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| - | <!-- RESULTS --> | + | <p>Hello</p> |
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| - | <div id="results">
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| - | <h1 class="cntr"> RESULTS </h1>
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| - | <h2> <b><u>Characterisation of the CpxR & split IFP1.4 stress-sensitive response </u> </b> </h2>
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| - | <h3> <b>Experiment 1: </b> Promoter characterisation and folding ability of fused GFP to CpxR via 10 amino acid 2 x (GGGGS) flexible linker </h3>
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| - | <p>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit ameh2t, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus</p>
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| - | <h3><b>Experiment 2: </b>CpxR dimerization & Dimerization Orientation </h3>
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| - | <u>Introduction</u> <br />
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| - | 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 E.Coli CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in E.Coli.</p>
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| - | <u>Aim</u> <br />
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| - | This experiment aimed to determine if and how CpxR dimerised in vivo in E.Coli. 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.
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| - | <u>Methods</u> <br />
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| - | 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 E.Coli 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>.
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| - | <u>Results</u> <br />
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| - | 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.
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| - | <u>Discussion</u> <br />
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| - | 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.
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| - | <img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" alt="Construct Comparison">
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| - | <h3><b> Experiment 3: </b>Signal induction by various concentrations of KCl & signal shutdown by centrifugation </h3>
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| - | <u>Aim</u> <br />
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| - | 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.
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| - | 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.
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| - | <u>Methods</u> <br />
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| - | 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>.
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| - | <u>Results</u> <br />
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| - | 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.
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| - | <img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" alt="GA1 Shutdown">
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| - | <h3><b> Experiment 4: </b>Visualization of the the CpxR split IFP1.4 activation by KCl stress </h3>
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| - | <u>Aim</u> <br />
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| - | <u>Results</u> <br />
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| - | <h2> <b><u>Characterisation of the split luciferase </u> </b> </h2>
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| - | <h3><b>Experiment 1: </b>CheY/CheZ fused to split Firefly/Renilla luciferase, and full Firefly/Renilla luciferase characterisation </h3>
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| - | <p><u>Introduction</u> <br \>
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| - | 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 Laboratory, we wanted to redo and adapt the experiment to test our own splits.<br \> <br \>
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| - | <u>Aim</u> <br \>
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| - | 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 \>
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| - | <u>Method</u> <br \>
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| - | 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 : BBa_K1486022 and Firefly : BBa_K325108 from Cambridge 2010 team) as positive controls and the non-fused splits (Renilla : BBa_K1486021 and Firefly : BBa_K1486018) as negative controls.<br \> <br \>
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| - | The bioluminescence assay was performed as described <a href="https://static.igem.org/mediawiki/2014/6/6d/Protocol_-_Bioluminescence_assay.pdf">here</a>. <br \>
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| - | The constructs were designed and assembled as described <a href="https://static.igem.org/mediawiki/2014/3/3b/Constructs_design_CheYCheZ.pdf">here</a>.<br \>.<br \> <br \>
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| - | <u>Results</u> <br \>
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| - | As shown in the graphs below (fig.1A and 1B), we couldn't really observe a high signal for our complementation 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 \>
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| - | <img src="https://static.igem.org/mediawiki/2014/f/f9/Firefly-CheYCheZexp.png" width="612">
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| - | 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 \>
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| - | <h2> <b><u>Microfluidic stuff ? </u> </b> </h2>
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| - | <h3><b>Experiment 1: </b></h3>
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| - | <p>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus</p>
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| - | <h2> <b><u>Yeast stuff ?</u> </b> </h2>
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| - | <h3><b>Experiment 1: </b></h3>
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| - | <p>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus</p>
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