
<br />
<br />
<a id="Yeast_exp2"></a><br />
<h3><b>The split-sfGFP strain stress-response</b></h3><br />
<p><br />
<br />
<br />
Having confirmed that our design for yeast transformations was correct via the previous experiment, we performed stress-response tests upon our PBS2-HOG1-splitGFP (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486029">BBa_K1486029</a>) and (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486035">BBa_K1486035</a>) strain to determine whether the pathway is reactive to stress as theorized. A co-transformation was performed using linear fragments produced in the same way as for the first experiment to obtain the strain. The N-terminal split of the superfolder GFP was tagged onto PBS2 and the C-terminal onto HOG1. The strain was then tested along with non-transformed cells in a 96 well plate with various stresses (e.g. Acetic Acid, Ethanol, Glucose...). The cells were centrifuged and resuspended in PBS before loading into the plate.<br />
</p><br />
<br />
<br />
<div class="cntr"><br />
<br />
<div class="cntr"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/1/1d/Splitgfpgraph.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<p><br />
The most reactive of stresses turned out to be Acetic Acid 3.6%(shown above) followed by Ethanol 10%. Other stresses tested did not seem to show conclusive results and we were unable to determine more reactants before the wiki freeze deadline. To further assess the fluorescence 10μl of our cells were spread on a glass slide and imaged with a x63 objective and a Green LP filter<br />
(Excitation BP 450-490, Dichroïc FT 510, Emission LP 515). Below is shown a comparison of before and after 3.6% Acetic Acid stress in the microscopy image merged with the fluorescence. <br />
</p><br />
<br />
<div class="cntr"><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/3b/Splitgfpyeast.jpg" data-lightbox="caca" data-title="Image of our split sfGFP expressing cells before and after the 3.6% acetic acid stress (x63 objective and a Green LP filter)"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3b/Splitgfpyeast.jpg" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<br />
<div class="cntr"><br />
<p><br />
For a more quantitative measure, the fluorescent cells to total cells ratio was calculated and illustrated below.<br />
<div class="cntr"><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Splitgfpcount.png" class="img-responsive"><br />
</div><br />
</p><br />
</div><br />
<br />
<p><br />
<br />
<br />
From the data shown above, we can see that fluorescence increases within cells in our split sfGFP strain in response to stress while it does not in non-transformed cells. We can thus conclude that the orientation of the split sfGFP constructs added to PBS2 and HOG1 allows proper re-assembly upon interaction of the two proteins. This result shows promise for the actual application of the project as a Biopad using yeast cells. <b>It is the first time a split gets succesfully implemented in the HOG1 pathway by direct attachment to proteins of the kinase cascade</b>. Detection of protein–protein interactions within the pathway were previously studied <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/yea.320080602/abstract"> using vesicle targeting</a>.<br />
<br />
<p/><br />
<br />
<br />
<br />
<br />
<a id="Yeast_exp3"></a><br />
<h3><b>The split-Luciferase strain stress-response</b></h3><br />
<p><br />
After determining that the pathway is reactive to certain stresses and that the split-GFP strain increases its fluorescence, we performed the same test in the obtained split-luciferase (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486030">BBa_K1486030</a>) and (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486036">BBa_K1486036</a>) strain. Unfortunately, due to the difficult nature of detecting an instantaneous luminescence signal and the unstable nature of coelenterazine-Luciferase reaction in different conditions(inhibition due to various molecules such as glucose), our attempts in detecting it on the plate reader failed. Further tests are planned, including an experiment in the microfluidic chips, to directly measure the effect of mechanical pressure, but will not be done before the wiki freeze. <br />
</p><br />
<br />
<br />
<a id="Microfluidics_experiments"></a><br />
<br />
<br />
<br />
<a id="Micro_exp1"></a><br />
<h2><b>Microfluidic achievements</b></h2><br />
<table class="table table-striped valign-middle table-center"><br />
<thead><br />
<tr><br />
<th></th><br />
<th>MITOMI</th><br />
<th>MITOMI modified</th><br />
<th>SmashColi</th><br />
<th>BioPad</th><br />
<th>FilterColi</th><br />
<th>CleanColi</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>Full chip</td><br />
<td class="cntr"><a target="_blank" href="https://static.igem.org/mediawiki/2014/a/ab/Mitomi11.png" data-lightbox="chips" data-title="MITOMI"><img src="https://static.igem.org/mediawiki/2014/a/ab/Mitomi11.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/6/64/Mitomimodif1.png" data-lightbox="chips" data-title="MITOMI Modified"><img src="https://static.igem.org/mediawiki/2014/6/64/Mitomimodif1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/1/15/Smash1.png" data-lightbox="chips" data-title="SmashColi"><img src="https://static.igem.org/mediawiki/2014/1/15/Smash1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/d/db/Biopad1.png" data-lightbox="chips" data-title="BioPad"><img src="https://static.igem.org/mediawiki/2014/d/db/Biopad1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/d/db/Filter1.png" data-lightbox="chips" data-title="FilterColi"><img src="https://static.igem.org/mediawiki/2014/d/db/Filter1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/b/b0/Screen_Shot_2014-10-16_at_22.15.39.png" data-lightbox="chips" data-title="CleanColi"><img src="https://static.igem.org/mediawiki/2014/b/b0/Screen_Shot_2014-10-16_at_22.15.39.png" width="70%"/></a><br /></td><br />
</tr><br />
<tr><br />
<td>Unit Cell</td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/0/0f/MitomiUnit1.png" data-lightbox="chips" data-title="MITOMI Unit"><img src="https://static.igem.org/mediawiki/2014/0/0f/MitomiUnit1.png" width="50%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/7/78/MitomimodifUnit.png" data-lightbox="chips" data-title="MITOMI Modified Unit"><img src="https://static.igem.org/mediawiki/2014/7/78/MitomimodifUnit.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/3/3a/Smahsunit1.png" data-lightbox="chips" data-title="SmashColi Unit"><img src="https://static.igem.org/mediawiki/2014/3/3a/Smahsunit1.png" width="30%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/6/60/Biopadunit1.png" data-lightbox="chips" data-title="BioPad Unit"><img src="https://static.igem.org/mediawiki/2014/6/60/Biopadunit1.png" width="70%"/></a><br /></td><br />
<td class="cntr"><a href="https://static.igem.org/mediawiki/2014/9/9e/FilterUnit.png" data-lightbox="chips" data-title="FilterColi Unit"><img src="https://static.igem.org/mediawiki/2014/9/9e/FilterUnit.png" width="70%"/></a><br /></td><br />
<td><span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Designed</td><br />
<td><span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
</tr><br />
<tr><br />
<td>Mold fabrication</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Fabrication of the chip</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
<tr><br />
<td>Application</td><br />
<td><span class="glyphicon glyphicon-ok glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-ok glyph-check"></span> </td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
<td> <span class="glyphicon glyphicon-time glyph-check"></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br/> <p>To start our microfluidic experiments, we used the <a target="_blank" href="http://link.springer.com/protocol/10.1007%2F978-1-61779-292-2_6">MITOMI chip</a> that was designed in the laboratory of Pr. Maerkl.</p><br/><br />
<br/><br />
<br />
<br />
<br />
<br />
<a id="Micro_exp2"></a><br />
<h3><b>Culturing <i>E. coli</i> with constitutive GFP on chip</b></h3><br />
<p>We loaded <i>E. coli</i>, which contained constitutive GFP, in the chip. By using LabVIEW, a protocol was launched overnight to ensure the growth of the cells (the protocol can be found <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#growth">here</a>).<br />
<br /></p><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/e/e4/Growth_small.gif" data-lightbox="pipi"><img src="<br />
https://static.igem.org/mediawiki/2014/e/e4/Growth_small.gif" width="50%"></a><br />
</div><br />
</p><br />
<p>The next morning, a scan of the chip was done to see the intensity of the GFP in the chip.<br /></p><br />
<div class=""><br />
<p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/f/fe/Gfp.png" data-lightbox="prout"><img src="<br />
https://static.igem.org/mediawiki/2014/f/fe/Gfp.png" width="50%" class="img-responsive"></a><br />
</div><br />
</p><br />
</div><br />
<br />
<br /><br />
<br />
<a id="Micro_exp3"></a><br />
<br />
<h3><b>CpxR linked with GFP on the N terminal, induced by arabinose in <i>E. coli</i></b></h3><br />
<p>The experiment that was done on wetbench to show that CpxR linked with GFP was expressed with an arabinose promoter was replicated on a MITOMI chip.<br />
LB medium with arabinose was flowed in the upper half whereas LB medium without arabinose was flowed in the lower half. We scanned every hour for 5h (to know how it was done click <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#date-08-21-2014">here</a>).</p><br />
<div class="row"><br />
<div class="col col-md-6"><a href="https://static.igem.org/mediawiki/2014/4/4e/Truc2.png" data-lightbox="chips" data-title="Scan of the microfluidic chip at t = 0min. No signal is detected."><img src="https://static.igem.org/mediawiki/2014/4/4e/Truc2.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 1.</strong> Scan of the microfluidic chip at t = 0min. No signal is detected<br />
</div></div><br />
<div class="col col-md-6"><br />
<a href="https://static.igem.org/mediawiki/2014/3/32/Truc3.png" data-lightbox="chips" data-title="Scan of the microfluidic chip at t = 300min."><img src="https://static.igem.org/mediawiki/2014/3/32/Truc3.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 2.</strong> Scan of the microfluidic chip at t = 300min.</div></div><br />
</div><br />
<p>We analysed the scans and obtained the following results.</p><br />
<p><a href="https://static.igem.org/mediawiki/2014/4/4c/Gfp_ara.png" data-lightbox="chips" data-title="Figure 3. Evolution of CpxR-GFP fluorescence over time"><img src="https://static.igem.org/mediawiki/2014/4/4c/Gfp_ara.png" alt="" class="img-responsive" /></a></p><br />
<br />
<div class="cntr"> <br />
<strong>Figure 3.&nbsp;</strong> Evolution of CpxR-GFP fluorescence over time<br />
</div> <br />
<br />
<br/><br />
<br />
<a id="Micro_exp4"></a><br />
<br />
<h3><b>On chip infrared detection</b></h3><br />
<p>As we focused part of our work on the IFP1.4, we needed confirmation of this signal detection in our microfluidic chips. Thus the aim of this experiment was to prove this fluorescence detection capability. Bacteria were loaded in the smash-coli chip. The first batch was KCl stressed and the second batch was unstressed. We then simply scanned the chip and analysed the results using ImageJ. For more details, please visit our <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics#date-10-03-2014">notebook</a>. </p><br />
<div class="row"><br />
<div class="col col-md-6"><a href="https://static.igem.org/mediawiki/2014/c/cf/No_KCl_red.png" data-lightbox="chips" data-title="Figure 1. Cy5 scan of a chamber containing non-stressed CpxR-IFP bacteria"><img src="https://static.igem.org/mediawiki/2014/c/cf/No_KCl_red.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 1.</strong> Cy5 scan of a chamber containing non-stressed CpxR-IFP bacteria.<br />
</div></div><br />
<div class="col col-md-6"><br />
<a href="https://static.igem.org/mediawiki/2014/c/c2/KCL_red.png" data-lightbox="chips" data-title="Figure 2. Cy5 scan of a chamber containing KCl-stressed CpxR-IFP bacteria"><img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_red.png" alt="" class="img-responsive" /></a><br /><br />
<div class="cntr"><br />
<strong>Figure 2.</strong> Cy5 scan of a chamber containing KCl-stressed CpxR-IFP bacteria.</div></div><br />
</div><br />
<p>We analysed the scans and obtained the following results.</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/2/24/KCl_stack.PNG" alt="" class="img-responsive" /></p><br />
<strong>Figure 3.&nbsp;</strong> Histogram of KCl stressed cells and non-stressed cells.<br />
<br />
<br/><br/><br />
<br />
<hr><br />
<br />
<h3>BioPad Detector: Detection of sfGFP</h3><br />
<br />
