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| | + | <script> |
| | + | /* |
| | + | URL OF THE PAGE |
| | + | */ |
| | + | var myurl = "https://2015.igem.org/Team:Toulouse/project/attract"; |
| | + | var elementId = "project"; |
| | + | </script> |
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| | </html>{{Toulouse/nav}}<html> | | </html>{{Toulouse/nav}}<html> |
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| | <div class="wrapper row0 bgded" style="background-image:url('https://static.igem.org/mediawiki/2015/f/f5/TLSE_bg_1.png')"> | | <div class="wrapper row0 bgded" style="background-image:url('https://static.igem.org/mediawiki/2015/f/f5/TLSE_bg_1.png')"> |
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| | <div class="shout"> | | <div class="shout"> |
| − | | + | <!-- ################################################################################################ --> |
| | <div class="shout-content clear"> | | <div class="shout-content clear"> |
| | <div class="maintitle"> | | <div class="maintitle"> |
| − | <center> <h3>Results</h3></center> | + | <center> <h3>Attract</h3> </center> |
| | </div> | | </div> |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/67/TLSE_BG.png"></center> | + | <center><img src=" https://static.igem.org/mediawiki/2015/5/57/TLSE_Attract_BG.png"></center> |
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| | <main class="container clear"> | | <main class="container clear"> |
| | + | |
| | + | <!-- container body --> |
| | + | <!-- ################################################################################################ --> |
| | + | |
| | + | <div class="title"> |
| | + | <h3>Content</h3> |
| | + | </div> |
| | + | <center> |
| | + | <div id="breadcrumb" class="clear" style="float: center;" > |
| | + | <ul> |
| | + | <li><a href="#part1">How to attract <i>Varroa destructor?</i></a></li> |
| | + | <li><a href="#part2">Butyrate attraction test</a></li> |
| | + | <li><a href="#part3">How to produce butyrate with <i>E.Coli</i>?</a></li> |
| | + | </ul> |
| | + | </div> |
| | + | |
| | + | <hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1);"> |
| | + | </center> |
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| − | <!-- ################################ ICI CONTENEUR OU TU PEUX T'AMUSER A METTRE LES DIVS ############################### -->
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| − |
| |
| − | <div class="title">
| |
| − | <h3>Attract</h3>
| |
| − | </div>
| |
| − |
| |
| − | <div class="subtitle">
| |
| − | <h3>Tests on varroas</h3>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | In the US patent which describes utilization of butyric acid in order to attract varroa mites, it is said that a concentration of 4 % (V/V) is used in their tests. In the final description it is specify that a butyric acid concentration more than 0.1% is efficient but previously it is assumed that an efficient amount of attractant may at the minimum be 0.00001 %.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | In order to verify results of this patent, we made an attraction test on varroas. Champollion University in Albi, welcome us in their lab to do this test but there were not a lot of varroas available so we chose to make only one test in order to have a significant result. So for this test we use a 4 % butyric acid concentration as it was made in the patent.
