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<p><a href="#Reconnect" class="link">Reconnect Nerves</a></p> | <p><a href="#Reconnect" class="link">Reconnect Nerves</a></p> | ||
+ | <p><a href="#Cell" class="link">Cell culture</a></p> | ||
<p><a href="#Fight" class="link">Fight Infections</a></p> | <p><a href="#Fight" class="link">Fight Infections</a></p> | ||
<p><a href="#Kill" class="link">Kill Switch</a></p> | <p><a href="#Kill" class="link">Kill Switch</a></p> | ||
<p><a href="#Membrane" class="link">Membrane</a></p> | <p><a href="#Membrane" class="link">Membrane</a></p> | ||
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<div id="indexRight"> | <div id="indexRight"> | ||
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<div class="block full bothContent"> | <div class="block full bothContent"> | ||
<div class="block dropDown" id="Reconnect"> | <div class="block dropDown" id="Reconnect"> | ||
− | <h4>RECONNECT NERVES<br><i>Click to see more</i></h4> | + | <h4>RECONNECT NERVES: DNA ASSEMBLY<br><i>Click to see more</i></h4> |
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<p><i>Achievements: </i><br> | <p><i>Achievements: </i><br> | ||
− | <ul style="text-align: left;"> | + | <ul style="text-align: left;list-style: disc;"> |
− | <li>Successfully cloned a biobrick coding for secretion of NGF in pET43.1a and iGEM plasmid backbone pSB1C3, creating a new part <a href= | + | <li>Successfully cloned a biobrick coding for secretion of NGF in pET43.1a and iGEM plasmid backbone pSB1C3, creating a new part <a href="http://parts.igem.org/Part:BBa_K2616000"style="font-weight: bold ; color:#85196a;" target="__blank"> BBa_K2616000</a>. </li> |
− | <li>Successfully sequenced <a href= | + | <li>Successfully sequenced <a href="http://parts.igem.org/Part:BBa_K2616000"style="font-weight: bold ; color:#85196a;" target="__blank"> BBa_K2616000</a> BBa_K2616000</a> in pSB1C3 and sent to iGEM registry. </li> |
<li>Successfully co-transformed <i>E. coli</i> with plasmid secreting proNGF and plasmid expressing the secretion system, creating bacteria <b>capable of secreting NGF</b> in the medium.</li> | <li>Successfully co-transformed <i>E. coli</i> with plasmid secreting proNGF and plasmid expressing the secretion system, creating bacteria <b>capable of secreting NGF</b> in the medium.</li> | ||
<li>Successfully characterized production of proNGF thanks to mass spectrometry and western blot.</li> | <li>Successfully characterized production of proNGF thanks to mass spectrometry and western blot.</li> | ||
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− | + | ||
</ul><br></p> | </ul><br></p> | ||
<p><i>Next steps:</i><br> | <p><i>Next steps:</i><br> | ||
− | <ul style="text-align: left;"> | + | <ul style="text-align: left;list-style: disc;"> |
<li><b>Purify</b> secreted proNGF, and characterize its effects on neuron growth thanks to our microfluidic device. </li> | <li><b>Purify</b> secreted proNGF, and characterize its effects on neuron growth thanks to our microfluidic device. </li> | ||
− | + | ||
</ul> | </ul> | ||
</p> | </p> | ||
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<div class="block full bothContent"> | <div class="block full bothContent"> | ||
<div class="block dropDown" id="Cell"> | <div class="block dropDown" id="Cell"> | ||
− | <h4>CELL CULTURE<br><i>Click to see more</i></h4> | + | <h4>RECONNECT NERVES: CELL CULTURE<br><i>Click to see more</i></h4> |
</div> | </div> | ||
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<p><i>Imaging was performed in collaboration with the BioImagerie Photonique platform of the Institut Pasteur. Data are presented as MEAN ± SEM. Significance between 2 different groups was determined using an Ordinary one-way ANOVA test on the software Prism6 (GraphPad). (ns: non-significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001) </i> </p> | <p><i>Imaging was performed in collaboration with the BioImagerie Photonique platform of the Institut Pasteur. Data are presented as MEAN ± SEM. Significance between 2 different groups was determined using an Ordinary one-way ANOVA test on the software Prism6 (GraphPad). (ns: non-significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001) </i> </p> | ||
<p>As an alternative to our recombinant proNGF for control experiments, we performed an <i>in vitro</i> neural primary culture with commercial NGF. For this, a pair of E18 Sprague Dawley cortexes were purchased from BrainBits.co.uk. We digested the tissue with manufacturer provided papain according to their protocol and seeded 40 000 dissociated neurons on our microfluidic chips with different conditions of culture for six days at 37°C, and 5% CO2. </p> | <p>As an alternative to our recombinant proNGF for control experiments, we performed an <i>in vitro</i> neural primary culture with commercial NGF. For this, a pair of E18 Sprague Dawley cortexes were purchased from BrainBits.co.uk. We digested the tissue with manufacturer provided papain according to their protocol and seeded 40 000 dissociated neurons on our microfluidic chips with different conditions of culture for six days at 37°C, and 5% CO2. </p> | ||
+ | |||
<p>On our two-chamber microfluidic devices, we seeded neurons only on one side. Fifteen chips were used in total. After six days, neurons are fixed with paraformaldehyde (PFA) 4% and stained with DAPI. For differentiated markers: MAP2 (coupled with Alexa Fluor 555), a cytoskeletal associated protein and Beta-III Tubulin (coupled with Alexa Fluor 488), one of the major components of microtubules and a neuron-specific marker were used.</p> | <p>On our two-chamber microfluidic devices, we seeded neurons only on one side. Fifteen chips were used in total. After six days, neurons are fixed with paraformaldehyde (PFA) 4% and stained with DAPI. For differentiated markers: MAP2 (coupled with Alexa Fluor 555), a cytoskeletal associated protein and Beta-III Tubulin (coupled with Alexa Fluor 488), one of the major components of microtubules and a neuron-specific marker were used.</p> | ||
− | <p>We can see in Figure 11 that we had contaminations on many of our microfluidic chips | + | <p>We can see in Figure 11 that we had contaminations on many of our microfluidic chips because we could not use antibiotic selection otherwise our bacteria would have suffered from it, and that most of our experiments could not be analyzed. However, no contaminations were apparent for eight of them. Two of these successful ones are displayed in Figure 12. </p> |
</div> | </div> | ||
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<img src="https://static.igem.org/mediawiki/2018/4/43/T--Pasteur_Paris--Figure_11.png"> | <img src="https://static.igem.org/mediawiki/2018/4/43/T--Pasteur_Paris--Figure_11.png"> | ||
− | <div class="legend"><b>Figure 11: </b> | + | <div class="legend"><b>Figure 11: </b> In orange, are displayed bacteria found inside one of our microfluidic devices.</div> |
</div> | </div> | ||
<br> | <br> | ||
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<div class="block half"> | <div class="block half"> | ||