<p> To get a first idea of how our BioPad was able to detect signal, we attempted to detect sfGFP signal emission by exciting our sfGFP with a 470 nm emitting LED and acquiring the images through our device. A sample of the video taken can be seen here bellow: </p><br />
<br />
https://static.igem.org/mediawiki/2014/2/2f/Gfptracking_EPFL.mp4<br />
<br />
<p><br />
To learn more about how the detector works check out our <a href="https://2014.igem.org/Team:EPF_Lausanne/Hardware" Hardware page!</a><br />
</p><br />
<br />
<!--<h4> References </h4><br />
<p><br />
<a id="ref1"></a>1: S.K. Hatzios, S. Ringgaard, B. M. Davis, M. K. Waldor (2012, August 15). Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition. <i>Plos One</i>.<br />
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<li><a href="#IFP_Exp" class="active">Characterization of spatiotemporal dynamics of the CpxR stress sensor</a><br />
<ul class="nav"><br />
<li><a href="#IFP_Exp1">AraC/PBad Promoter & GFP fused to CpxR</a></li><br />
<li><a href="#IFP_Exp2">CpxR dimerization & Orientation Elucidation</a></li><br />
<li><a href="#IFP_Exp3">Titration of KCl & signal shutdown </a></li><br />
<li><a href="#IFP_Exp4"></b>Microscopy of KCl stress </a></li><br />
<li><a href="#IFP_Exp5"></b>Mechanical stress on microfluidic chip</a></li><br />
<br />
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<li><a href="#Characterisation_of_the_split_luciferase">CheY/CheZ - split Firefly/Renilla luciferase & full Firefly/Renilla luciferase</a></li><br />
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<ul class="nav"><br />
<li><a href="#Yeast_exp1">Confirmation of successful transformation via the Renilla Luciferase tag</a></li><br />
<li><a href="#Yeast_exp2">The split-GFP strain stress-response </a></li><br />
<li><a href="#Yeast_exp3">The split-Luciferase strain stress response</a></li><br />
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<li><a href="#Microfluidics_experiments" class="active">Microfluidics</a><br />
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<li><a href="#Micro_exp1">Chips</a></li><br />
<li><a href="#Micro_exp2">Culturing <i>E.coli</i> </a></li><br />
<li><a href="#Micro_exp3">sfGFP-CpxR fusion under pBAD</a></li><br />
<li><a href="#Micro_exp4">On chip infrared detection</a></li><br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/File:Gfptracking_EPFL.mp4File:Gfptracking EPFL.mp42014-10-18T00:44:38Z<p>Robert Baldwin: </p>
<hr />
<div></div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/OverviewTeam:EPF Lausanne/Overview2014-10-17T23:15:23Z<p>Robert Baldwin: </p>
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<!-- PROJECT --><br />
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<h1 class="cntr">Overview</h1><br />
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<h2 class="section-heading" id="title_intro">Introduction</h2><br />
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<!--Our team has been working on showing that biologically engineered organisms can detect and process signals quickly and efficiently.--><br />
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Biological responses can be quick and precise! This is the message that our team wants to convey! With this in mind, our team brought forward a novel idea: <br />
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Combining Protein Complementation techniques with biosensors and microfluidics, allows fast spatiotemporal analysis of bacterial/yeast responses to stimuli.<br />
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Touch_bacteria_EPFL_Ted.png" alt="touch bacteria" width="190" class="pull-left img-left img-border" /><br />
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Biological responses can be quick! This is the message that the 2014 EPFL iGEM team want to convey. <br />
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<br /><br /><i>The BioPad project is intended to be a solid proof-of-concept of the combination of biosensors with protein complementation techniques to achieve fast spatiotemporal analysis of bacterial or yeast response to mechanical stimuli.</i> <br />
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Our team explored this hypothesis by engineering two stress related pathways in <i>E. coli</i> and <i>S. cerevisiae</i> with in mind the development of a BioPad: a biological touchscreen consisting of a microfluidic chip, touch responsive bacteria/yeast, and a signal detector. Learn more about <a href="#howitworks">how the BioPad works !</a> </p><br />
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The pathway engineered in <i>E. coli</i>, the Cpx Pathway, is a two-component (CpxA and CpxR) regulatory system responsive to envelope stress. It controls the expression of "survival" genes whose products act in the periplasm to maintain membrane integrity. CpxA intermembrane kinase activates and autophosphorylates when sensing misfolded protein (due to stress for example). CpxA phosphorylates CpxR which homodimerizes, acting as a transcription factor. A full description of the pathway is available <a href="https://2014.igem.org/Team:EPF_Lausanne/Envelope_stress_responsive_bacteria">Envelope Stress Responsive Bacteria page !</a></p><br />
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<p class="lead"><br />
The pathway engineered in <i>E. coli</i>, the Cpx Pathway, is a two-component regulatory system responsive to envelope stress. A full description of the pathway is available <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Envelope_stress_responsive_bacteria#cpx">here.</a><br />
<br/><br />
In <i>S. cerevisiae</i> we modified the HOG Pathway - a MAPKK pathway responsive to osmotic stress. For more information concerning the HOG Pathway click <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Yeast">here.</a><br />
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<a href="https://static.igem.org/mediawiki/2014/f/fd/CpxR-HOG.jpg" data-lightbox="cpxr" data-title="EPFL microfluidic chips"><br />
<img src="https://static.igem.org/mediawiki/2014/f/fd/CpxR-HOG.jpg" alt="touch response" class="img-responsive" />Hello</a><br />
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<div class="pull-left img-left"><br />
<a href="https://static.igem.org/mediawiki/2014/d/d8/EPFLmicrofluidics.JPG" data-lightbox="image-1" data-title="EPFL microfluidic chips"><img src="https://static.igem.org/mediawiki/2014/d/d8/EPFLmicrofluidics.JPG" width="200" class="img-border"></a><br />
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<p class="lead"><br />
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Our project also includes an extensive <b>microfluidics section</b>. Our self designed chips helped us improve precision, safety, and quantification methods used throughout the project. To learn more about the microfluidic components of our project check out <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Microfluidics">this link.</a></p><br />
<br />
<div class="pull-right img-right"><br />
<img src="http://www.raspberrypi.org/wp-content/uploads/2011/07/RaspiModelB.png" alt="first" width="200" class="img-border"><br />
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<p class="lead"><br />
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Last but not least, we designed a <b>novel signal detector</b> ! To make signal detection more practical we developed an automatised cheap tracking system made of a mini-computer (Raspberry Pi) and a mini-HD camera. More details concerning this the BioPad detector can be found <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Hardware">here.</a><br />
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<h2 id="CpxPathway">The Cpx Pathway in <i> E.Coli </i></h2><br />
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<p class="lead"><br />
The natural function of the Cpx two component regulatory system in bacteria is to control the expression of ‘survival’ genes whose products act in the periplasm to maintain membrane integrity. This ensures continued bacterial growth even in environments with harmful extracytoplasmic stresses. The Cpx two component regulatory system belongs to the class I histidine kinases and includes three main proteins: CpxA, CpxR, and CpxP. For more detailed information about the Cpx pathway check out our <a href="https://2014.igem.org/Team:EPF_Lausanne/Envelope_stress_responsive_bacteria">Envelope Stress Responsive Bacteria page !</a></p><br />
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<h2 class="section-heading" id="howitworks">How the BioPad works - <i>E. coli</i></h2><br />
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<div class="pull-right img-right"><br />
<a href="https://static.igem.org/mediawiki/2014/b/b7/Puce_petit.jpg" data-lightbox="image-0" data-title="A potential BioPad"><br />
<img src="https://static.igem.org/mediawiki/2014/b/b7/Puce_petit.jpg" alt="Potential biopad" height="200" class="img-border" /></a><br />
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<p class="lead"><br />
Our BioPad is a self-designed PDMS microfluidic chip, made of thousands of compartments representing “pixels”. Each 300μm x 300μm x 50μm compartment contains a few layers of <i>E. coli</i>. When the surface of the chip is touched, a deformation on the chip - and thus of the compartments – leads to cellular membrane shear stress and protein aggregation/misfolding in the periplasm.<br/> <br/></p><br />
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<p class="lead"><br />
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The histidine kinase sensor CpxA auto-phosphorylates and transfers its phosphate to its corresponding relay protein, CpxR, resulting in its dimerization. We engineered the pathway, by fusing split reporter protein fragments to the CpxR (IFP1.4). This allows the two fragments to remain inactive until physical interaction of CpxR (stimulated by envelope stress) leads to the proper folding of IFP1.4 and reconstitution of its fluorescent properties. As the reconstitution of the split fragments of IFP1.4 are reversible, the system can be shutdown upon stress removal (CpxA changes conformation to become a phosphatase and induces CpxR’s dissociation).<br/> <br/> <br />
<br />
The BioPad also includes a signal detector. The BioPad Detector is composed of an inexpensive credit card-sized single-board computer called Raspberry Pi, a highly sensitive digital camera with appropriate light filters, and a light-emitting source. It identifies and processes the position of the light/fluorescence emitted by the BioPad. The information about the position of the light relative to chip is then used to control the associated electronic device. <br/> <br />
<br />
<br />
<!--<br />
Our self-designed PDMS microfluidic chip, the BioPad, is made of hundreds of compartments representing "pixels." Each 30µm x 30µm x 3µm compartment contains a few layers of <i>E. coli</i>. When the surface of the chip is touched, a deformation of the chip - and thus of the compartments - leads to cellular membrane shear stress and protein aggregation/misfolding in the periplasm. <br />
<br /><br />
<br />
<br />
The aggregated/misfolded proteins are then sensed by the histidine kinase CpxA sensor, which auto-phosphorylates and transfers its phosphate to its corresponding relay protein, CpxR. Upon phosphorylation, CpxR homo-dimerizes. <br />
<br />
<br /><br />
Our engineered bacteria contain CpxR proteins fused to split fluorescent protein fragments (split IFP1.4) via a 10-amino acid, 2x GGGGS flexible linker. This allows us to detect CpxR dimerization, synonymous periplasmic stress and touch. Moreover, the split protein fragments are reversible. Therefore, when stress is removed, CpxA changes conformation and dephosphorylates CpxR allowing it to dissociate. The signal is shutdown and darkness returns The BioPad Detector (composed of an inexpensive CMOS called Raspberry Pi, a highly sensitive digital camera with appropriate light filters, and a light emitting source) identifies and processes the position of the light/fluorescence emitted by the BioPad. This information about the position of the light relative to chip is then used to control the associated electronic device.<br />
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<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/1/15/Screen_Shot_2014-10-12_at_3.29.30_PM.png" data-lightbox="image-0" data-title="Fluorescence"><br />
<img src="https://static.igem.org/mediawiki/2014/1/15/Screen_Shot_2014-10-12_at_3.29.30_PM.png" alt="touch bacteria" height="200" class="img-border" /></a><br />
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<!--<hr /><br />
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<h2 id="thehogpathway">The HOG Pathway</h2><br />