| |
| − | </p>
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| − | </div>
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| − |
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| − | <center>
| |
| − | <table>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>
| |
| | | | |
| | + | <!-- FIRST PARAGRAPH --> |
| | + | <center><div class="subtitle" > |
| | + | <h3>About varroasis</h3> |
| | + | </div></center> |
| | | | |
| − | <div style="one-half;padding:10px;"> | + | <div class="group center"> <!-- FIRST PARAGRAPH --> |
| − | <img src="https://static.igem.org/mediawiki/2015/4/40/TLSE_Results_Attract_1.PNG" style="width:80%;" /> | + | <p align="justify" style="font-size:15px;"> |
| − | | + | Varroasis occurs with the <i>Varroa destructor</i> entrance in the hive, carried by |
| | + | infected bees: |
| | + | the mite can begin its parasitism and infest the brood. When the queen |
| | + | gives birth |
| | + | to new larvaes in honeycombs, the fertilized adult female varroa mite |
| | + | will come into |
| | + | it before capping, and lay her eggs. The larvaes will develop, |
| | + | increasing the overall |
| | + | infection that affects bee population [1]. To tackle this issue, |
| | + | it is necessary to attract |
| | + | varroa carried by honeybees before they come into the hive. |
| | + | </p> |
| | + | </div> |
| | + | |
| | + | |
| | + | |
| | + | <div class="group center"> |
| | + | <br> |
| | + | <img src="https://static.igem.org/mediawiki/2015/ 0/ 04/ TLSE_Attract_fig1. png" /> |
| | + | </div> |
| | + | <div id="part1"></div><!-- ANCHOR 1 --> |
| | + | <div class="group center"> |
| | + | <br> |
| | + | <p>Figure 1 : <i>Varroa destructor</i> life cycle, |
| | + | adapted from B. Alexander</p> |
| | + | </div> |
| | + | |
| | + | <div> |
| | + | |
| | + | <div class="subtitle" > |
| | + | <h3>How to attract <i>Varroa destructor</i>?</h3> |
| | </div> | | </div> |
| | | | |
| − | </td>
| + | |
| − | | + | |
| − | <td>
| + | |
| − | <div style="one-half;padding:10px;">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/e/ea/TLSE_Results_Attract_2.PNG" style="width:90%;" />
| + | |
| − | | + | |
| − | </div>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | </center>
| + | |
| − | | + | |
| − | | + | |
| − | <center>
| + | |
| − | <p class="legend">
| + | |
| − | Figure 1: Butyric acid test pie chart and statistical test
| + | |
| − | </p>
| + | |
| − | </center>
| + | |
| − | | + | |
| − | | + | |
| | <div class="group center"> | | <div class="group center"> |
| − | | + | <p align="justify" style="font-size:15px;"> |
| − | Thanks to this test we demonstrate that a solution of 4 % in butyric acid concentration attracts varraos. But in “Cytotoxicity” part we show that this concentration is totally lethal for bacteria, so our goal is to produce at least a concentration of butyric acid of 0,00001 % because of the explanation above.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | | | |
| − |
| + | Just before capping, bee larvaes produce a wide range of molecules, |
| − | <div class="group">
| + | those molecules warn the mite about the upcoming capping and motivate |
| − | <p class="text">
| + | it to enter the cell [2]. |
| − | In a second time it will be interesting to do another attraction test with the right concentration we could produce.
| + | Of all these molecules, scientific studies have shown that one can |
| | + | significantly attract varroa: |
| | + | <i>butyrate</i> [3]. |
| | </p> | | </p> |
| − | </div>
| |
| − |
| |
| − | <div class="subtitle">
| |
| − | <h3>Cloning butyrate genes</h3>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group">
| |
| − | <p class="text">
| |
| − | When we ordered synthesized genes we chose to have directly regulated ways, so we had ccr genes behind lacI ready for circadian circle. So we had to clone ccr with all genes necessary for butyrate production, in order to have the construction below:
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <center>
| |
| − | <div style="one-half;padding:10px;">
| |
| − | <img src="https://static.igem.org/mediawiki/2015/8/84/TLSE_Results_Attract_3.PNG" style="width:60%;" />
| |
| | </div> | | </div> |
| − |
| |
| − | <p class="legend">
| |
| − | Figure 2: All genes necessary to butyrate production, 5220 Kb. First arrow represents promoter, others genes, green circle RBS and red circle terminator. Purple gene comes from Streptomyces collinus, blue genes form Clostridium acetobutylicum and yellow genes form Escherichia coli.
| |
| − | </p>
| |
| − | </center>
| |
| − |
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | This construction is a biobrick format so we digested it by EcoRI and PstI to confirm it is integrated into pSB1C3 as we can see in figure 2.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <center>
| |
| − | <div style="one-half;padding:10px;">
| |
| − | <img src="https://static.igem.org/mediawiki/2015/1/15/TLSE_Results_Attract_4.PNG" style="width:18%;" />
| |
| − | </div>
| |
| − |
| |
| − | <p class="legend">
| |
| − | Figure 3: Gel electrophoresis for verification of butyrate biobrick
| |
| − | </p>
| |
| − | </center>
| |
| | | | |
| | | | |
| | | | |
| | <div class="group center"> | | <div class="group center"> |
| − | <p class="text">
| + | <div class="one_half first"> |
| − | The first band fits 5000Kb that matches with Biobrick and the second band fits 2000Kb that matches with pSB1C3. So now we have all genes necessary to butyrate production we can test it.