<img src="https://static.igem.org/mediawiki/2018/e/e6/T--Pasteur_Paris--Figure_12B.png"> | <img src="https://static.igem.org/mediawiki/2018/e/e6/T--Pasteur_Paris--Figure_12B.png"> | ||
+ | </div> | ||
+ | <div class="block full"> | ||
<div class="legend"><b>Figure 12: </b> Sprague Dawley E18 cortex neurons after six days of incubation at 37°C, and 5% CO2. Blue: DAPI stained nuclei, Green: Anti-Beta-III Tubulin coupled to Alexa Fluor 488, Yellow: Co-localization of anti-Beta-III Tubulin and MAP2. <b>(A)</b> Neurons were put in culture in Neurobasal, B27, GlutaMAX medium. <b>(B)</b> Neurons were put in culture in DMEM FBS 10% medium. </div> | <div class="legend"><b>Figure 12: </b> Sprague Dawley E18 cortex neurons after six days of incubation at 37°C, and 5% CO2. Blue: DAPI stained nuclei, Green: Anti-Beta-III Tubulin coupled to Alexa Fluor 488, Yellow: Co-localization of anti-Beta-III Tubulin and MAP2. <b>(A)</b> Neurons were put in culture in Neurobasal, B27, GlutaMAX medium. <b>(B)</b> Neurons were put in culture in DMEM FBS 10% medium. </div> | ||
</div> | </div> | ||
<div class="block full"> | <div class="block full"> | ||
− | <p>As we can see, we succeeded in growing the cells inside our device in the presence of Neurobasal, B27 and GlutaMAX medium. It is possible to see neurons passing through one chamber to the other in this experiment. Unfortunately, the PDMS of the microfluidic chips detached from the bottom of the glass culture dish, leading to the growth of cells not inside of the microchannels, but | + | <p>As we can see, we succeeded in growing the cells inside our device in the presence of Neurobasal, B27 and GlutaMAX medium. It is possible to see neurons passing through one chamber to the other in this experiment. Unfortunately, the PDMS of the microfluidic chips detached from the bottom of the glass culture dish, leading to the growth of cells not inside of the microchannels, but below them (Figure 13). </p> |
</div> | </div> | ||
+ | |||
+ | <div class="block full"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/c/c2/T--Pasteur_Paris--Figure_13bis.png" style="width:400px"> | ||
+ | </div> | ||
+ | |||
+ | <div class="block full"> | ||
+ | <div class="legend"><b>Figure 13: </b> PDMS detachment from the glass bottom culture dish, we can observe the axonal development under the microchannels. Neurons were put in culture with Neurobasal, B27, GlutaMAX, and commercial NGF 50 ng/mL.</div> | ||
+ | </div> | ||
<div class="block full"> | <div class="block full"> | ||
− | |||
<p>We also tested the action of commercial NGF on our culture. Neurons were put in culture in the presence of commercial NGF at different concentrations: 50 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL and 900 ng/mL. The optimal concentration was determined by modeling of NGF diffusion inside the medium. It was possible to capture the cells passing through one of the chambers of the microfluidic chip to the other side during a time-lapsed using phase-contrast microscopy recorded for the first 48h of culture at the <i>BioImagerie Photonique </i> platform, proving that our device was working as expected (Video 1).</p> | <p>We also tested the action of commercial NGF on our culture. Neurons were put in culture in the presence of commercial NGF at different concentrations: 50 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL and 900 ng/mL. The optimal concentration was determined by modeling of NGF diffusion inside the medium. It was possible to capture the cells passing through one of the chambers of the microfluidic chip to the other side during a time-lapsed using phase-contrast microscopy recorded for the first 48h of culture at the <i>BioImagerie Photonique </i> platform, proving that our device was working as expected (Video 1).</p> | ||
</div> | </div> | ||
− | <div class="block | + | <div class="block full"> |
− | + | <video width="70%" height="auto" controls> | |
+ | <source src="https://static.igem.org/mediawiki/2018/b/b9/T--Pasteur_Paris--vidéo.mp4" type="video/mp4"> | ||
+ | Your browser does not support the video tag. | ||
+ | </video> | ||
+ | </div> | ||
+ | |||
<div class="legend"><b>Video 1: </b> A video excerpt of a 48h time-lapsed in phase contrast. Neuron entering the microchannel are visible. Medium of culture: Neurobasal, B27, GlutaMAX and commercial NGF at a concentration of 50 ng/mL. </div> | <div class="legend"><b>Video 1: </b> A video excerpt of a 48h time-lapsed in phase contrast. Neuron entering the microchannel are visible. Medium of culture: Neurobasal, B27, GlutaMAX and commercial NGF at a concentration of 50 ng/mL. </div> | ||
− | + | ||
<div class="block full"> | <div class="block full"> | ||
− | <p>Because we were running out of fresh microfluidic chips, and since we already proved that our device was working as expected, we switched and put our next cultures in a 96-well plate for 10 days at 37°C, testing the influence of the different concentrations of NGF on the growth of the cells. </p> | + | <p>Because we were running out of fresh microfluidic chips, and since we already proved that our device was working as expected, we switched and put our next cultures in a 96-well plate for 10 days at 37°C, testing the influence of the different concentrations of NGF on the growth of the cells (Figure 14). </p> |
− | < | + | </div> |
+ | <div class="block full"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/1/18/T--Pasteur_Paris--Figure_14_ABC.png"> | ||
+ | </div> | ||
+ | <div class="block full"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/c/c1/T--Pasteur_Paris--Figure_14_DEF.png"> | ||
+ | </div> | ||
+ | <div class="legend"><b>Figure 14: </b> Best representations of the imaging of the 96-well plate for each condition. A2: 0 ng/mL commercial NGF, B3: 50 ng/mL commercial NGF, C4: 250 ng/mL commercial NGF, D4: 500 ng/mL commercial NGF, E4: 750 ng/mL commercial NGF, F4: 900 ng/mL commercial NGF. </div> | ||
− | <div class=" | + | <div class="block full"> |
+ | <p>Images were analyzed on ImageJ, and following results are shown in Figure 15. </p> | ||
+ | </div> | ||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/0/05/T--Pasteur_Paris--Figure_15_A.jpg"> | ||
+ | </div> | ||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/c/cd/T--Pasteur_Paris--Figure_15_B.jpg"> | ||
+ | </div> | ||
<div class="block full"> | <div class="block full"> | ||
− | <p>As we can see in Figure | + | <div class="legend"><b>Figure 15: </b> <b>(A)</b> Percentage area of <FONT face="raleway">β</FONT>-III Tubulin in each well and <b>(B)</b> number of stained nuclei in each well with no NGF, 50 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL and 900 ng/mL of commercial NGF added in our medium Neurobasal, B27, GlutaMAX. Each condition was compared to the control group without NGF. <i>(ns: non-significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).</i></div> |
+ | </div> | ||
+ | |||
+ | <div class="block full"> | ||