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<p class="lead">The HOG (High Osmolarity Glycerol) pathway is a MAPK (Mitogen activated protein kinase) pathway which yeast cells use to coordinate intracellular activities to optimise survival and proliferation in not only hyper-osmotic stress but also heat shock, nitrogen stress and oxidative stress. It is represented below.</p><br />
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<br /><br />
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<img src="https://static.igem.org/mediawiki/2014/6/6d/Hog_pathway_copy.jpg" width="750" alt="HOG_pathway_description"><br />
<br /><br />
<p> The pathway includes five main proteins:</p><br />
<ul style="padding-left:80px"><br />
<li><p class="lead">Sho1/Sln1 – Membrane proteins which are classed as STREs (STress Response Elements) which sense the stress and initiate the pathway</p></li><br />
<li><p class="lead">Ste11 – The MAPKKK which phosphorylates PBS2</p></li><br />
<li><p class="lead">PBS2 – The MAPKK which phosphorylates HOG1</p></li><br />
<li><p class="lead">HOG1 – The MAPK which localizes to the nucleus upon phosphorylation and induces target gene transcription</p></li><br />
</ul><br />
<br>--><br />
<br><br />
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<h2 id="howweengineered">Engineering the HOG pathway in <i>S. cerevisiae</i></h2><br />
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<br />
<br />
<p class="lead"><br />
<br/><br />
The <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Yeast">HOG pathway</a> controls the response to osmotic stress in yeast cells.<br />
Like bacteria, our modified <i>S. cerevisiae</i> cells can be loaded into the PDMS microfluidic chips we have designed. Thus cells are confined in small compartments made of a rather soft material. When the surface of the chip is touched, it leads to a deformation of the chip and its chambers. Since the HOG pathway is reactive to turgor pressure, the mechanical pressure applied activates it. Upon induction of the pathway, which is a classical MAP kinase pathway, PBS2 – a MAPKK – is phosphorylated and binds HOG1 – a MAPK – and in turn phosphorylates it.<br />
</p><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/7/7b/Schema_split.png" data-lightbox="img1" data-title="Schema of Hog1/Pbs2 split sfGFP and split rLuc"><br />
<img src="https://static.igem.org/mediawiki/2014/7/7b/Schema_split.png" width="55%" alt="" class="pull-left img-left img-border"></a><br />
<br />
<p class="lead"><br />
Therefore, we have fused these two kinases to split fluorescent and luminescent proteins, via a 13-amino acid flexible linker, by homologous recombination. This allows us to detect the phosphorylation of Hog1 by Pbs2 in response to osmotic pressure or touch. We have used split sfGFP and split Renilla luciferase tags on the C-terminals of both proteins.<br />
</p><br />
<br />
<p class="lead"><br />
The split sfGFP is irreversible and was made to show the interaction between our two Pbs2 and Hog1 while we use the reversible split luciferase tags to assess the activation and inactivation of the pathway. In fact, when stress is removed, the signal should decline. The <b>BioTouch Detector </b> is programmed to identify and process the light position and can transmit the information to a computer.<br />
</p><br />
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<hr><br />
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<br />
<h2 class="section-heading" id="thebiopad">The BioPad Detector</h2><br />
<p class="lead"><br />
<br /><br />
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<!-- ENGINEERING DETECTOR --><br />
<br />
The signals induced by the BioTouch Chip are then processed by our self designed detection system: the BioTouch Detector. The BioTouch Detector is mainly made of a cheap computer (Raspberry Pi), a highly sensitive digital camera with appropriate light filters, and a light emitting source. The BioTouch Detector locates signals from various sources (infrared fluorescence, green fluorescence and luminescence), processes them and sends back the relative positions of the signals with respect to the BioTouch Pad. Thanks to this position, we are able to extract information such as giving a computer operating system that the position represents the position of the mouse on a screen, that the well at the given position is a suitable antibiotic candidate, or that a gene of interest has been activated. We therefore effectively control a computer or any other electronic device through a living interface: the BioTouch Pad.<br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/HardwareTeam:EPF Lausanne/Hardware2014-10-17T23:13:54Z<p>Robert Baldwin: </p>
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<h1 class="cntr">Hardware</h1><br />
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<br/><br/><br />
<br />
<br />
<p class="lead"><br />
<br />
<br />
Having successfully engineered touch responsive bacteria, the next major step to build a functional BioPad is to detect and process the emitted signals. The microfluidic chip containing our engineered organisms has the advantage of being small and portable. We aimed to keep these characteristics all along the project. That is why, instead of using a big and cumbersome device to detect signals, we opted for a small and cheap Raspberry Pi. Let the adventure for building the BioPad Detector begin !<br /><br /><br />
<br />
</p><br />
<br />
<h3 id="raspberry">Raspberry Pi</h3><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" alt="Raspberry Pi" class="pull-left img-left img-border" style="width: 30%;" /></a><br />
<br />
<br /><br />
<p>The Raspberry Pi is a small and cheap (40.- CHF) single-board computer. The raspberry Pi will be used to monitor the light emitted by each chamber of the microfluidic chip. We will be able to detect and process all emitted signals through this small device !<br /><br /></p><br />
<br />
<br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<p>When brainstorming how to build the detector, we initially drafted the follwing setup: </p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" alt="Raspberry Pi" class="img-border img-responsive" /></a></div><br />
<br />
<br /><br />
<br /><br />
<br />
<p>As seen in the picture above, the camera is directly linked to the Raspberry Pi and is able to get a clear view of the whole chip. Our final device thus needed a small and high-resolution camera able to easily track signal emission from our touch responsive organisms (including signals emitted in the infrared spectrum). <br /></p><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" alt="Raspberry Pi" class="pull-right img-right img-border" style="width: 30%;" /></a><br />
<br />
<p>The camera best suited to the characteristics above was the Raspberry Pi NoIR. The Raspberry Pi NoIR is a 5 MegaPixel, 1080p, 20mm x 25mm x 9mm CMOS camera. It is especially good for low intensity signals. Moreover, NoIR stands for No Infrared Filter, meaning that with this camera we are able to see near infrared wavelength.<br /></p><br />
<br />
<p>The near-infrared spectrum correspond to wavelengths between 700 and 1000nm. We will use the ability of the Raspberry Pi NoIR to detect near-infrared to track the IFP signal emitted by our CpxR - split IFP1.4 stress responsive cells. The emission of IFP in these wavelengths is especially useful for us, as few things emit auto-fluorescence in the infrared spectrum. This drastically increases the precision of our device as it reduces background noise.<br /></p><br />
<br />
<p>Taking in account all the information above, the main idea driving the way we plan to detect signals through our BioPad detector is to collect the entire light spectrum including near infrared wavelengths and then use a filter to eliminate the visible spectrum.</p><br />
<br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" alt="Raspberry Pi Plan" class="img-border img-responsive" /></a><br />
</div><br />
<br /><br />
<br />
<h3 id="lighttracking">Light tracking</h3><br />
<br />
<p>To track the signal dynamics in the chambers via our detector, we wrote a custom C++ code using OpenCV able to specifically detect the exact position of the signal as well as its nature and intensity. The entire code as well as all the supporting files can be found here: <a href="https://github.com/arthurgiroux/igemtracking/" target="_blank">https://github.com/arthurgiroux/igemtracking/</a>. An extract of the main code is given here:</p><br />
<br />
<script src="http://gist-it.sudarmuthu.com/https://github.com/arthurgiroux/igemtracking/blob/master/src/tracking.cpp"></script><br />
<br />
<br/><br/><br/><br />
<br />
<p> Check out the result of our program here:</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/c/cd/Lighttracking2.png" alt="" class="img-responsive" /></p><br />
<br />
<div class="pull-left img-left" style="width: 30%"><br />
<a href=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" data-lightbox="raspberry"><img src=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" alt="YCrCb" class="img-border" /></a><br /><br />
<figcaption class="cntr">Representation of the YCrCb color space</figcaption><br />
</div><br />
<p>To detect the nature of the signal, we used a fairly novel way of processing images and videos. The most common color space used in programming is RGB - pixel colors can be split into three components (Red, Green, and Blue) each taking a value between 0 and 255.</p><br />
<br />
<p>We used another color space, better adapted for this application as we were especially interested in light intensity and color nuances. We therefore chose the YcrCb color space.</p><br />
<br/><br />
<br />
<p>In the YCrCb color space, each pixel is decomposed into three components: </p><br />
<br />
<ul><br />
<li>Y – the luma value (intensity)</li><br />
<li>Cr – the red difference</li><br />
<li>Cb – the blue difference</li><br />
</ul><br />
<br />
<br />
<br />
<p><br />
This allows us to extract the necessary information for our application.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3 id="lenses">Lenses</h3><br />
<br />
<p>The Raspberry Pi camera that we use has a fixed lens which is not adapted to what we want to do, as we cannot change the focus, the aperture or the zoom.</p><br />
<br />
<p>We searched for a different lens which would allow us more control, and found that the easiest way was to remove the initial Raspberry Pi lens, put a CS mount on it and attach a much bigger lens.</p><br />
<br />
<p>The first thing we did was to unplug the camera module from the PCB. Then, the lens was carefully removed by unscrewing it and the new lens was mounted. </p><br />
<br />
INSERT IMAGE<br />
<br />
<p>You will then see the camera sensor (CMOS).</p><br />
<br />
<p>The CS mount was screwed on the board and the lens plugged in.</p><br />
<br />
<p>You can see here what the lens sees: <br /></p><br />
<br />
IMAGE!!!<br />
<br />
<p>We can clearly see the chambers of the microfluidic chip.</p><br />
<br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/HardwareTeam:EPF Lausanne/Hardware2014-10-17T23:11:51Z<p>Robert Baldwin: </p>
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<h1 class="cntr">Hardware</h1><br />
<br />
<br/><br/><br />
<br />
<br />
<p class="lead"><br />
<br />
<br />
Having successfully engineered touch responsive bacteria, the next major step to build a functional BioPad is to detect and process the emitted signals. The microfluidic chip containing our engineered organisms has the advantage of being small and portable. We aimed to keep these characteristics all along the project. That is why, instead of using a big and cumbersome device to detect signals, we opted for a small and cheap Raspberry Pi. Let the adventure for building the BioPad Detector begin !<br /><br /><br />
<br />
</p><br />
<br />
<h3 id="raspberry">Raspberry Pi</h3><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" alt="Raspberry Pi" class="pull-left img-left img-border" style="width: 30%;" /></a><br />
<br />
<br /><br />
<p>The Raspberry Pi is a small and cheap (40.- CHF) single-board computer. The raspberry Pi will be used to monitor the light emitted by each chamber of the microfluidic chip. We will be able to detect and process all emitted signals through this small device !<br /><br /></p><br />
<br />
<br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<p>When brainstorming how to build the detector, we initially drafted the follwing setup: </p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" alt="Raspberry Pi" class="img-border img-responsive" /></a></div><br />
<br />
<br /><br />
<br /><br />
<br />
<p>As seen in the picture above, the camera is directly linked to the Raspberry Pi and is able to get a clear view of the whole chip. Our final device thus needed a small and high-resolution camera able to easily track signal emission from our touch responsive organisms (including signals emitted in the infrared spectrum). <br /></p><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" alt="Raspberry Pi" class="pull-right img-right img-border" style="width: 30%;" /></a><br />