| + | <p align="justify" style="font-size:15px;"> |
| − | </p>
| + | <br> |
| − | </div> | + | Butyrate is a volatile acid which is non-toxic for honeybees |
| | + | nor the human being, because it is already present at physiologic |
| | + | concentrations in the digestive tract. Moreover this molecule |
| | + | is naturally |
| | + | produced by some bacterial strains like <i>Clostridium</i>, |
| | + | which is an asset |
| | + | for this synthetic biology project [4].</p><div id="part2"></div> <!-- ANCHOR 2 --><p align="justify" style="font-size:15px;"> Therefore we decided to |
| | + | modify <i>E. coli</i> |
| | + | so it will synthesize |
| | + | butyrate in order to attract varroa. |
| | + | </p> |
| | + | </div> |
| | | | |
| − | | + | |
| − | | + | |
| − | <h3>Test of butyrate production</h3>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <div class="group">
| + | |
| − | <p class="text">
| + | |
| − | In order to test butyrate production, we cultivated ApiColi in micro-aerobic condition, then we sampled supernatant that we filtrated for NMR analysis. All protocols are well described in “Protocols” part.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="group">
| + | |
| − | <p class="text">
| + | |
| − | We tested our genetic construction in BW25113 but we were unable to detect butyrate or a significant difference from our control for others products. So we tested to produce butyrate with a strain which is deleted for phosphate acetyltransferase, as it is explained in “Metabolic Engineering of Escherichia coli for Production of Butyric Acid” (1) and the figure below.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <center>
| + | |
| − | <div style="one-half;padding:10px;">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/4/45/TLSE_Results_Attract_5.PNG" style="width:55%;" />
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <p class="legend">
| + | |
| − | Figure 4: Exogenous acetate system. In red there are deleted genes. Reference (1)
| + | |
| − | </p>
| + | |
| − | </center>
| + | |
| − |
| + | |
| − | <div class="group">
| + | |
| − | <p class="text">
| + | |
| − | We did not use same enzymes for butyrate production, as we explained in “Attract” part, but this figure could be helpful to know which genes have to be deleted for butyrate production.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − |
| + | |
| − | <div class="group">
| + | |
| − | <p class="text">
| + | |
| − | Indeed, in the article they deleted three others genes to produce butyrate, but we could not deleted them because of lack of time so we tested with only pta deletion.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − |
| + | |
| − | <center>
| + | |
| − | <div style="one-half;padding:10px;">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/e/e9/TLSE_Results_Attract_6.PNG" style="width:70%;" />
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <p class="legend">
| + | |
| − | Figure 5: Test of butyrate production in E.coli strain deleted for pta gene. Culture in micro-aerobic condition in falcon, result after 28.5 hour culture.