+ | <p>As we can see in Figure 15 <b>(A)</b>, it was possible to observe a difference in the percentage of area taken by the <FONT face="raleway">β</FONT>-III Tubulin. Indeed, it is possible to observe a significant increase in this percentage when commercial NGF is put at a concentration of 250 ng/mL or higher. The concentration of NGF seems to influence the growth of axons. It was possible to observe the same significant increase of cell number at a concentration of 250 ng/mL or higher (Figure 15 <b>B</b>). We can also see that at a concentration of 900 ng/mL, it seems that both the percentage of area taken by the <FONT face="raleway">β</FONT>-III Tubulin and the number of cells decrease, even though it is not significant, we can hypothesize that at a high concentration of NGF, the receptor p75<sup>NTR</sup> is getting internalized, resulting in a decreasing number of available receptors. </p> | ||
<p>It seems that commercial NGF has a dose-response effect on both the growth of neuronal axons and / or the survival of the cells. To determine in which category the NGF was affecting, we standardize the percentage area of <FONT face="raleway">β</FONT>-III Tubulin compared to the number of cells. </p> | <p>It seems that commercial NGF has a dose-response effect on both the growth of neuronal axons and / or the survival of the cells. To determine in which category the NGF was affecting, we standardize the percentage area of <FONT face="raleway">β</FONT>-III Tubulin compared to the number of cells. </p> | ||
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</div> | </div> | ||
− | <div class="block | + | <div class="block full"> |
− | + | <img src="https://static.igem.org/mediawiki/2018/6/6f/T--Pasteur_Paris--Figure_16.jpg" style="max-width:35em;"> | |
− | <div class="legend"><b>Figure | + | </div> |
+ | <div class="legend"><b>Figure 16:</b> Ratio of the percentage area of <FONT face="raleway">β</FONT>-III Tubulin on the number of stained nucleus. <i>(ns: non-significant, * : p<0.05, ** : p<0.01, *** : p<0.001, **** : p<0.0001).</i> </div> | ||
− | |||
<div class="block full"> | <div class="block full"> | ||
− | <p>As we can see in figure | + | <p>As we can see in figure 16, we have a decreasing amount of <FONT face="raleway">β</FONT>-III Tubulin per nuclei each time the concentration of NGF gets higher. We can see a significant decrease of this ratio when the NGF is at 500 ng/mL and higher, which is not an expected result and an opposite result from the images that we occurred from the platform. </p> |
− | <p>We assumed from the start that all of our cells put in culture were neuronal cells, which might not be the case. We know that the NGF has an effect of the survival of the cells <sup>[1], [2]</sup> (Figure | + | <p>We assumed from the start that all of our cells put in culture were neuronal cells, which might not be the case. We know that the NGF has an effect of the survival of the cells <sup>[1], [2]</sup> (Figure 15 <b>B</b>). We did not have the suitable marker to differentiate the neuronal cells from the other types of cells, and should have stained the cells with NeuN, a neuronal nuclear antigen used as a biomarker for neurons. Therefore, the standardization we did with the number of cells is not an accurate one. We can still appreciate the qualitative results we had (Figure 14 and 15 <b>A</b>) and are positive on the effect NGF has on axon’s growth as well as cell survival.</p> |
− | <p>After having collected the data on the effect of commercial NGF, we decided to put in culture our cells in the presence of our bacterial lysate to test the effect of our proNGF. We put in culture for 2 days 30 000 cells with or without commercial NGF at 500 ng/mL and 900 ng/mL as well as our bacterial lysate in different dilutions. Since we wanted to inactivate as much bacterial proteins as possible ( | + | <p>After having collected the data on the effect of commercial NGF, we decided to put in culture our cells in the presence of our bacterial lysate to test the effect of our proNGF ( produced with <a href="http://parts.igem.org/Part:BBa_K2616000"> Bba_K2616000 </a> ). We put in culture for 2 days 30 000 cells with or without commercial NGF at 500 ng/mL and 900 ng/mL as well as our bacterial lysate in different dilutions. Since we wanted to inactivate as much bacterial proteins as possible (endotoxins), we checked the denaturation temperature for our proNGF, 70°C, and heat-inactivated the lysate at 60°C for 5 minutes before putting it in culture. Due to lack of time, only one well per condition was analyzed. </p> </div> |
<div class="block half"> | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/d/d1/T--Pasteur_Paris--Figure17A.jpg" style="max-width:30em;"></div> | ||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/d/d1/T--Pasteur_Paris--Figure_17B.jpg"style="max-width:30em;"></div> | ||
− | <div class="legend"><b> Figure | + | <div class="legend"><b> Figure 17:</b> <b>(A)</b> Percentage area of <FONT face="raleway">β</FONT>-III Tubulin in each well and <b>(B)</b> percentage area of nucleus in each well with no commercial NGF, 500 ng/mL or 900 ng/mL or bacterial lysate at 1/5, 1/10, 1/20 or 1/30 added in our medium Neurobasal, B27, GlutaMAX. </div> |
− | |||
<div class="block full"> | <div class="block full"> | ||
− | <p>We can see in Figure | + | <p>We can see in Figure 17 that our lysate seems to increase the percentage area of the <FONT face="raleway">β</FONT>-III Tubulin compared to the control without NGF. Our results with the commercial NGF seem to be equivalent to the results we had from our first experiment (Figure 15), with a decrease of axons at a concentration of 900 ng/mL. We can hypothesize that the lysate does have an effect on axon’s growth from the increasing percentage area of <FONT face="raleway">β</FONT>-III Tubulin, increase similar to the one we observe in our first experiment (Figure 15) and that the activity of our proNGF could be equivalent to commercial NGF with a concentration between 500 ng/mL and 900 ng/mL.</p> |
− | <p>We also could see an influence of the commercial NGF on the survival of the cells, similar to our first experiment (Figure | + | <p>We also could see an influence of the commercial NGF on the survival of the cells, similar to our first experiment (Figure 15). Our lysate, put at a concentration of 1/10 and higher, seems to have the same effect (Figure 18). </p> |
+ | |||
+ | </div> | ||
+ | |||
+ | <div class="block full"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/f/f1/T--Pasteur_Paris--Figure_18.jpg"style="max-width:50em;"></div> | ||
+ | <div class="legend"><b>Figure 18: </b> Image of the whole well of the 96-well plate. Neurons were put in culture in Neurobasal, B27, GlutaMAX, and our lysate at a concentration of 1/10 medium.</div> | ||
+ | |||
+ | |||
+ | <div class="block full"> | ||
<p>Of course, those data require further statistical tests, since we only had time to analyze one well per condition, and for only 2 days of culture due to French customs administrative delays that came with the order of the E18 cortex pair from the USA. Still, in those 2 days of culture, we have been able to observe a difference in both the percentage area of <FONT face="raleway">β</FONT>-III Tubulin and nuclei counts. </p> | <p>Of course, those data require further statistical tests, since we only had time to analyze one well per condition, and for only 2 days of culture due to French customs administrative delays that came with the order of the E18 cortex pair from the USA. Still, in those 2 days of culture, we have been able to observe a difference in both the percentage area of <FONT face="raleway">β</FONT>-III Tubulin and nuclei counts. </p> | ||