<br />
<p>The camera best suited to the characteristics above was the Raspberry Pi NoIR. The Raspberry Pi NoIR is a 5 MegaPixel, 1080p, 20mm x 25mm x 9mm CMOS camera. It is especially good for low intensity signals. Moreover, NoIR stands for No Infrared Filter, meaning that with this camera we are able to see near infrared wavelength.<br /></p><br />
<br />
<p>The near-infrared spectrum correspond to wavelengths between 700 and 1000nm. We will use the ability of the Raspberry Pi NoIR to detect near-infrared to track the IFP signal emitted by our CpxR - split IFP1.4 stress responsive cells. The emission of IFP in these wavelengths is especially useful for us, as few things emit auto-fluorescence in the infrared spectrum. This drastically increases the precision of our device as it reduces background noise.<br /></p><br />
<br />
<p>Taking in account all the information above, the main idea driving the way we plan to detect signals through our BioPad detector is to collect the entire light spectrum including near infrared wavelengths and then use a filter to eliminate the visible spectrum.</p><br />
<br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" alt="Raspberry Pi Plan" class="img-border img-responsive" /></a><br />
</div><br />
<br /><br />
<br />
<h3 id="lighttracking">Light tracking</h3><br />
<br />
<p>To track the signal dynamics in the chambers via our detector, we wrote a custom C++ code using OpenCV able to specifically detect the exact position of the signal as well as its nature and intensity. The entire code as well as all the supporting files can be found here: <a href="https://github.com/arthurgiroux/igemtracking/" target="_blank">https://github.com/arthurgiroux/igemtracking/</a>. An extract of the main code is given here:</p><br />
<br />
<script src="http://gist-it.sudarmuthu.com/https://github.com/arthurgiroux/igemtracking/blob/master/src/tracking.cpp"></script><br />
<br />
<br/><br/><br/><br />
<br />
<p> Check out the result of our program here:</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/c/cd/Lighttracking2.png" alt="" class="img-responsive" /></p><br />
<br />
<div class="pull-left img-left" style="width: 30%"><br />
<a href=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" data-lightbox="raspberry"><img src=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" alt="YCrCb" class="img-border" /></a><br /><br />
<figcaption class="cntr">Representation of the YCrCb color space</figcaption><br />
</div><br />
<p>To detect the nature of the signal, we used a fairly novel way of processing images and videos. The most common color space used in programming is RGB - pixel colors can be split into three components (Red, Green, and Blue) each taking a value between 0 and 255.</p><br />
<br />
<p>On the other, we used another color space, better adapted for this application as we were especially interested in light intensity and color nuances. We therefore chose the YcrCb color space.</p><br />
<br/><br />
<br />
<p>In the YCrCb color space, each pixel is decomposed into three components: </p><br />
<br />
<ul><br />
<li>Y – the luma value (intensity)</li><br />
<li>Cr – the red difference</li><br />
<li>Cb – the blue difference</li><br />
</ul><br />
<br />
<br />
<br />
<p><br />
This allows us to extract the necessary information for our application.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3 id="lenses">Lenses</h3><br />
<br />
<p>The Raspberry Pi camera that we use has a fixed lens which is not adapted to what we want to do, as we cannot change the focus, the aperture or the zoom.</p><br />
<br />
<p>We searched for a different lens which would allow us more control, and found that the easiest way was to remove the initial Raspberry Pi lens, put a CS mount on it and attach a much bigger lens.</p><br />
<br />
<p>The first thing we did was to unplug the camera module from the PCB. Then, the lens was carefully removed by unscrewing it and the new lens was mounted. </p><br />
<br />
INSERT IMAGE<br />
<br />
<p>You will then see the camera sensor (CMOS).</p><br />
<br />
<p>The CS mount was screwed on the board and the lens plugged in.</p><br />
<br />
<p>You can see here what the lens sees: <br /></p><br />
<br />
IMAGE!!!<br />
<br />
<p>We can clearly see the chambers of the microfluidic chip.</p><br />
<br />
<br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/HardwareTeam:EPF Lausanne/Hardware2014-10-17T23:11:30Z<p>Robert Baldwin: </p>
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<h1 class="cntr">Hardware</h1><br />
<br />
<br/><br/><br />
<br />
<br />
<p class="lead"><br />
<br />
<br />
Having successfully engineered touch responsive bacteria, the next major step to build a functional BioPad is to detect and process the emitted signals. The microfluidic chip containing our engineered organisms has the advantage of being small and portable. We aimed to keep these characteristics all along the project. That is why, instead of using a big and cumbersome device to detect signals, we opted for a small and cheap Raspberry Pi. Let the adventure for building the BioPad Detector begin !<br /><br /><br />
<br />
</p><br />
<br />
<h3 id="raspberry">Raspberry Pi</h3><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" alt="Raspberry Pi" class="pull-left img-left img-border" style="width: 30%;" /></a><br />
<br />
<br /><br />
<p>The Raspberry Pi is a small and cheap (40.- CHF) single-board computer. The raspberry Pi will be used to monitor the light emitted by each chamber of the microfluidic chip. We will be able to detect and process all emitted signals through this small device !<br /><br /></p><br />
<br />
<br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<p>When brainstorming how to build the detector, we initially drafted the follwing setup: </p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" alt="Raspberry Pi" class="img-border img-responsive" /></a></div><br />
<br />
<br /><br />
<br /><br />
<br />
<p>As seen in the picture above, the camera is directly linked to the Raspberry Pi and is able to get a clear view of the whole chip. Our final device thus needed a small and high-resolution camera able to easily track signal emission from our touch responsive organisms (including signals emitted in the infrared spectrum). <br /></p><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" alt="Raspberry Pi" class="pull-right img-right img-border" style="width: 30%;" /></a><br />
<br />
<p>The camera best suited to the characteristics above was the Raspberry Pi NoIR. The Raspberry Pi NoIR is a 5 MegaPixel, 1080p, 20mm x 25mm x 9mm CMOS camera. The Raspberry Pi NoIR is especially good for low intensity signals. Moreover, NoIR stands for No Infrared Filter, meaning that with this camera we are able to see near infrared wavelength.<br /></p><br />
<br />
<p>The near-infrared spectrum correspond to wavelengths between 700 and 1000nm. We will use the ability of the Raspberry Pi NoIR to detect near-infrared to track the IFP signal emitted by our CpxR - split IFP1.4 stress responsive cells. The emission of IFP in these wavelengths is especially useful for us, as few things emit auto-fluorescence in the infrared spectrum. This drastically increases the precision of our device as it reduces background noise.<br /></p><br />
<br />
<p>Taking in account all the information above, the main idea driving the way we plan to detect signals through our BioPad detector is to collect the entire light spectrum including near infrared wavelengths and then use a filter to eliminate the visible spectrum.</p><br />
<br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" alt="Raspberry Pi Plan" class="img-border img-responsive" /></a><br />
</div><br />
<br /><br />
<br />
<h3 id="lighttracking">Light tracking</h3><br />
<br />
<p>To track the signal dynamics in the chambers via our detector, we wrote a custom C++ code using OpenCV able to specifically detect the exact position of the signal as well as its nature and intensity. The entire code as well as all the supporting files can be found here: <a href="https://github.com/arthurgiroux/igemtracking/" target="_blank">https://github.com/arthurgiroux/igemtracking/</a>. An extract of the main code is given here:</p><br />
<br />
<script src="http://gist-it.sudarmuthu.com/https://github.com/arthurgiroux/igemtracking/blob/master/src/tracking.cpp"></script><br />
<br />
<br/><br/><br/><br />
<br />
<p> Check out the result of our program here:</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/c/cd/Lighttracking2.png" alt="" class="img-responsive" /></p><br />
<br />
<div class="pull-left img-left" style="width: 30%"><br />
<a href=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" data-lightbox="raspberry"><img src=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" alt="YCrCb" class="img-border" /></a><br /><br />
<figcaption class="cntr">Representation of the YCrCb color space</figcaption><br />
</div><br />
<p>To detect the nature of the signal, we used a fairly novel way of processing images and videos. The most common color space used in programming is RGB - pixel colors can be split into three components (Red, Green, and Blue) each taking a value between 0 and 255.</p><br />
<br />
<p>On the other, we used another color space, better adapted for this application as we were especially interested in light intensity and color nuances. We therefore chose the YcrCb color space.</p><br />
<br/><br />
<br />
<p>In the YCrCb color space, each pixel is decomposed into three components: </p><br />
<br />
<ul><br />
<li>Y – the luma value (intensity)</li><br />
<li>Cr – the red difference</li><br />
<li>Cb – the blue difference</li><br />
</ul><br />
<br />
<br />
<br />
<p><br />
This allows us to extract the necessary information for our application.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3 id="lenses">Lenses</h3><br />
<br />
<p>The Raspberry Pi camera that we use has a fixed lens which is not adapted to what we want to do, as we cannot change the focus, the aperture or the zoom.</p><br />
<br />
<p>We searched for a different lens which would allow us more control, and found that the easiest way was to remove the initial Raspberry Pi lens, put a CS mount on it and attach a much bigger lens.</p><br />
<br />
<p>The first thing we did was to unplug the camera module from the PCB. Then, the lens was carefully removed by unscrewing it and the new lens was mounted. </p><br />
<br />
INSERT IMAGE<br />
<br />
<p>You will then see the camera sensor (CMOS).</p><br />
<br />
<p>The CS mount was screwed on the board and the lens plugged in.</p><br />
<br />
<p>You can see here what the lens sees: <br /></p><br />
<br />
IMAGE!!!<br />
<br />
<p>We can clearly see the chambers of the microfluidic chip.</p><br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/HardwareTeam:EPF Lausanne/Hardware2014-10-17T23:10:52Z<p>Robert Baldwin: </p>
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<h1 class="cntr">Hardware</h1><br />
<br />
<br/><br/><br />
<br />
<br />
<p class="lead"><br />
<br />
<br />
Having successfully engineered touch responsive bacteria, the next major step to build a functional BioPad is to detect and process the emitted signals. The microfluidic chip containing our engineered organisms has the advantage of being small and portable. We aimed to keep these characteristics all along the project. That is why, instead of using a big and cumbersome device to detect signals, we opted for a small and cheap Raspberry Pi. Let the adventure for building the BioPad Detector begin !<br /><br /><br />
<br />
</p><br />
<br />
<h3 id="raspberry">Raspberry Pi</h3><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" alt="Raspberry Pi" class="pull-left img-left img-border" style="width: 30%;" /></a><br />
<br />
<br /><br />
<p>The Raspberry Pi is a small and cheap (40.- CHF) single-board computer. The raspberry Pi will be used to monitor the light emitted by each chamber of the microfluidic chip. We will be able to detect and process all emitted signals through this small device !<br /><br /></p><br />
<br />
<br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<p>When brainstorming how to build the detector, we initially drafted the follwing setup: </p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" alt="Raspberry Pi" class="img-border img-responsive" /></a></div><br />
<br />
<br /><br />
<br /><br />
<br />