| + | |
| − | </p>
| + | |
| − | </center>
| + | |
| − | | + | |
| − | <center>
| + | |
| − | <div style="one-half;padding:10px;">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/f/fd/TLSE_Results_Attract_7.PNG" style="width:70%;" />
| + | |
| − | </div>
| + | |
| − | </center>
| + | |
| − |
| + | |
| − |
| + | |
| − | <div class="group">
| + | |
| − | <p class="text">
| + | |
| − | Unfortunately we could not detect butyric acid on NMR analysis, but there are differences for others fermentation products. Our bacteria produced less formate and a little more actetate but the most diiference is that Ethanol is less produced. It would be possible that Acetyl-CoA is transformed in Acetoactyl-CoA thanks to enzyme we added. Then Acetyl-CoA would be less available to be transformed into ethanol. We could not detect these intermediate metabolites to confirm that hypothesis because they are intracellular. In a further experiment it would be useful to measure intracellular metabolite to see if you produced intermediate products.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − |
| + | |
| − | <div class="group">
| + | |
| − | <p class="text">
| + | |
| − | In any case, our genetic construction modified the fermentative balance. Moreover, it would be very interesting to test our genetic construction with a strain deleted for all genes indicated in figure 4.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − |
| + | |
| − |
| + | |
| − | <div class="title">
| + | |
| − | <h3>Eradicate</h3>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div class="subtitle">
| + | <img src="https://static.igem.org/mediawiki/2015/e/e6/TLSE_Attract_fig2.png"> |
| − | <h3>Tests on verroas</h3>
| + | <p> Figure 2: Results of butyrate attraction |
| − | </div>
| + | test with quadrants method |
| − |
| + | |
| − | | + | |
| − | <div class="group">
| + | |
| − | <p class="text"> | + | |
| − | In order to determine which concentrations of formic acid we have to produce we tested different concentrations of formic acid on varroas as we explained in “Protocol” part.
| + | |
| | </p> | | </p> |
| − | </div> | + | |
| − |
| + | </div> |
| − | <center>
| + | </div> |
| − | <div style="one-half;padding:10px;">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/e/e7/TLSE_Results_Eradicate_1.PNG" style="width:70%;" />
| + | |
| − | </div> | + | |
| | | | |
| − | <p class="legend"> | + | <div class="subtitle" > |
| − | Figure 6: Mortality of varroas as a function of time for different formic acid concentrations
| + | <h3>Butyrate attraction test</h3> |
| − | </p> | + | |
| − | </center> | + | |
| − |
| + | |
| − |
| + | |
| − | <center>
| + | |
| − | <div style="one-half;padding:10px;">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/d/d2/TLSE_Results_Eradicate_2.PNG" style="width:70%;" />
| + | |
| | </div> | | </div> |
| | | | |
| − | <p class="legend"> | + | |
| − | Figure 7: Histogram representing mortality of varroas after 2 hours and after 7 hours
| + | <div class="group center"> <!-- FIRST PARAGRAPH --> |
| − | </p> | + | |
| − | </center>
| + | <div class=" one_half first"> |
| − |
| + | |
| − |
| + | <img src="https://static.igem.org/mediawiki/2015/b/b8/TLSE_Attract_fig3.png"> |
| − | | + | <p>Figure 3: Butyrate attraction test using |
| − | <p class="text"> | + | T tube, with varroa mite in the middle |
| − | Thanks to figure 6, it is possible to see a dose-dependent between formic acid concentration and varroa mortality. So, with 10mM all varroas died before three hours but as we explain in “Protocols” part varroas stop moving for less concentrations. Figure 7 shows that even with 50µM around 30 % varroas died after 7 hours. Moreover, we would like to have a project which respects bee, so we would like to produce as little as possible formic acid. It is for this reason we set to produce at least 50µmol.L<sup>-1</sup> during the night (7hours).
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | <div class="subtitle">
| + | |
| − | <h3>Test of formate production</h3>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div class="group">
| |
| − | <p class="text">
| |
| − | For formate production we synthesized directly genes coding for pyruvate formate lyase and the activate protein so we could test formate production without cloning step, as it is explained in “Eradicate” part.