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<div class="block title"><h1 id="References">REFERENCES</h1></div> | <div class="block title"><h1 id="References">REFERENCES</h1></div> | ||
<div class="block full"> | <div class="block full"> | ||
− | <ul style="text-align: left;"> | + | <ul style="text-align: left;list-style: disc;"> |
<li style="list-style-type: decimal;">Matsumoto, T., Numakawa, T., Yokomaku, D., Adachi, N., Yamagishi, S., | <li style="list-style-type: decimal;">Matsumoto, T., Numakawa, T., Yokomaku, D., Adachi, N., Yamagishi, S., | ||
Numakawa, Y., Kunugi, H., and Taguchi, T. (2006). <i>Brain-derived neurotrophicfactor-induced potentiation of glutamate and GABA release: Different dependency on signaling pathways and neuronal activity.</i>Mol. Cell. Neurosci. 31, 70–84 <br><br></li> | Numakawa, Y., Kunugi, H., and Taguchi, T. (2006). <i>Brain-derived neurotrophicfactor-induced potentiation of glutamate and GABA release: Different dependency on signaling pathways and neuronal activity.</i>Mol. Cell. Neurosci. 31, 70–84 <br><br></li> | ||
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</div> | </div> | ||
+ | |||
+ | <div class="block separator-mark"></div> | ||
+ | </div> | ||
+ | |||
+ | <div class="block full" style="background-color: transparent;"> | ||
+ | <div class="block title"> | ||
+ | <h3>Summary</h3> | ||
+ | </div> | ||
+ | <p><i>Achievements: </i><br> | ||
+ | <ul style="text-align: left;list-style: disc;"> | ||
+ | <li>Successfully <b>observed axon growth</b> in microfluidic chip in presence of commercial NGF.</li> | ||
+ | <li>Successfully observed <b>activity of our proNGF</b> in invitro cellular culture compared to commercial NGF with a concentration between 500 ng/mL and 900 ng/mL.</li> | ||
+ | </ul><br></p> | ||
+ | <p><i>Next steps:</i><br> | ||
+ | <ul style="text-align: left;list-style: disc;"> | ||
+ | <li><b>Statistical analysis</b> of our <i>in vitro</i> culture in presence of bacterial lysate. </li> | ||
+ | <li><b>Global proof of concept</b> in a microfluidic device containing neurons in one of the chamber, and our engineered bacteria in the other.</li> | ||
+ | |||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
</div> | </div> | ||
</div> | </div> | ||
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<div class="block one-third"> | <div class="block one-third"> | ||
<img src="https://static.igem.org/mediawiki/2018/f/f1/T--Pasteur_Paris--96-culture-wells-2.jpg"> | <img src="https://static.igem.org/mediawiki/2018/f/f1/T--Pasteur_Paris--96-culture-wells-2.jpg"> | ||
− | <div class="legend"><b>Figure 17: </b>Biofilm culture fixed with Bouin's solution in 96-well | + | <div class="legend"><b>Figure 17: </b>Biofilm culture fixed with Bouin's solution in 96-well microtiter plate</div> |
</div> | </div> | ||
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</div> | </div> | ||
<i><p>Achievements:<br></i> | <i><p>Achievements:<br></i> | ||
− | <ul style="text-align: left;"> | + | <ul style="text-align: left;list-style: disc;"> |
<li>Successfully cloned a biobrick coding for RIP secretion in pBR322 and in pSB1C3, creating a new part <a href="http://parts.igem.org/Part:BBa_K2616001"> Bba_K2616001 </a>. | <li>Successfully cloned a biobrick coding for RIP secretion in pBR322 and in pSB1C3, creating a new part <a href="http://parts.igem.org/Part:BBa_K2616001"> Bba_K2616001 </a>. | ||
<li>Successfully sequenced <a href="http://parts.igem.org/Part:BBa_K2616001"> Bba_K2616001 </a> in pSB1C3 and sent to iGEM registry. | <li>Successfully sequenced <a href="http://parts.igem.org/Part:BBa_K2616001"> Bba_K2616001 </a> in pSB1C3 and sent to iGEM registry. | ||
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</ul><br></p> | </ul><br></p> | ||
<p><i>Next steps:<br></i> | <p><i>Next steps:<br></i> | ||
− | <ul style="text-align: left;"> | + | <ul style="text-align: left;list-style: disc;"> |
<li>Clone the sensor device with inducible RIP production upon <i>S. aureus</i> detection.</li> | <li>Clone the sensor device with inducible RIP production upon <i>S. aureus</i> detection.</li> | ||
<li>Improve the characterization of RIP effect on biofilm formation with a more standardized assay.</li> | <li>Improve the characterization of RIP effect on biofilm formation with a more standardized assay.</li> | ||
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<div class="block full"> | <div class="block full"> | ||
− | + | ||
<div class="block title"> | <div class="block title"> | ||
<h1 style="padding-top: 50px;">KILL SWITCH</h1> | <h1 style="padding-top: 50px;">KILL SWITCH</h1> | ||
+ | <h2><a href="http://parts.igem.org/Part:BBa_K2616002"> BBa_K2616002</a></h2><br><br> | ||
</div> | </div> | ||
+ | <br> | ||
+ | <div class="block full"> | ||
+ | <p>The sequence designed codes for two different proteins: CcdB toxin and CcdA antitoxine. The antitoxin production is under an constitutive promoter (PLac) and the toxin production under a thermosensitive one (PcspA). </p></div> | ||
+ | <div class="block full"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/3/33/T--Pasteur_Paris--BBa_K2616002.png"> | ||
+ | </div> | ||
<p>We gene synthesized the genetic construct of our kill-switch commercially. Once we received the sequence, called Seq9, in a commercial plasmid, we transformed competent bacteria <i>E. coli</i> DH5<FONT face="Raleway">α</FONT>. After bacterial culture and plasmid DNA extraction, we digested our DNA with restriction enzymes, extracted the inserts from the gel, and ligated it into linearized pSB1C3 for iGEM submission and expression in BL21(DE3).</p> | <p>We gene synthesized the genetic construct of our kill-switch commercially. Once we received the sequence, called Seq9, in a commercial plasmid, we transformed competent bacteria <i>E. coli</i> DH5<FONT face="Raleway">α</FONT>. After bacterial culture and plasmid DNA extraction, we digested our DNA with restriction enzymes, extracted the inserts from the gel, and ligated it into linearized pSB1C3 for iGEM submission and expression in BL21(DE3).</p> | ||
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</div> | </div> | ||
<p><i><p>Achievements:<br></i> | <p><i><p>Achievements:<br></i> | ||
− | <ul style="text-align: left; | + | <ul style="text-align: left;list-style: disc;"> |
<li>Successfully cloned the biobrick <a href="http://parts.igem.org/Part:BBa_K2616002"style="font-weight: bold ; color:#85196a;"target="_blank"> Bba_K2616002</a> coding for toxin/antitoxin (CcdB/CcdA) system in pSB1C3, creating a <b>new part</b>.</li> | <li>Successfully cloned the biobrick <a href="http://parts.igem.org/Part:BBa_K2616002"style="font-weight: bold ; color:#85196a;"target="_blank"> Bba_K2616002</a> coding for toxin/antitoxin (CcdB/CcdA) system in pSB1C3, creating a <b>new part</b>.</li> | ||