<p>As seen in the picture above, the camera is directly linked to the Raspberry Pi and is able to get a clear view of the whole chip. Our final device thus needed a small and high-resolution camera able to easily track signal emission from our touch responsive organisms (including signals emitted in the infrared spectrum). <br /></p><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" alt="Raspberry Pi" class="pull-right img-right img-border" style="width: 30%;" /></a><br />
<br />
<p>The camera best suited to the characteristics above was the Raspberry Pi NoIR. The Raspberry Pi NoIR is a 5 MegaPixel, 1080p, 20mm x 25mm x 9mm CMOS camera. No IR is especially good for low intensity signals. Moreover, NoIR stands for No Infrared Filter, meaning that with this camera we are able to see near infrared wavelength.<br /></p><br />
<br />
<p>The near-infrared spectrum correspond to wavelengths between 700 and 1000nm. We will use the ability of the Raspberry Pi NoIR to detect near-infrared to track the IFP signal emitted by our CpxR - split IFP1.4 stress responsive cells. The emission of IFP in these wavelengths is especially useful for us, as few things emit auto-fluorescence in the infrared spectrum. This drastically increases the precision of our device as it reduces background noise.<br /></p><br />
<br />
<p>Taking in account all the information above, the main idea driving the way we plan to detect signals through our BioPad detector is to collect the entire light spectrum including near infrared wavelengths and then use a filter to eliminate the visible spectrum.</p><br />
<br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" alt="Raspberry Pi Plan" class="img-border img-responsive" /></a><br />
</div><br />
<br /><br />
<br />
<h3 id="lighttracking">Light tracking</h3><br />
<br />
<p>To track the signal dynamics in the chambers via our detector, we wrote a custom C++ code using OpenCV able to specifically detect the exact position of the signal as well as its nature and intensity. The entire code as well as all the supporting files can be found here: <a href="https://github.com/arthurgiroux/igemtracking/" target="_blank">https://github.com/arthurgiroux/igemtracking/</a>. An extract of the main code is given here:</p><br />
<br />
<script src="http://gist-it.sudarmuthu.com/https://github.com/arthurgiroux/igemtracking/blob/master/src/tracking.cpp"></script><br />
<br />
<br/><br/><br/><br />
<br />
<p> Check out the result of our program here:</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/c/cd/Lighttracking2.png" alt="" class="img-responsive" /></p><br />
<br />
<div class="pull-left img-left" style="width: 30%"><br />
<a href=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" data-lightbox="raspberry"><img src=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" alt="YCrCb" class="img-border" /></a><br /><br />
<figcaption class="cntr">Representation of the YCrCb color space</figcaption><br />
</div><br />
<p>To detect the nature of the signal, we used a fairly novel way of processing images and videos. The most common color space used in programming is RGB - pixel colors can be split into three components (Red, Green, and Blue) each taking a value between 0 and 255.</p><br />
<br />
<p>On the other, we used another color space, better adapted for this application as we were especially interested in light intensity and color nuances. We therefore chose the YcrCb color space.</p><br />
<br/><br />
<br />
<p>In the YCrCb color space, each pixel is decomposed into three components: </p><br />
<br />
<ul><br />
<li>Y – the luma value (intensity)</li><br />
<li>Cr – the red difference</li><br />
<li>Cb – the blue difference</li><br />
</ul><br />
<br />
<br />
<br />
<p><br />
This allows us to extract the necessary information for our application.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3 id="lenses">Lenses</h3><br />
<br />
<p>The Raspberry Pi camera that we use has a fixed lens which is not adapted to what we want to do, as we cannot change the focus, the aperture or the zoom.</p><br />
<br />
<p>We searched for a different lens which would allow us more control, and found that the easiest way was to remove the initial Raspberry Pi lens, put a CS mount on it and attach a much bigger lens.</p><br />
<br />
<p>The first thing we did was to unplug the camera module from the PCB. Then, the lens was carefully removed by unscrewing it and the new lens was mounted. </p><br />
<br />
INSERT IMAGE<br />
<br />
<p>You will then see the camera sensor (CMOS).</p><br />
<br />
<p>The CS mount was screwed on the board and the lens plugged in.</p><br />
<br />
<p>You can see here what the lens sees: <br /></p><br />
<br />
IMAGE!!!<br />
<br />
<p>We can clearly see the chambers of the microfluidic chip.</p><br />
<br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/HardwareTeam:EPF Lausanne/Hardware2014-10-17T23:08:13Z<p>Robert Baldwin: </p>
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<h1 class="cntr">Hardware</h1><br />
<br />
<br/><br/><br />
<br />
<br />
<p class="lead"><br />
<br />
<br />
Having successfully engineered touch responsive bacteria, the next major step to build a functional BioPad is to detect and process the emitted signals. The microfluidic chip containing our engineered organisms has the advantage of being small and portable. We aimed to keep these characteristics all along the project. That is why, instead of using a big and cumbersome device to detect signals, we opted for a small and cheap Raspberry Pi. Let the adventure for building the BioPad Detector begin !<br /><br /><br />
<br />
</p><br />
<br />
<h3 id="raspberry">Raspberry Pi</h3><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" alt="Raspberry Pi" class="pull-left img-left img-border" style="width: 30%;" /></a><br />
<br />
<br /><br />
<p>The Raspberry Pi is a small and cheap (40.- CHF) single-board computer. The raspberry Pi will be used to monitor the light emitted by each chamber of the microfluidic chip. We will be able to detect and process all emitted signals through this small device !<br /><br /></p><br />
<br />
<br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<p>When brainstorming how to build the detector, we initially drafted the follwing setup: </p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" alt="Raspberry Pi" class="img-border img-responsive" /></a></div><br />
<br />
<br /><br />
<br /><br />
<br />
<p>As seen in the picture above, the camera is directly linked to the Raspberry Pi and is able to get a clear view of the whole chip. Our final device thus needed a small and high-resolution camera able to easily track signal emission from our touch responsive organisms (including signals emitted in the infrared spectrum). <br /></p><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" alt="Raspberry Pi" class="pull-right img-right img-border" style="width: 30%;" /></a><br />
<br />
<p>The camera best suited to the characteristics above was the Raspberry Pi NoIR. The Raspberry Pi NoIR is a 5 MegaPixel, 1080p, 20mm x 25mm x 9mm CMOS camera. No IR is especially good for low intensity signals. Moreover, NoIR stands for No Infrared Filter, meaning that with this camera we are able to see near infrared wavelength.<br /></p><br />
<br />
<p>The Pi NoIR can track near infrared wavelengths corresponding to wavelengths between 700 and 1000nm. We will use this feature of the camera to track the IFP signal emitted by our CpxR - split IFP1.4 stress responsive cells. The emission of IFP in these wavelengths is especially useful for us, as few things emit auto-fluorescence in the infrared spectrum. This drastically increases the precision of our device as it reduces background noise.<br /></p><br />
<br />
<p>Taking in account all the information above, the main idea driving the way we plan to detect signals through our BioPad detector is to collect the entire light spectrum including near infrared wavelengths and then use a filter to eliminate the visible spectrum.</p><br />
<br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" alt="Raspberry Pi Plan" class="img-border img-responsive" /></a><br />
</div><br />
<br /><br />
<br />
<h3 id="lighttracking">Light tracking</h3><br />
<br />
<p>To track the signal dynamics in the chambers via our detector, we wrote a custom C++ code using OpenCV able to specifically detect the exact position of the signal as well as its nature and intensity. The entire code as well as all the supporting files can be found here: <a href="https://github.com/arthurgiroux/igemtracking/" target="_blank">https://github.com/arthurgiroux/igemtracking/</a>. An extract of the main code is given here:</p><br />
<br />
<script src="http://gist-it.sudarmuthu.com/https://github.com/arthurgiroux/igemtracking/blob/master/src/tracking.cpp"></script><br />
<br />
<br/><br/><br/><br />
<br />
<p> Check out the result of our program here:</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/c/cd/Lighttracking2.png" alt="" class="img-responsive" /></p><br />
<br />
<div class="pull-left img-left" style="width: 30%"><br />
<a href=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" data-lightbox="raspberry"><img src=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" alt="YCrCb" class="img-border" /></a><br /><br />
<figcaption class="cntr">Representation of the YCrCb color space</figcaption><br />
</div><br />
<p>To detect the nature of the signal, we used a fairly novel way of processing images and videos. The most common color space used in programming is RGB - pixel colors can be split into three components (Red, Green, and Blue) each taking a value between 0 and 255.</p><br />
<br />
<p>On the other, we used another color space, better adapted for this application as we were especially interested in light intensity and color nuances. We therefore chose the YcrCb color space.</p><br />
<br/><br />
<br />
<p>In the YCrCb color space, each pixel is decomposed into three components: </p><br />
<br />
<ul><br />
<li>Y – the luma value (intensity)</li><br />
<li>Cr – the red difference</li><br />
<li>Cb – the blue difference</li><br />
</ul><br />
<br />
<br />
<br />
<p><br />
This allows us to extract the necessary information for our application.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3 id="lenses">Lenses</h3><br />
<br />
<p>The Raspberry Pi camera that we use has a fixed lens which is not adapted to what we want to do, as we cannot change the focus, the aperture or the zoom.</p><br />
<br />
<p>We searched for a different lens which would allow us more control, and found that the easiest way was to remove the initial Raspberry Pi lens, put a CS mount on it and attach a much bigger lens.</p><br />
<br />
<p>The first thing we did was to unplug the camera module from the PCB. Then, the lens was carefully removed by unscrewing it and the new lens was mounted. </p><br />
<br />
INSERT IMAGE<br />
<br />
<p>You will then see the camera sensor (CMOS).</p><br />
<br />
<p>The CS mount was screwed on the board and the lens plugged in.</p><br />
<br />
<p>You can see here what the lens sees: <br /></p><br />
<br />
IMAGE!!!<br />
<br />
<p>We can clearly see the chambers of the microfluidic chip.</p><br />
<br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/HardwareTeam:EPF Lausanne/Hardware2014-10-17T23:07:36Z<p>Robert Baldwin: </p>
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<h1 class="cntr">Hardware</h1><br />
<br />
<br/><br/><br />
<br />
<br />
<p class="lead"><br />
<br />
<br />
Having successfully engineered touch responsive bacteria, the next major step to build a functional BioPad is to detect and process the emitted signals. The microfluidic chip containing our engineered organisms has the advantage of being small and portable. We aimed to keep these characteristics all along the project. That is why, instead of using a big and cumbersome device to detect signals, we opted for a small and cheap Raspberry Pi. Let the adventure for building the BioPad Detector begin !<br /><br /><br />
<br />
</p><br />
<br />
<h3 id="raspberry">Raspberry Pi</h3><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" alt="Raspberry Pi" class="pull-left img-left img-border" style="width: 30%;" /></a><br />
<br />
<br /><br />
<p>The Raspberry Pi is a small and cheap (40.- CHF) single-board computer. The raspberry Pi will be used to monitor the light emitted by each chamber of the microfluidic chip. We will be able to detect and process all emitted signals through this small device !<br /><br /></p><br />
<br />
<br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<p>When brainstorming how to build the detector, we initially drafted the follwing setup: </p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" alt="Raspberry Pi" class="img-border img-responsive" /></a></div><br />
<br />
<br /><br />
<br /><br />
<br />