| |
| | </p> | | </p> |
| − | </div> | + | </div> |
| − | | + | |
| − | <center>
| + | <div class="one_half"> |
| − | <div style="one-half;padding:10px;"> | + | <p align="justify" style="font-size:15px;"> |
| − | <img src="https://static.igem.org/mediawiki/2015/5/5c/TLSE_Results_Eradicate_3.PNG" style="width:70%;" />
| + | To check adequacy and relevance of this study (Figure 2), |
| − | </div> | + | an experiment using a glass T-tube has been developed (Figure 3). |
| − | | + | In the first branch, there is a cotton soaked with 50 µL of water, |
| − | <p class="legend"> | + | in the second a cotton with 50 µL of butyrate at 4%, and finally the |
| − | Figure 8: Substrate and products concentration for formate production in a micro-aerobic culture
| + | last one contains the varroa. </ p> <div id=" part3"> <!-- ANCHOR 3 --> </div> <p align=" justify" style="font-size:15px;"> Butyrate being very volatile, our |
| | + | system |
| | + | used a pump to renew air, producing a concentration gradient as seen <a href="https://2015.igem.org/Team:Toulouse/Results#varrotest">here</a> |
| | </p> | | </p> |
| − | </center> | + | </div> |
| − | | + | |
| − | | + | |
| − | <center>
| + | |
| − | <div style="one-half;padding:10px;">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/7/7b/TLSE_Results_Eradicate_4.PNG" style="width:70%;" />
| + | |
| | </div> | | </div> |
| | | | |
| − | <p class="legend"> | + | <div class="subtitle" > |
| − | Figure 9: Summary of formate production test after 3 days cultivation in micro-aerobic condition
| + | <h3>How to produce butyrate with <i>E.coli</i>?</h3> |
| − | </p> | + | |
| − | </center> | + | |
| − |
| + | |
| − | <center>
| + | |
| − | <div style="one-half;padding:10px;"> | + | |
| − | <img src="https://static.igem.org/mediawiki/2015/3/3c/TLSE_Results_Eradicate_5.PNG" style="width:65%;" />
| + | |
| | </div> | | </div> |
| − | </center>
| |
| − |
| |
| − | <div class="group">
| |
| − | <p class="text">
| |
| − | Figure 8 shows that the only difference between ApiColi and the control is for formate production, so we plot the specific histogram for formate. Figure 9 indicates that formate production increased significantly. So we increased formate production by 10 %.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group">
| |
| − | <p class="text">
| |
| − | Our goal is to produce 50µM of formic acid in 7 hours that matches 77mM of formate. We produce around 25mM in 36 hours (Figure 8), namely around 5mM in 7 hours. So our production is in the same order of magnitude of our target production.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group">
| |
| − | <p class="text">
| |
| − | Thanks to our device considerations and all results summarized below, we show that it will be possible to reach our target production with optimization.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | <div class="title">
| |
| − | <h3>Device: physical tests</h3>
| |
| − | </div>
| |
| − |
| |
| − | <div class="subtitle">
| |
| − | <h3>TPX Bag</h3>
| |
| − | </div>
| |
| − |
| |
| − | <div class="subsubtitle">
| |
| − | <h3>Growth tests</h3>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | The second step was to know if bacteria can grow inside a small bag of TPX®. Thus, the strain <i>E. coli</i> BW 25113 has been inoculated in a small bag and disposed inside a biological sample tube which has been incubate at 37 °C. The monitoring of OD at 600 nm has been performed over 10 days.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <center>
| |
| − | <img src="https://static.igem.org/mediawiki/2015/2/21/TLSE_Results_Device_Growth1.jpg" style="width:30%;"/>
| |
| − | </center>
| |
| − | <div class="group center">
| |
| − | <p class="legend">
| |
| − | Figure 1: Growth test of <i>E. coli</i> BW 25113 inside a small bag of TPX® (Tubes n°1 and n°2). <br>
| |
| − | Both tubes contain a small bag of TPX® in which bacteria are growing (t = 17 hours, 37 °C).
| |
| − | </p></div>
| |
| − |
| |
| − | <center>
| |
| − | <img src="https://static.igem.org/mediawiki/2015/5/54/TLSE_Results_Device_Growth2.jpg" style="width:15%;"/>
| |
| − | </center>
| |
| − | <div class="group center">
| |
| − | <p class="legend">
| |
| − | Figure 2: Growth test of <i>E. coli</i> BW 25113 in a culture tube (Tube 3). <br>
| |
| − | The culture tube, contains bacteria which are growing (t = 17 hours, 37 °C, 130 rpm) in parallel of biological sample tubes shown above. It represents the control: <i>E. coli</i> BW 25113 are growing under aerobic condition with agitation.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <br>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | The graph below shows the monitoring of OD at 600 nm. <b>The strain <i>E. coli</i> BW 25113 is growing over 10 days (Tube n°1') in the TPX® bag </b>.