<li>Successfully sequenced <a href="http://parts.igem.org/Part:BBa_K2616002"style="font-weight: bold ; color:#85196a;"target="_blank"> BBa_K2616002</a> in pSB1C3 and sent it to iGEM registry.</li> | <li>Successfully sequenced <a href="http://parts.igem.org/Part:BBa_K2616002"style="font-weight: bold ; color:#85196a;"target="_blank"> BBa_K2616002</a> in pSB1C3 and sent it to iGEM registry.</li> | ||
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</ul><br></p> | </ul><br></p> | ||
<p><i>Next steps:</i><br> | <p><i>Next steps:</i><br> | ||
− | <ul style="text-align: left;"> | + | <ul style="text-align: left;list-style: disc;"> |
<li>Find a system that kills bacteria when released in the environment rather than just stopping their growth.</li> | <li>Find a system that kills bacteria when released in the environment rather than just stopping their growth.</li> | ||
</ul> | </ul> | ||
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<p>The <b> membrane filter </b> is a key element of our prosthesis system, allowing the <b> confinement </b> of the genetically modified bacteria and the <b> conduction of neuron impulses </b>. We tested two types of membranes: Sterlitech Polycarbonate <b> Gold-Coated </b> Membrane Filters (pores diameter of 0.4 micrometers) and Sterlitech <b> Alumina Oxide </b> Membrane Filters (pores diameter of 0.2 micrometers).<br> | <p>The <b> membrane filter </b> is a key element of our prosthesis system, allowing the <b> confinement </b> of the genetically modified bacteria and the <b> conduction of neuron impulses </b>. We tested two types of membranes: Sterlitech Polycarbonate <b> Gold-Coated </b> Membrane Filters (pores diameter of 0.4 micrometers) and Sterlitech <b> Alumina Oxide </b> Membrane Filters (pores diameter of 0.2 micrometers).<br> | ||
Sterlitech Alumina Oxide Membrane Filters were <b> coated </b> with different types of <b> biocompatible conductive polymers: PEDOT:PSS </b> (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate), <b> PEDOT:Cl </b> and <b> PEDOT:Ts </b> .<br> | Sterlitech Alumina Oxide Membrane Filters were <b> coated </b> with different types of <b> biocompatible conductive polymers: PEDOT:PSS </b> (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate), <b> PEDOT:Cl </b> and <b> PEDOT:Ts </b> .<br> | ||
− | To characterize the potential of the different types of membranes to be integrated into our prosthesis system, <b> we designed a PDMS well chip for that exact purpose </b>, a modified <b>culture well </b>. The bottom of the well is a membrane, and a platinum wire touching the membrane electrically connects the inside of the well with the exterior.<br></p> | + | To characterize the potential of the different types of membranes to be integrated into our prosthesis system, <b> we designed a PDMS (polydimethylsiloxane) well chip for that exact purpose </b>, a modified <b>culture well </b>. The bottom of the well is a membrane, and a platinum wire touching the membrane electrically connects the inside of the well with the exterior.<br></p> |
</div> | </div> | ||
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<div class="block full"> | <div class="block full"> | ||
<h2 style="text-align: left;">Confinement</h2> | <h2 style="text-align: left;">Confinement</h2> | ||
− | <p> | + | <p> The first requirement for the membrane is that it needs to meet is the ability to <b> retain bacteria </b> of the size of <i>E. coli</i> (1-2 micrometers). Theoretically, confinement should be garanteed because the membrane's pore size is smaller than <i>E. coli</i>. Three experiments were conducted on membrane microchannel chips to <b> prove that the gold-coated membranes can retain bacteria </b>. </p> |
<h3> First experiment </h3> | <h3> First experiment </h3> | ||
<p> The optical density of an <i>E. coli</i> liquid culture was measured. Liquid culture was poured in a microchannel chip on one side, and optical density of the liquid that flowed to the other side of the microchannnels was measured. </p> | <p> The optical density of an <i>E. coli</i> liquid culture was measured. Liquid culture was poured in a microchannel chip on one side, and optical density of the liquid that flowed to the other side of the microchannnels was measured. </p> | ||
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<p> OD (600 nm) of liquid after flowing through the chip: 0.41. </p> | <p> OD (600 nm) of liquid after flowing through the chip: 0.41. </p> | ||
<h4 style="text-align: left;"> Interpretation </h4> | <h4 style="text-align: left;"> Interpretation </h4> | ||
− | <p> We expected a much lower OD after liquid flow through the chip, so this suggests the presence of a leak in the chip, that | + | <p> We expected a much lower OD after liquid flow through the chip, so this suggests the presence of a leak in the chip, that allowed the liquid culture to flow without retaining the bacteria.</p> |
</div> | </div> | ||
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<p> A few drops of RFP expressing DH5alpha <i>E. coli</i> liquid culture were poured in a membrane microchannel chip. The chip was then observed under a microscope. </p> | <p> A few drops of RFP expressing DH5alpha <i>E. coli</i> liquid culture were poured in a membrane microchannel chip. The chip was then observed under a microscope. </p> | ||
<h4 style="text-align: left;"> Interpretation </h4> | <h4 style="text-align: left;"> Interpretation </h4> | ||
− | <p> Membrane is located on the right side, and liquid culture was poured on that side, before the membrane. Bacteria was still able to flow to the left side, but they were not following the microchannels, instead they were just flowing in a single direction, suggesting the membrane | + | <p> Membrane is located on the right side, and liquid culture was poured on that side, before the membrane. Bacteria was still able to flow to the left side, but they were not following the microchannels, instead they were all just flowing in a single direction, suggesting the membrane lifted the microfluidic chip from below and thus caused massive leakings in the microfluidic circuitry. </p> |
</div> | </div> | ||
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<p> A few drops of E. coli liquid culture were poured in a membrane microchannel chip. The chip was then observed under a microscope. </p> | <p> A few drops of E. coli liquid culture were poured in a membrane microchannel chip. The chip was then observed under a microscope. </p> | ||
<h4 style="text-align: left;"> Interpretation </h4> | <h4 style="text-align: left;"> Interpretation </h4> | ||