<p>As seen in the picture above, the camera is directly linked to the Raspberry Pi and is able to get a clear view of the whole chip. Our final device thus needed a small and high-resolution camera able to easily track signal emission from our touch responsive organisms (including signals emitted in the infrared spectrum). <br /></p><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" alt="Raspberry Pi" class="pull-right img-right img-border" style="width: 30%;" /></a><br />
<br />
<p>The camera best suited to the characteristics above was the Raspberry Pi NoIR. The Raspberry Pi NoIR is a 5 MegaPixel, 1080 pixels, 20mm x 25mm x 9mm CMOS camera. No IR is especially good for low intensity signals. Moreover, NoIR stands for No Infrared Filter, meaning that with this camera we are able to see near infrared wavelength.<br /></p><br />
<br />
<p>The Pi NoIR can track near infrared wavelengths corresponding to wavelengths between 700 and 1000nm. We will use this feature of the camera to track the IFP signal emitted by our CpxR - split IFP1.4 stress responsive cells. The emission of IFP in these wavelengths is especially useful for us, as few things emit auto-fluorescence in the infrared spectrum. This drastically increases the precision of our device as it reduces background noise.<br /></p><br />
<br />
<p>Taking in account all the information above, the main idea driving the way we plan to detect signals through our BioPad detector is to collect the entire light spectrum including near infrared wavelengths and then use a filter to eliminate the visible spectrum.</p><br />
<br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" alt="Raspberry Pi Plan" class="img-border img-responsive" /></a><br />
</div><br />
<br /><br />
<br />
<h3 id="lighttracking">Light tracking</h3><br />
<br />
<p>To track the signal dynamics in the chambers via our detector, we wrote a custom C++ code using OpenCV able to specifically detect the exact position of the signal as well as its nature and intensity. The entire code as well as all the supporting files can be found here: <a href="https://github.com/arthurgiroux/igemtracking/" target="_blank">https://github.com/arthurgiroux/igemtracking/</a>. An extract of the main code is given here:</p><br />
<br />
<script src="http://gist-it.sudarmuthu.com/https://github.com/arthurgiroux/igemtracking/blob/master/src/tracking.cpp"></script><br />
<br />
<br/><br/><br/><br />
<br />
<p> Check out the result of our program here:</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/c/cd/Lighttracking2.png" alt="" class="img-responsive" /></p><br />
<br />
<div class="pull-left img-left" style="width: 30%"><br />
<a href=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" data-lightbox="raspberry"><img src=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" alt="YCrCb" class="img-border" /></a><br /><br />
<figcaption class="cntr">Representation of the YCrCb color space</figcaption><br />
</div><br />
<p>To detect the nature of the signal, we used a fairly novel way of processing images and videos. The most common color space used in programming is RGB - pixel colors can be split into three components (Red, Green, and Blue) each taking a value between 0 and 255.</p><br />
<br />
<p>On the other, we used another color space, better adapted for this application as we were especially interested in light intensity and color nuances. We therefore chose the YcrCb color space.</p><br />
<br/><br />
<br />
<p>In the YCrCb color space, each pixel is decomposed into three components: </p><br />
<br />
<ul><br />
<li>Y – the luma value (intensity)</li><br />
<li>Cr – the red difference</li><br />
<li>Cb – the blue difference</li><br />
</ul><br />
<br />
<br />
<br />
<p><br />
This allows us to extract the necessary information for our application.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3 id="lenses">Lenses</h3><br />
<br />
<p>The Raspberry Pi camera that we use has a fixed lens which is not adapted to what we want to do, as we cannot change the focus, the aperture or the zoom.</p><br />
<br />
<p>We searched for a different lens which would allow us more control, and found that the easiest way was to remove the initial Raspberry Pi lens, put a CS mount on it and attach a much bigger lens.</p><br />
<br />
<p>The first thing we did was to unplug the camera module from the PCB. Then, the lens was carefully removed by unscrewing it and the new lens was mounted. </p><br />
<br />
INSERT IMAGE<br />
<br />
<p>You will then see the camera sensor (CMOS).</p><br />
<br />
<p>The CS mount was screwed on the board and the lens plugged in.</p><br />
<br />
<p>You can see here what the lens sees: <br /></p><br />
<br />
IMAGE!!!<br />
<br />
<p>We can clearly see the chambers of the microfluidic chip.</p><br />
<br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/HardwareTeam:EPF Lausanne/Hardware2014-10-17T23:05:49Z<p>Robert Baldwin: </p>
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<br />
<h1 class="cntr">Hardware</h1><br />
<br />
<br/><br/><br />
<br />
<br />
<p class="lead"><br />
<br />
<br />
Having successfully engineered touch responsive bacteria, the next major step to build a functional BioPad is to detect and process the emitted signals. The microfluidic chip containing our engineered organisms has the advantage of being small and portable. We aimed to keep these characteristics all along the project. That is why, instead of using a big and cumbersome device to detect signals, we opted for a small and cheap Raspberry Pi. Let the adventure for building the BioPad Detector begin !<br /><br /><br />
<br />
</p><br />
<br />
<h3 id="raspberry">Raspberry Pi</h3><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" alt="Raspberry Pi" class="pull-left img-left img-border" style="width: 30%;" /></a><br />
<br />
<br /><br />
<p>The Raspberry Pi is a small and cheap (40.- CHF) single-board computer. The raspberry Pi will be used to monitor the light emitted by each chamber of the microfluidic chip. We will be able to detect and process all emitted signals through this small device !<br /><br /></p><br />
<br />
<br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<p>When brainstorming how to build the detector, we initially drafted the follwing setup: </p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" alt="Raspberry Pi" class="img-border img-responsive" /></a></div><br />
<br />
<br /><br />
<br /><br />
<br />
<p>As seen in the picture above, the camera is directly linked to the Raspberry Pi and is able to get a clear view of the whole chip. Our final device thus needed a small and high-resolution camera able to easily track signal emission from our touch responsive organisms (including signals emitted in the infrared spectrum). <br /></p><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" alt="Raspberry Pi" class="pull-right img-right img-border" style="width: 30%;" /></a><br />
<br />
<pThe camera best suited to the characteristics above was the Raspberry Pi NoIR. The Raspberry Pi NoIR is a 5 MegaPixel, 1080 pixels, 20mm x 25mm x 9mm CMOS camera. No IR is especially good for low intensity signals. Moreover, NoIR stands for No Infrared Filter, meaning that with this camera we are able to see near infrared wavelength.<br /></p><br />
<br />
<p>The Pi NoIR can track near infrared corresponds to wavelengths between 700 and 1000nm. We will use this feature of the camera to track the IFP signal emitted by our CpxR - split IFP1.4 stress responsive cells. The emission of IFP in these wavelengths is especially useful for us, as few things emit auto-fluorescence in the infrared spectrum. This drastically increases the precision of our device as it reduces background noise.<br /></p><br />
<br />
<p>Taking in account all the information above, the main idea driving the way we plan to detect signals through our BioPad detector is to collect the entire light spectrum including near infrared wavelengths and then use a filter to eliminate the visible spectrum.</p><br />
<br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" alt="Raspberry Pi Plan" class="img-border img-responsive" /></a><br />
</div><br />
<br /><br />
<br />
<h3 id="lighttracking">Light tracking</h3><br />
<br />
<p>To track the signal dynamics in the chambers via our detector, we wrote a custom C++ code using OpenCV able to specifically detect the exact position of the signal as well as its nature and intensity. The entire code as well as all the supporting files can be found here: <a href="https://github.com/arthurgiroux/igemtracking/" target="_blank">https://github.com/arthurgiroux/igemtracking/</a>. An extract of the main code is given here:</p><br />
<br />
<script src="http://gist-it.sudarmuthu.com/https://github.com/arthurgiroux/igemtracking/blob/master/src/tracking.cpp"></script><br />
<br />
<br/><br/><br/><br />
<br />
<p> Check out the result of our program here:</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/c/cd/Lighttracking2.png" alt="" class="img-responsive" /></p><br />
<br />
<div class="pull-left img-left" style="width: 30%"><br />
<a href=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" data-lightbox="raspberry"><img src=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" alt="YCrCb" class="img-border" /></a><br /><br />
<figcaption class="cntr">Representation of the YCrCb color space</figcaption><br />
</div><br />
<p>To detect the nature of the signal, we used a fairly novel way of processing images and videos. The most common color space used in programming is RGB - pixel colors can be split into three components (Red, Green, and Blue) each taking a value between 0 and 255.</p><br />
<br />
<p>On the other, we used another color space, better adapted for this application as we were especially interested in light intensity and color nuances. We therefore chose the YcrCb color space.</p><br />
<br/><br />
<br />
<p>In the YCrCb color space, each pixel is decomposed into three components: </p><br />
<br />
<ul><br />
<li>Y – the luma value (intensity)</li><br />
<li>Cr – the red difference</li><br />
<li>Cb – the blue difference</li><br />
</ul><br />
<br />
<br />
<br />
<p><br />
This allows us to extract the necessary information for our application.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3 id="lenses">Lenses</h3><br />
<br />
<p>The Raspberry Pi camera that we use has a fixed lens which is not adapted to what we want to do, as we cannot change the focus, the aperture or the zoom.</p><br />
<br />
<p>We searched for a different lens which would allow us more control, and found that the easiest way was to remove the initial Raspberry Pi lens, put a CS mount on it and attach a much bigger lens.</p><br />
<br />
<p>The first thing we did was to unplug the camera module from the PCB. Then, the lens was carefully removed by unscrewing it and the new lens was mounted. </p><br />
<br />
INSERT IMAGE<br />
<br />
<p>You will then see the camera sensor (CMOS).</p><br />
<br />
<p>The CS mount was screwed on the board and the lens plugged in.</p><br />
<br />
<p>You can see here what the lens sees: <br /></p><br />
<br />
IMAGE!!!<br />
<br />
<p>We can clearly see the chambers of the microfluidic chip.</p><br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/HardwareTeam:EPF Lausanne/Hardware2014-10-17T21:59:06Z<p>Robert Baldwin: </p>
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<h1 class="cntr">Hardware</h1><br />
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<p class="lead"><br />
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Having successfully engineered touch responsive bacteria, the next major step to build a functional BioPad is to detect and process the emitted signals. The microfluidic chip containing our engineered organisms has the advantage of being small and portable. We aimed to keep these characteristics all along the project. That is why, instead of using a big and cumbersome device to detect signals, we opted for a small and cheap Raspberry Pi. Let the adventure for building the BioPad Detector begin !<br /><br /><br />
<br />
</p><br />
<br />
<h3 id="raspberry">Raspberry Pi</h3><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" alt="Raspberry Pi" class="pull-left img-left img-border" style="width: 30%;" /></a><br />
<br />
<br /><br />
<p>The Raspberry Pi is a small and cheap (40.- CHF) single-board computer. The raspberry Pi will be used to monitor the light emitted by each chamber of the microfluidic chip. We will be able to detect and process all emitted signals through this small device !<br /><br /></p><br />
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<div class="clearfix"></div><br />
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<br />
<p>When brainstorming how to build the detector, we initially drafted the follwing setup: </p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" alt="Raspberry Pi" class="img-border img-responsive" /></a></div><br />