| |
| − | </p>
| |
| − | </div>
| |
| − | <center> <img src= "https://static.igem.org/mediawiki/2015/0/0f/Tlse_2015_Monitoring_of_E._coli_BW25113_%28Growth_culture_test_in_TPX%29.PNG"></center>
| |
| − | <div class="group center">
| |
| − | <p class="legend">
| |
| − | Figure 3: Growth test in TPX® bag by monitoring OD at 600 nm over 7 days (tubes n°1 and 2) over 10 days (tube n°1'). <br>
| |
| − |
| |
| − | </p></div>
| |
| | | | |
| | <div class="group center"> | | <div class="group center"> |
| − | | + | |
| − | The picture below represents the strain of <i>E. coli</i> BW25113 that grow, previously identified as Tubes n°1', n°1, n°2 and n°3 after 10 days of culture for tube 1' and 7 days for tubes 1, 2 and 3.
| + | <p align=" justify" style="font-size:15px;"> |
| | + | In this project, an <i>Escherichia coli</i> strain is used for its known |
| | + | simplicity of genetic manipulation and its adequacy with butyrate |
| | + | synthesis. Indeed, among the five enzymes of the butyrate pathway, |
| | + | two enzymes are naturally produced by the bacteria. The following |
| | + | engineered butyrate pathway has been designed: |
| | + | </p> <br> |
| | + | </div> |
| | | | |
| − | </p>
| + | |
| − | </div>
| + | <div class="group center"> < !-- CENTERED FIGURE --> |
| − | < center><img src="https://static.igem.org/mediawiki/2015/ 2/ 2c/ Tlse_2015_Petridish_Growth_test_in_TPX.png" style=" width:35%;"/></ center> | + | <img src="https://static.igem.org/mediawiki/2015/ 0/ 02/ TLSE_Attract_fig4.png" /> |
| − |
| + | </div> |
| | + | <div class="group center"> |
| | + | <figcaption>Figure 4: Engineered butyrate pathway</figcaption> |
| | + | |
| | + | </div> |
| | + | |
| | <div class="group center"> | | <div class="group center"> |
| − | | + | |
| − | Figure 4: Colonies of <i>E. coli</i> BW 25113 on Petri dishes after an overnight incubation at 37°C. <br>
| + | <p align=" justify" style="font-size:15px;"> |
| − |
| + | <br> |
| − | </p></div>
| + | The initial substrate is glucose which is decomposed into |
| | + | acetyl-CoA during glycolysis. Finally, butyrate pathway |
| | + | begin with acetyl-CoA: five genes are required with two |
| | + | homologous and three heterologous genes. |
| | + | </p> |
| | | | |
| − | <div class="group center"> | + | </div> |
| − | <p class="text">
| + | <br> |
| − | Bacteria are still alive after 10 days or 7 days depending on the tube while growing in TPX® bags. In all the culture tubes, the number of cells is about 4.10<sup>6</sup>. Thus, the strain can survive over 10 days in the TPX® bag.
| + | |
| | | | |
| − | </p>
| |
| − | </div>
| |
| | | | |
| − | <div class="subsubtitle"> | + | <div style="font-size:15px;"> |
| − | <h3>Gas diffusion tests</h3>
| + | <ul> |
| − | </div>
| + | <li><b><i>atoB</i></b> present in <i>E.coli</i>, coding for acetyl-CoA |
| − |
| + | acetyltransferase, an acetyltransferase catalyzing the combination |
| − | <div class="group center">
| + | of two acetyl-CoA. |
| − | <p class="text">
| + | |
| − | In order to know if our test that we described in “Protocol” test
| + | |
| − | functions, we made a control with acids solutions in a Falcon and
| + | |
| − | we sample gas in balance.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | For butyric acid we did not detect gas butyric acid by NMR,
| + | |
| − | the solution we used to solubilize gas should be not basic enough.