− | <p> The membrane is located on the left side, and liquid culture was poured on that side, before the membrane. Bacteria this time wasn't able to flow to the right side, the membrane stopped their progression. It is clear on figure 24, that the left side is crowded with bacteria, and the right side is empty (apart from a few PDMS impurities). Final conclusion on the membrane microchannel chips is, that although the integration method of the membrane filter in the chip is complicated and a bit improvised, some chips apparently do fulfill their purpose, demonstrating this way the confinement of the bacteria with a membrane. Leaks observed in previous experiments were also probably caused by membrane filters that were not correctly stretching across the whole chip.</p> | + | <p> The membrane is located on the left side, and liquid culture was poured on that side, before the membrane. Bacteria this time wasn't able to flow to the right side, <b> the membrane stopped their progression </b>. It is clear on figure 24, that the left side is crowded with bacteria, and the right side is empty (apart from a few PDMS impurities). Final conclusion on the membrane microchannel chips is, that although the integration method of the membrane filter in the chip is complicated and a bit improvised, <b> some chips apparently do fulfill their purpose </b>, demonstrating this way the confinement of the bacteria with a membrane. Leaks observed in previous experiments were also probably caused by membrane filters that were not correctly stretching across the whole chip.</p> |
</div> | </div> | ||
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<div class="block full"> | <div class="block full"> | ||
<h2 style="text-align: left;"> Polymer coating </h2> | <h2 style="text-align: left;"> Polymer coating </h2> | ||
− | <p> Bare alumina oxide membranes were coated with different polymers to enhance their conductivity values and their biocompatibility. </p> | + | <p> Bare alumina oxide membranes were coated with different polymers to enhance their conductivity values and their biocompatibility. </p> |
</div> | </div> | ||
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<div class="block half"> | <div class="block half"> | ||
<img src="https://static.igem.org/mediawiki/2018/f/f7/T--Pasteur_Paris--Alumina-oxide-membrane-micro.jpg" style="width:400px"> | <img src="https://static.igem.org/mediawiki/2018/f/f7/T--Pasteur_Paris--Alumina-oxide-membrane-micro.jpg" style="width:400px"> | ||
− | <div class="legend"><b>Figure 26: </b> Scanning electron microscopy of bare alumina oxide membranes</div> | + | <div class="legend"><b>Figure 26: </b> Scanning electron microscopy of bare alumina oxide membranes</div> |
</div> | </div> | ||
<div class="block full"> | <div class="block full"> | ||
<h3> PEDOT:PSS coating</h3> | <h3> PEDOT:PSS coating</h3> | ||
− | <p> The color of the alumina oxide membranes changed radically from light grey to black, suggesting the deposit of PEDOT:PSS on the membrane, as expected. Scanning electron microscopy of a PEDOT:PSS-coated membrane revealed cluster-like formation of PEDOT:PSS deposits. It is thought that the lack of uniformity of the coating won't give the expected results in matters of biocompatibility and conductivity. </p> | + | <p> The color of the alumina oxide membranes changed radically from <b> light grey to black </b>, suggesting the deposit of PEDOT:PSS on the membrane, as expected. Scanning electron microscopy (courtesy of Bruno Bresson, Sciences et Ingénierie de la Matière Molle Physico-chimie des Polymères et Milieux Dispersés) of a PEDOT:PSS-coated membrane revealed <b> cluster-like formation of PEDOT:PSS deposits </b>. It is thought that the lack of uniformity of the coating won't give the expected results in matters of biocompatibility and conductivity. </p> |
</div> | </div> | ||
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<div class="block full"> | <div class="block full"> | ||
<h3> PEDOT:Cl and PEDOT:Ts coating</h3> | <h3> PEDOT:Cl and PEDOT:Ts coating</h3> | ||
− | <p> The color of the alumina oxide membranes changed radically from light grey to black with green shades for PEDOT:Cl and blue shades for PEDOT:Ts, suggesting the deposit of the polymers on the membranes, as expected. Scanning electron microscopy reveals a uniform thickening of the membrane's surface, suggesting a uniform PEDOT:Cl coating of the membrane. We expect better results from the PEDOT:Cl-coated and PEDOT:Ts-coated membranes than PEDOT:PSS-coated ones. </p> | + | <p> The color of the alumina oxide membranes changed radically from <b> light grey to black with green shades for PEDOT:Cl and blue shades for PEDOT:Ts </b>, suggesting the deposit of the polymers on the membranes, as expected. Scanning electron microscopy reveals a uniform thickening of the membrane's surface, suggesting a <b> uniform </b> PEDOT:Cl coating of the membrane. We expect better results from the PEDOT:Cl-coated and PEDOT:Ts-coated membranes than PEDOT:PSS-coated ones. </p> |
</div> | </div> | ||
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<div class="block full"> | <div class="block full"> | ||
<h2 style="text-align: left;">Conductivity</h2> | <h2 style="text-align: left;">Conductivity</h2> | ||
− | <p> The membranes used in our system should possess good electric conductive capabilities for nerve influx conduction. The goal here is to evaluate the conductivity of the membranes.</p> | + | <p> The membranes used in our system should possess good electric conductive capabilities for nerve influx conduction. The goal here is to <b> evaluate the conductivity of the membranes </b>.</p> |
</div> | </div> | ||
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</div> | </div> | ||
− | <div class="block two-third"> | + | <div class="block two-third"> |
− | <p>The conductivity of the membranes was measured on a self-made device. It consists of a culture well made of PDMS (polydimethylsiloxane), with a membrane filter at its bottom and a platinum wire linking the conductive membrane filter with the exterior.</p> | + | <p>The conductivity of the membranes was measured on a <b> self-made device </b>. It consists of a culture well made of PDMS (polydimethylsiloxane), with a <b> membrane filter </b> at its bottom and a <b> platinum wire </b> linking the conductive membrane filter with the exterior.</p> |
</div> | </div> | ||
<div class ="block full"> | <div class ="block full"> | ||
<h3>Platinum wire</h3> | <h3>Platinum wire</h3> | ||
− | <p>As in the end we | + | <p>As in the end we were going to measure the conductivity of the system biofilm+membrane+platinum wire, we wanted to simplify the measurements and <b> neglect the impact of the platinum wire </b>. Function generator was set on sine. The physical quantities measured here are Eg, the generator's tension amplitude and Ep, the voltage difference between the two extremities of a platinum wire. the quantity calculated here is 20*log(Ep/Eg) for different frequencies. </p> |
<h4 style="text-align: left;"> Results </h4> | <h4 style="text-align: left;"> Results </h4> | ||
<img src="https://static.igem.org/mediawiki/2018/c/c4/T--Pasteur_Paris--Conductivity-platinum-wire.jpg" style="width:500px"> | <img src="https://static.igem.org/mediawiki/2018/c/c4/T--Pasteur_Paris--Conductivity-platinum-wire.jpg" style="width:500px"> | ||
<div class="legend"><b>Figure 33: </b> Conductivity of a platinum wire for different frequencies </div> | <div class="legend"><b>Figure 33: </b> Conductivity of a platinum wire for different frequencies </div> | ||