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<p>As seen in the picture above, the camera is directly linked to the Raspberry Pi and is able to get a clear view of the whole chip. Our final thus needed a small and high-resolution camera able to easily track signal emission from our touch responsive organisms (including signals emitted in the infrared spectrum). <br /></p><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" alt="Raspberry Pi" class="pull-right img-right img-border" style="width: 30%;" /></a><br />
<br />
<pThe camera best suited to the characteristics above was the Raspberry Pi NoIR. The Raspberry Pi NoIR is a 5 MegaPixel, 1080 pixels, 20mm x 25mm x 9mm CMOS camera. No IR is especially good for low intensity signals. Moreover, NoIR stands for No Infrared Filter, meaning that with this camera we are able to see near infrared wavelength.<br /></p><br />
<br />
<p>The Pi NoIR can track near infrared corresponds to wavelengths between 700 and 1000nm. We will use this feature of the camera to track the IFP signal emitted by our CpxR - split IFP1.4 stress responsive cells. The emission of IFP in these wavelengths is especially useful for us, as few things emit auto-fluorescence in the infrared spectrum. This drastically increases the precision of our device as it reduces background noise.<br /></p><br />
<br />
<p>Taking in account all the information above, the main idea driving the way we plan to detect signals through our BioPad detector is to collect the entire light spectrum including near infrared wavelengths and then use a filter to eliminate the visible spectrum.</p><br />
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<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" alt="Raspberry Pi Plan" class="img-border img-responsive" /></a><br />
</div><br />
<br /><br />
<br />
<h3 id="lighttracking">Light tracking</h3><br />
<br />
<p>To track the signal dynamics in the chambers via our detector, we wrote a custom C++ code using OpenCV able to specifically detect the exact position of the signal as well as its nature and intensity. The entire code as well as all the supporting files can be found here: <a href="https://github.com/arthurgiroux/igemtracking/" target="_blank">https://github.com/arthurgiroux/igemtracking/</a>. An extract of the main code is given here:</p><br />
<br />
<script src="http://gist-it.sudarmuthu.com/https://github.com/arthurgiroux/igemtracking/blob/master/src/tracking.cpp"></script><br />
<br />
<br/><br/><br/><br />
<br />
<p> Check out the result of our program here:</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/c/cd/Lighttracking2.png" alt="" class="img-responsive" /></p><br />
<br />
<div class="pull-left img-left" style="width: 30%"><br />
<a href=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" data-lightbox="raspberry"><img src=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" alt="YCrCb" class="img-border" /></a><br /><br />
<figcaption class="cntr">Representation of the YCrCb color space</figcaption><br />
</div><br />
<p>To detect the nature of the signal, we used a fairly novel way of processing images and videos. The most common color space used in programming is RGB - pixel colors can be split into three components (Red, Green, and Blue) each taking a value between 0 and 255.</p><br />
<br />
<p>On the other, we used another color space, better adapted for this application as we were especially interested in light intensity and color nuances. We therefore chose the YcrCb color space.</p><br />
<br/><br />
<br />
<p>In the YCrCb color space, each pixel is decomposed into three components: </p><br />
<br />
<ul><br />
<li>Y – the luma value (intensity)</li><br />
<li>Cr – the red difference</li><br />
<li>Cb – the blue difference</li><br />
</ul><br />
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<p><br />
This allows us to extract the necessary information for our application.<br />
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</p><br />
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<br />
<h3 id="lenses">Lenses</h3><br />
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<p>The Raspberry Pi camera that we use has a fixed lens which is not adapted to what we want to do, as we cannot change the focus, the aperture or the zoom.</p><br />
<br />
<p>We searched for a different lens which would allow us more control, and found that the easiest way was to remove the initial Raspberry Pi lens, put a CS mount on it and attach a much bigger lens.</p><br />
<br />
<p>The first thing we did was to unplug the camera module from the PCB. Then, the lens was carefully removed by unscrewing it and the new lens was mounted. </p><br />
<br />
INSERT IMAGE<br />
<br />
<p>You will then see the camera sensor (CMOS).</p><br />
<br />
<p>The CS mount was screwed on the board and the lens plugged in.</p><br />
<br />
<p>You can see here what the lens sees: <br /></p><br />
<br />
IMAGE!!!<br />
<br />
<p>We can clearly see the chambers of the microfluidic chip.</p><br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/HardwareTeam:EPF Lausanne/Hardware2014-10-17T21:58:40Z<p>Robert Baldwin: </p>
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<h1 class="cntr">Hardware</h1><br />
<br />
<br/><br/><br />
<br />
<br />
<p class="lead"><br />
<br />
<br />
Having successfully engineered touch responsive bacteria, the next major step to build a functional BioPad is to detect and process the emitted signals. The microfluidic chip containing our engineered organisms has the advantage of being small and portable. We aimed to keep these characteristics all along the project. That is why, instead of using a big and cumbersome device to detect signals, we opted for a small and cheap Raspberry Pi. Let the adventure for building the BioPad Detector begin !<br /><br /><br />
<br />
</p><br />
<br />
<h3 id="raspberry">Raspberry Pi</h3><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" alt="Raspberry Pi" class="pull-left img-left img-border" style="width: 30%;" /></a><br />
<br />
<br /><br />
<p>The Raspberry Pi is a small and cheap (40.- CHF) single-board computer. The raspberry Pi will be used to monitor the light emitted by each chamber of the microfluidic chip. We will be able to detect and process all emitted signals through this small device !<br /><br /></p><br />
<br />
<br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<p>When brainstorming how to build the detector, we initially drafted the follwing setup: </p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" alt="Raspberry Pi" class="img-border img-responsive" /></a></div><br />
<br />
<br /><br />
<br /><br />
<br />
<p>As seen in the picture above, the camera is directly linked to the Raspberry Pi and is able to get a clear view of the whole chip. Our final thus needed a small and high-resolution camera able to easily track signal emission from our touch responsive organisms (including signals emitted in the infrared spectrum). <br /></p><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" alt="Raspberry Pi" class="pull-right img-right img-border" style="width: 30%;" /></a><br />
<br />
<pThe camera best suited to the characteristics above was the Raspberry Pi NoIR. The Raspberry Pi NoIR is a 5 MegaPixel, 1080 pixels, 20mm x 25mm x 9mm CMOS camera. No IR is especially good for low intensity signals. Moreover, NoIR stands for No Infrared Filter, meaning that with this camera we are able to see near infrared wavelength.<br /></p><br />
<br />
<p>The Pi NoIR can track near infrared corresponds to wavelengths between 700 and 1000nm. We will use this feature of the camera to track the IFP signal emitted by our CpxR - split IFP1.4 stress responsive cells. The emission of IFP in these wavelengths is especially useful for us, as few things emit auto-fluorescence in the infrared spectrum. This drastically increases the precision of our device as it reduces background noise.<br /></p><br />
<br />
<p>Taking in account all the information above, the main idea driving the way we plan to detect signals through our BioPad detector is to collect the entire light spectrum including near infrared wavelengths and then use a filter to eliminate the visible spectrum.</p><br />
<br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" alt="Raspberry Pi Plan" class="img-border img-responsive" /></a><br />
</div><br />
<br /><br />
<br />
<h3 id="lighttracking">Light tracking</h3><br />
<br />
<p>To track the signal dynamics in the chambers via our detector, we wrote a custom C++ code using OpenCV able to specifically detect the exact position of the signal as well as its nature and intensity. The entire code as well as all the supporting files can be found here: <a href="https://github.com/arthurgiroux/igemtracking/" target="_blank">https://github.com/arthurgiroux/igemtracking/</a>. An extract of the main code is given here:</p><br />
<br />
<script src="http://gist-it.sudarmuthu.com/https://github.com/arthurgiroux/igemtracking/blob/master/src/tracking.cpp"></script><br />
<br />
<br/><br/><br/><br />
<br />
<p> Check out the result of our program here:</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/c/cd/Lighttracking2.png" alt="" class="img-responsive" /></p><br />
<br />
<div class="pull-left img-left" style="width: 30%"><br />
<a href=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" data-lightbox="raspberry"><img src=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" alt="YCrCb" class="img-border" /></a><br /><br />
<figcaption class="cntr">Representation of the YCrCb color space</figcaption><br />
</div><br />
<p>To detect the nature of the signal, we used a fairly novel way of processing images and videos. The most common color space used in programming is RGB - pixel colors can be split into three components (Red, Green, and Blue) each taking a value between 0 and 255.</p><br />
<br />
<p>On the other, we used another color space, better adapted for this application as we were especially interested in light intensity and color nuances. We therefore chose the YcrCb color space.</p><br />
<br />
<p>In the YCrCb color space, each pixel is decomposed into three components: </p><br />
<br />
<ul><br />
<li>Y – the luma value (intensity)</li><br />
<li>Cr – the red difference</li><br />
<li>Cb – the blue difference</li><br />
</ul><br />
<br />
<br />
<br />
<p><br />
This allows us to extract the necessary information for our application.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3 id="lenses">Lenses</h3><br />
<br />
<p>The Raspberry Pi camera that we use has a fixed lens which is not adapted to what we want to do, as we cannot change the focus, the aperture or the zoom.</p><br />
<br />
<p>We searched for a different lens which would allow us more control, and found that the easiest way was to remove the initial Raspberry Pi lens, put a CS mount on it and attach a much bigger lens.</p><br />
<br />
<p>The first thing we did was to unplug the camera module from the PCB. Then, the lens was carefully removed by unscrewing it and the new lens was mounted. </p><br />
<br />
INSERT IMAGE<br />
<br />
<p>You will then see the camera sensor (CMOS).</p><br />
<br />
<p>The CS mount was screwed on the board and the lens plugged in.</p><br />
<br />
<p>You can see here what the lens sees: <br /></p><br />
<br />
IMAGE!!!<br />
<br />
<p>We can clearly see the chambers of the microfluidic chip.</p><br />
<br />
<br />
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</html><br />
{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/HardwareTeam:EPF Lausanne/Hardware2014-10-17T21:58:20Z<p>Robert Baldwin: </p>
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<h1 class="cntr">Hardware</h1><br />
<br />
<br/><br/><br />
<br />
<br />
<p class="lead"><br />
<br />
<br />
Having successfully engineered touch responsive bacteria, the next major step to build a functional BioPad is to detect and process the emitted signals. The microfluidic chip containing our engineered organisms has the advantage of being small and portable. We aimed to keep these characteristics all along the project. That is why, instead of using a big and cumbersome device to detect signals, we opted for a small and cheap Raspberry Pi. Let the adventure for building the BioPad Detector begin !<br /><br /><br />
<br />
</p><br />
<br />
<h3 id="raspberry">Raspberry Pi</h3><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" alt="Raspberry Pi" class="pull-left img-left img-border" style="width: 30%;" /></a><br />
<br />
<br /><br />
<p>The Raspberry Pi is a small and cheap (40.- CHF) single-board computer. The raspberry Pi will be used to monitor the light emitted by each chamber of the microfluidic chip. We will be able to detect and process all emitted signals through this small device !<br /><br /></p><br />
<br />
<br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<p>When brainstorming how to build the detector, we initially drafted the follwing setup: </p><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" alt="Raspberry Pi" class="img-border img-responsive" /></a></div><br />
<br />
<br /><br />
<br /><br />
<br />
<p>As seen in the picture above, the camera is directly linked to the Raspberry Pi and is able to get a clear view of the whole chip. Our final thus needed a small and high-resolution camera able to easily track signal emission from our touch responsive organisms (including signals emitted in the infrared spectrum). <br /></p><br />
<br />
<a href="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" alt="Raspberry Pi" class="pull-right img-right img-border" style="width: 30%;" /></a><br />
<br />
<pThe camera best suited to the characteristics above was the Raspberry Pi NoIR. The Raspberry Pi NoIR is a 5 MegaPixel, 1080 pixels, 20mm x 25mm x 9mm CMOS camera. No IR is especially good for low intensity signals. Moreover, NoIR stands for No Infrared Filter, meaning that with this camera we are able to see near infrared wavelength.<br /></p><br />
<br />
<p>The Pi NoIR can track near infrared corresponds to wavelengths between 700 and 1000nm. We will use this feature of the camera to track the IFP signal emitted by our CpxR - split IFP1.4 stress responsive cells. The emission of IFP in these wavelengths is especially useful for us, as few things emit auto-fluorescence in the infrared spectrum. This drastically increases the precision of our device as it reduces background noise.<br /></p><br />
<br />
<p>Taking in account all the information above, the main idea driving the way we plan to detect signals through our BioPad detector is to collect the entire light spectrum including near infrared wavelengths and then use a filter to eliminate the visible spectrum.</p><br />
<br />
<br /><br />
<div class="cntr"><br />
<a href="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" alt="Raspberry Pi Plan" class="img-border img-responsive" /></a><br />
</div><br />
<br /><br />
<br />
<h3 id="lighttracking">Light tracking</h3><br />
<br />
<p>To track the signal dynamics in the chambers via our detector, we wrote a custom C++ code using OpenCV able to specifically detect the exact position of the signal as well as its nature and intensity. The entire code as well as all the supporting files can be found here: <a href="https://github.com/arthurgiroux/igemtracking/" target="_blank">https://github.com/arthurgiroux/igemtracking/</a>. An extract of the main code is given here:</p><br />
<br />
<script src="http://gist-it.sudarmuthu.com/https://github.com/arthurgiroux/igemtracking/blob/master/src/tracking.cpp"></script><br />
<br />
<br/><br/><br/><br />
<br />
<p> Check out the result of our program here:</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/c/cd/Lighttracking2.png" alt="" class="img-responsive" /></p><br />
<br />
<p>To detect the nature of the signal, we used a fairly novel way of processing images and videos. The most common color space used in programming is RGB - pixel colors can be split into three components (Red, Green, and Blue) each taking a value between 0 and 255.</p><br />
<br />
<p>On the other, we used another color space, better adapted for this application as we were especially interested in light intensity and color nuances. We therefore chose the YcrCb color space.</p><br />
<br />
<p>In the YCrCb color space, each pixel is decomposed into three components: </p><br />
<br />
<ul><br />
<li>Y – the luma value (intensity)</li><br />
<li>Cr – the red difference</li><br />
<li>Cb – the blue difference</li><br />
</ul><br />
<br />
<div class="pull-left img-left" style="width: 30%"><br />
<a href=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" data-lightbox="raspberry"><img src=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" alt="YCrCb" class="img-border" /></a><br /><br />
<figcaption class="cntr">Representation of the YCrCb color space</figcaption><br />
</div><br />
<br />
<p><br />
This allows us to extract the necessary information for our application.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3 id="lenses">Lenses</h3><br />
<br />
<p>The Raspberry Pi camera that we use has a fixed lens which is not adapted to what we want to do, as we cannot change the focus, the aperture or the zoom.</p><br />
<br />
<p>We searched for a different lens which would allow us more control, and found that the easiest way was to remove the initial Raspberry Pi lens, put a CS mount on it and attach a much bigger lens.</p><br />
<br />
<p>The first thing we did was to unplug the camera module from the PCB. Then, the lens was carefully removed by unscrewing it and the new lens was mounted. </p><br />
<br />
INSERT IMAGE<br />
<br />
<p>You will then see the camera sensor (CMOS).</p><br />
<br />
<p>The CS mount was screwed on the board and the lens plugged in.</p><br />
<br />
<p>You can see here what the lens sees: <br /></p><br />
<br />
IMAGE!!!<br />
<br />
<p>We can clearly see the chambers of the microfluidic chip.</p><br />
<br />
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{{CSS/EPFL_bottom}}</div>Robert Baldwinhttp://2014.igem.org/Team:EPF_Lausanne/HardwareTeam:EPF Lausanne/Hardware2014-10-17T21:57:55Z<p>Robert Baldwin: </p>
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<h1 class="cntr">Hardware</h1><br />
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Having successfully engineered touch responsive bacteria, the next major step to build a functional BioPad is to detect and process the emitted signals. The microfluidic chip containing our engineered organisms has the advantage of being small and portable. We aimed to keep these characteristics all along the project. That is why, instead of using a big and cumbersome device to detect signals, we opted for a small and cheap Raspberry Pi. Let the adventure for building the BioPad Detector begin !<br /><br /><br />
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<h3 id="raspberry">Raspberry Pi</h3><br />
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<a href="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/e/ec/121220065212-raspberry-pi-close-up-horizontal-gallery.jpg" alt="Raspberry Pi" class="pull-left img-left img-border" style="width: 30%;" /></a><br />
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<p>The Raspberry Pi is a small and cheap (40.- CHF) single-board computer. The raspberry Pi will be used to monitor the light emitted by each chamber of the microfluidic chip. We will be able to detect and process all emitted signals through this small device !<br /><br /></p><br />
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<p>When brainstorming how to build the detector, we initially drafted the follwing setup: </p><br />
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<a href="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/3/37/Raspberry_.png" alt="Raspberry Pi" class="img-border img-responsive" /></a></div><br />
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<p>As seen in the picture above, the camera is directly linked to the Raspberry Pi and is able to get a clear view of the whole chip. Our final thus needed a small and high-resolution camera able to easily track signal emission from our touch responsive organisms (including signals emitted in the infrared spectrum). <br /></p><br />
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<a href="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/6/61/1567kit_LRG.jpg" alt="Raspberry Pi" class="pull-right img-right img-border" style="width: 30%;" /></a><br />
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<pThe camera best suited to the characteristics above was the Raspberry Pi NoIR. The Raspberry Pi NoIR is a 5 MegaPixel, 1080 pixels, 20mm x 25mm x 9mm CMOS camera. No IR is especially good for low intensity signals. Moreover, NoIR stands for No Infrared Filter, meaning that with this camera we are able to see near infrared wavelength.<br /></p><br />
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<p>The Pi NoIR can track near infrared corresponds to wavelengths between 700 and 1000nm. We will use this feature of the camera to track the IFP signal emitted by our CpxR - split IFP1.4 stress responsive cells. The emission of IFP in these wavelengths is especially useful for us, as few things emit auto-fluorescence in the infrared spectrum. This drastically increases the precision of our device as it reduces background noise.<br /></p><br />
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<p>Taking in account all the information above, the main idea driving the way we plan to detect signals through our BioPad detector is to collect the entire light spectrum including near infrared wavelengths and then use a filter to eliminate the visible spectrum.</p><br />
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<a href="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" data-lightbox="raspberry"><img src="https://static.igem.org/mediawiki/2014/0/04/Raspberry_plan.png" alt="Raspberry Pi Plan" class="img-border img-responsive" /></a><br />
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<h3 id="lighttracking">Light tracking</h3><br />
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<p>To track the signal dynamics in the chambers via our detector, we wrote a custom C++ code using OpenCV able to specifically detect the exact position of the signal as well as its nature and intensity. The entire code as well as all the supporting files can be found here: <a href="https://github.com/arthurgiroux/igemtracking/" target="_blank">https://github.com/arthurgiroux/igemtracking/</a>. An extract of the main code is given here:</p><br />
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<script src="http://gist-it.sudarmuthu.com/https://github.com/arthurgiroux/igemtracking/blob/master/src/tracking.cpp"></script><br />
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<p> Check out the result of our program here:</p><br />
<p><img src="https://static.igem.org/mediawiki/2014/c/cd/Lighttracking2.png" alt="" class="img-responsive" /></p><br />
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<p>To detect the nature of the signal, we used a fairly novel way of processing images and videos. The most common color space used in programming is RGB - pixel colors can be split into three components (Red, Green, and Blue) each taking a value between 0 and 255.</p><br />
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<p>On the other, we used another color space, better adapted for this application as we were especially interested in light intensity and color nuances. We therefore chose the YcrCb color space.</p><br />
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<a href=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" data-lightbox="raspberry"><img src=" https://static.igem.org/mediawiki/2014/b/b8/YCbCr.GIF" alt="YCrCb" class="img-border" /></a><br /><br />
<figcaption class="cntr">Representation of the YCrCb color space</figcaption><br />
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<p>In the YCrCb color space, each pixel is decomposed into three components: </p><br />
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<li>Y – the luma value (intensity)</li><br />
<li>Cr – the red difference</li><br />
<li>Cb – the blue difference</li><br />
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This allows us to extract the necessary information for our application.<br />
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<h3 id="lenses">Lenses</h3><br />
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<p>The Raspberry Pi camera that we use has a fixed lens which is not adapted to what we want to do, as we cannot change the focus, the aperture or the zoom.</p><br />
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<p>We searched for a different lens which would allow us more control, and found that the easiest way was to remove the initial Raspberry Pi lens, put a CS mount on it and attach a much bigger lens.</p><br />
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<p>The first thing we did was to unplug the camera module from the PCB. Then, the lens was carefully removed by unscrewing it and the new lens was mounted. </p><br />
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<p>You will then see the camera sensor (CMOS).</p><br />
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<p>The CS mount was screwed on the board and the lens plugged in.</p><br />
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<p>You can see here what the lens sees: <br /></p><br />
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IMAGE!!!<br />
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<p>We can clearly see the chambers of the microfluidic chip.</p><br />
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