<h4 style="text-align: left;"> Interpretation </h4> | <h4 style="text-align: left;"> Interpretation </h4> | ||
− | <p> Voltage difference calculated is extremely low, indicating a very good conductivity for the platinum wires, so its resistance (in low frequencies) | + | <p> Voltage difference calculated is extremely low, indicating a very good conductivity for the platinum wires, so its resistance (in low frequencies) <b> could be neglected </b> at first glance when it would be used in PDMS well chips. Resistance increases in higher frequencies, because of the skin-effect in metals: the strip transforms into an antenna. But as we were going to use only low frequencies, this didn't affect us. </p> |
</div> | </div> | ||
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<div class ="block full"> | <div class ="block full"> | ||
<h3>Frequency impact on membrane conductivity</h3> | <h3>Frequency impact on membrane conductivity</h3> | ||
− | <p> Before measuring the conductivity of multiple membranes, we | + | <p> Before measuring the conductivity of multiple membranes, we needed to have an overview of the impact of the frequency on the conductivity of a membrane. We tested two gold-coated membranes.Function generator was set on sine. The physical quantities measured here are Eg, the generator's tension amplitude and Ep, the voltage difference between the extremity of the platinum wire outside the well chip and a point on the edge of the membrane of the chip. the quantity calculated here is 20*log(Ep/Eg) for different frequencies. </p> |
<h4 style="text-align: left;"> Results </h4> | <h4 style="text-align: left;"> Results </h4> | ||
<img src="https://static.igem.org/mediawiki/2018/6/6e/T--Pasteur_Paris--Gold-membrane-well-chip-conductivity.jpg" style="width:500px"> | <img src="https://static.igem.org/mediawiki/2018/6/6e/T--Pasteur_Paris--Gold-membrane-well-chip-conductivity.jpg" style="width:500px"> | ||
<div class="legend"><b>Figure 34: </b> Conductivity of a platinum wire for different frequencies </div> | <div class="legend"><b>Figure 34: </b> Conductivity of a platinum wire for different frequencies </div> | ||
<h4 style="text-align: left;"> Interpretation </h4> | <h4 style="text-align: left;"> Interpretation </h4> | ||
− | <p> Voltage difference calculated is very low, indicating a very good conductivity for the gold-coated membrane. Technically, we measured the conductivity of the system membrane+platinum wire, but we showed that the wire's conductivity could be neglected. Resistance increases in higher frequencies, again because of the skin-effect in metals. But as we | + | <p> Voltage difference calculated is very low, indicating a very good conductivity for the gold-coated membrane. Technically, we measured the conductivity of the system membrane+platinum wire, but we showed that the wire's conductivity could be neglected. Resistance increases in higher frequencies, again because of the skin-effect in metals. But as we were going to use only low frequencies, this doesn't affect us, and moreover,<b> the frequency response is flat for wide range of low frequencies </b>. </p> |
</div> | </div> | ||
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<div class ="block full"> | <div class ="block full"> | ||
<h3>Membrane conductivity</h3> | <h3>Membrane conductivity</h3> | ||
− | <p> We measured the conductivity of 6 membranes on PDMS well chips (2 gold-coated, 1 bare alumina oxide, 1 PEDOT:PSS-coated, 1 PEDOT:Cl-coated, 1 PEDOT:Ts-coated). Here we show the electric circuit that we used for the following experiments. </p> | + | <p> We measured the <b> conductivity of 6 membranes on PDMS well chips </b> (2 gold-coated, 1 bare alumina oxide, 1 PEDOT:PSS-coated, 1 PEDOT:Cl-coated, 1 PEDOT:Ts-coated). Here we show the electric circuit that we used for the following experiments. </p> |
<img src="https://static.igem.org/mediawiki/2018/b/bb/T--Pasteur_Paris--Electrical_circuit_1.pdf" style="width:500px"> | <img src="https://static.igem.org/mediawiki/2018/b/bb/T--Pasteur_Paris--Electrical_circuit_1.pdf" style="width:500px"> | ||
<div class="legend"><b>Figure 35: </b> Electric circuit used for the different conductivity measurements </div> | <div class="legend"><b>Figure 35: </b> Electric circuit used for the different conductivity measurements </div> | ||
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<div class="legend"><b>Figure 36: </b> Conductivity of a platinum wire for different frequencies (mean value and standard deviation for each membrane) </div> | <div class="legend"><b>Figure 36: </b> Conductivity of a platinum wire for different frequencies (mean value and standard deviation for each membrane) </div> | ||
<h4 style="text-align: left;"> Interpretation </h4> | <h4 style="text-align: left;"> Interpretation </h4> | ||
− | <p> Bare alumina oxide and PEDOT:PSS-coated membranes show similar | + | <p> Bare alumina oxide and PEDOT:PSS-coated membranes show similar conductivities, indicating the <b> incomplete coating of PEDOT:PSS </b> on alumina oxide membranes. On the opposite, PEDOT:Cl and PEDOT:Ts exhibit on average better conductivities, but in the same time, the coating of these membranes revealed by electron microscopy seemed to have covered the alumina oxide membranes in a more uniform way, <b> ensuring enhanced conductive capabilities </b>. These results can be criticized because of the high deviation and because the membranes conductivity was measured after several biofilms were grown on them, which may have affected the measurements. </p> |
</div> | </div> | ||
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<div class="block full"> | <div class="block full"> | ||
<h2 style="text-align: left;">Biocompatibility and biofilm conductivity</h2> | <h2 style="text-align: left;">Biocompatibility and biofilm conductivity</h2> | ||
− | <p> One last important property of the membranes to measure | + | <p> One last important property of the membranes to measure was the <b> capability of bacteria to form a biofilm </b> on them, as in our prosthesis system, the membrane is going to be directly in contact with the genetically modified biofilm, as well as the human body. </p> |
− | <p> We used the last section of the following | + | <p> We used the last section of the following <a href="https://static.igem.org/mediawiki/2018/b/b5/T--Pasteur_Paris--PDMS-well-chip.pdf">protocol</a> to form biofilms in our PDMS well chips and to measure the biofilm growth.</p> |
<h4 style="text-align: left;"> Results: biofilm growth </h4> | <h4 style="text-align: left;"> Results: biofilm growth </h4> | ||
<p> Biofilm growth was measured 4 times in total. For each series of measurements, the measured optical densities were divided by the optical density of the base liquid culture, to normalize the measures.</p> | <p> Biofilm growth was measured 4 times in total. For each series of measurements, the measured optical densities were divided by the optical density of the base liquid culture, to normalize the measures.</p> | ||
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<div class="legend"><b>Figure 39: </b>Estimated biofilm conductivity </div> | <div class="legend"><b>Figure 39: </b>Estimated biofilm conductivity </div> | ||
<h4 style="text-align: left;"> Interpretation </h4> | <h4 style="text-align: left;"> Interpretation </h4> | ||
− | <p> As told by the membrane manufacturer, biofilm formation on gold membranes seems indeed to be more difficult than on other membranes. However we expected PEDOT:PSS-coated membranes to stimulate more the growth of biofilm, but perhaps | + | <p> As told by the membrane manufacturer, biofilm formation on gold membranes seems indeed to be more difficult than on other membranes. However we expected PEDOT:PSS-coated membranes to stimulate more the growth of biofilm, but perhaps this may be just another indicator of the incomplete coating. Surprisingly, <b> PEDOT:Cl tends to allow better formation of biofilms</b>. We realized only after the experiments the need for a control biofilm culture without membrane. </p> |
<p> Conductivity with a biofilm is better with gold membranes, although the conductivity of gold membranes themselves isn't the best. This may be explained by the fact, that because of the thinner biofilm formation on gold membranes, the electrical wires touched not only the biofilm, but also the membrane, bypassing the biofilm and leading to imprecise measurements. </p> | <p> Conductivity with a biofilm is better with gold membranes, although the conductivity of gold membranes themselves isn't the best. This may be explained by the fact, that because of the thinner biofilm formation on gold membranes, the electrical wires touched not only the biofilm, but also the membrane, bypassing the biofilm and leading to imprecise measurements. </p> | ||
− | <p> Approximate biofilm conductivity is therefore probably wrong for the gold membrane. However, it is interesting to notice that the biofilm conductivity measured for the bare alumina oxyde, PEDOT:Ts-coated one and PEDOT:PSS-coated give more or less the same value, suggesting that with more measurements, adapted equipement and better methods it would be indeed possible to measure the biofilm's conductivity with our PDMS well chips. </p> | + | <p> Approximate biofilm conductivity is therefore probably wrong for the gold membrane. However, it is interesting to notice that the biofilm conductivity measured for the bare alumina oxyde, PEDOT:Ts-coated one and PEDOT:PSS-coated give more or less the same value, suggesting that with <b> more measurements, adapted equipement and better methods it would be indeed possible to measure the biofilm's conductivity with our PDMS well chips </b>. </p> |
</div> | </div> | ||
+ | <div class="block separator-mark"></div> | ||
+ | </div> | ||
+ | |||
+ | <div class="block full" style="background-color: transparent;"> | ||
+ | <div class="block title"> | ||
+ | <h3>Summary</h3> | ||
+ | </div> | ||
+ | <p><i>Achievements: </i><br> | ||
+ | <ul style="text-align: left;list-style: disc;"> | ||
+ | <li> Successfully demonstrated the <b> confinement of bacteria </b> by a membrane filter. </li> | ||
+ | <li> Successfully <b> coated </b> alumina oxide membranes with PEDOT:Cl and PEDOT:Ts .</li> | ||
+ | <li> Partially <b> coated </b> alumina oxide membranes with PEDOT:PSS.</li> | ||
+ | <li> Successfully demonstrated the <b> enhanced conductivity </b> induced by the PEDOT:Cl and PEDOT:Ts coating. </li> | ||
+ | <li> Successfully <b> enhanced biocompatibilty </b> with PEDOT:Cl coating. </li></ul><br></p> | ||
+ | <p><i>Next steps:</i><br> | ||
+ | <ul style="text-align: left;list-style: disc;"> | ||
+ | <li> Enhance <b> measurement precision </b> for membrane conductivity with and without biofilm.</li> | ||
+ | <li> Improve <b> PEDOT:PSS coating </b> to form a uniform layer.</li> | ||
+ | </ul> | ||
+ | </p> | ||
Latest revision as of 14:55, 10 November 2018
RECONNECT NERVES: DNA ASSEMBLY
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Summary
Achievements:
- Successfully cloned a biobrick coding for secretion of NGF in pET43.1a and iGEM plasmid backbone pSB1C3, creating a new part BBa_K2616000.
- Successfully sequenced BBa_K2616000 BBa_K2616000 in pSB1C3 and sent to iGEM registry.
- Successfully co-transformed E. coli with plasmid secreting proNGF and plasmid expressing the secretion system, creating bacteria capable of secreting NGF in the medium.
- Successfully characterized production of proNGF thanks to mass spectrometry and western blot.
Next steps:
- Purify secreted proNGF, and characterize its effects on neuron growth thanks to our microfluidic device.
RECONNECT NERVES: CELL CULTURE
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Summary
Achievements:
- Successfully observed axon growth in microfluidic chip in presence of commercial NGF.
- Successfully observed activity of our proNGF in invitro cellular culture compared to commercial NGF with a concentration between 500 ng/mL and 900 ng/mL.
Next steps:
- Statistical analysis of our in vitro culture in presence of bacterial lysate.
- Global proof of concept in a microfluidic device containing neurons in one of the chamber, and our engineered bacteria in the other.
FIGHT INFECTIONS
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Summary
Achievements:
- Successfully cloned a biobrick coding for RIP secretion in pBR322 and in pSB1C3, creating a new part Bba_K2616001 .
- Successfully sequenced Bba_K2616001 in pSB1C3 and sent to iGEM registry.
- Successfully cultivated S. aureus biofilms in 96-well plates with different supernatants. Although there was a high variability in our results, and we used several protocols to overcome it, in one case, we were able to observe a reduction in biofilm formation in the presence of our RIP.
Next steps:
- Clone the sensor device with inducible RIP production upon S. aureus detection.
- Improve the characterization of RIP effect on biofilm formation with a more standardized assay.
KILL SWITCH
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Summary
Achievements:
- Successfully cloned the biobrick Bba_K2616002 coding for toxin/antitoxin (CcdB/CcdA) system in pSB1C3, creating a new part.
- Successfully sequenced BBa_K2616002 in pSB1C3 and sent it to iGEM registry.
- Successfully observed normal growth of our engineered bacteria at 25°C and 37°C and absence of growth at 18°C and 20°C, showing the efficiency of the kill switch.
Next steps:
- Find a system that kills bacteria when released in the environment rather than just stopping their growth.
MEMBRANE
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Summary
Achievements:
- Successfully demonstrated the confinement of bacteria by a membrane filter.
- Successfully coated alumina oxide membranes with PEDOT:Cl and PEDOT:Ts .
- Partially coated alumina oxide membranes with PEDOT:PSS.
- Successfully demonstrated the enhanced conductivity induced by the PEDOT:Cl and PEDOT:Ts coating.
- Successfully enhanced biocompatibilty with PEDOT:Cl coating.
Next steps:
- Enhance measurement precision for membrane conductivity with and without biofilm.
- Improve PEDOT:PSS coating to form a uniform layer.