| + | |
| − | So we made a test with a TPX® bag containing butyric acid into a
| + | |
| − | solution of sodium bicarbonate. As a control we sampled directly a
| + | |
| − | solution of 4% (V/V) of butyric acid.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <center>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/a/a0/TLSE_Device_Physical_tests_image1.png" style="width:60%;"/>
| + | |
| − | </center>
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="legend">
| + | |
| − |
| + | |
| − | Figure 1: NMR Spectrum of butyric acid liquid control
| + | |
| − | in red and butyric acid liquid which passed through
| + | |
| − | TPX bag in blue. <sup>*</sup> Blue curve is zoomed 1340 times more
| + | |
| − | than red curve. Each condition was tested in two replicates.
| + | |
| − | </p></div>
| + | |
| − |
| + | |
| − |
| + | |
| − | <center>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/a/ab/TLSE_Device_Physical_tests_table1.PNG" style="width:60%;"/>
| + | |
| − | </center>
| + | |
| − | <div class="group center">
| + | |
| − | <p class="legend">
| + | |
| − |
| + | |
| − | Table 1: Concentrations of butyric acid corresponding to
| + | |
| − | NMR spectrum
| + | |
| − | </p></div>
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | Thanks to these results, TPX® allows butyric acid
| + | |
| − | to pass outside the bag. We detect only a small quantity but
| + | |
| − | an optimization of test could be made or a plastic with
| + | |
| − | bigger porous could be use.
| + | |
| − | <br><br>
| + | |
| − | For formic acid we were able to detect it
| + | |
| − | in gas, probably because its pka is lower than butyric acid.
| + | |
| − | </p> | + | |
| − | </div>
| + | |
| − | <center>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/8/8d/TLSE_Device_Physical_tests_image2.png" style="width:60%;"/>
| + | |
| − | </center>
| + | |
| − | | + | |
| − | <div class="group center">
| + | |
| − | <p class="legend">
| + | |
| − | Figure 2: NMR spectrum of formic acid gas control in red and formic
| + | |
| − | acid gas which passed through TPX bag in blue. At the left top
| + | |
| − | internal standard shows that it is the same scale for both curves.
| + | |
| − | Each condition was tested in two replicates.
| + | |
| − | </p></div>
| + | |
| − | <center>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/9/90/TLSE_Device_Physical_tests_table2.PNG" style="width:60%;"/>
| + | |
| − | </center>
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="legend">
| + | |
| − |
| + | |
| − | Table 2: Concentrations of formic acid corresponding to NMR spectrum
| + | |
| − | </p></div>
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | According to these results, TPX® allows 56% of formic acid
| + | |
| − | to pass outside the bag in gas phase. We show that formic acid can
| + | |
| − | go through TPX plastic. And with a better test, as we proposed
| + | |
| − | above this percentage could increase.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <div class="subsubtitle">
| + | |
| − | <h3>Safety tests</h3>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | Based on the protocol specified in <a href="https://2015.igem.org/Team:Toulouse/Experiments#sterilTPX1">here</a>, the bacteria’s impermeability of <b>TPX®</b>, has been tested inoculating the strain
| + | |
| − | E. coli BW 25113 in M9 defined medium. To summarize, the strain (in M9 medium) has been inoculated inside the small bag of <b>TPX®</b>. Then, the inoculated bag has
| + | |
| − | been immersed in a glass measuring cylinder containing M9 medium. The OD600 nm monitoring of the external medium has been performed. <br><br> | + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <center>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/0/00/TLSE_Results_Device_Safety.jpg" style="width:30%;"/>
| + | |
| − | </center>
| + | |
| − | <div class="group center">
| + | |
| − | <p class="legend">
| + | |
| − |
| + | |
| − | Figure 1: Measuring cylinders used for the safety test of the TPX® polymer. <br>
| + | |
| − | The first cylinder, on the right contains the small TPX® bag with E. coli BW 25113 after 27 hours of growth at 37 °C. On the right, the negative control cylinder contains a small bag of TPX® without bacteria and immersed in M9 medium after 27 hours of growth at 37 °C.
| + | |
| − | </p></div> | + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | Over this time, no bacteria went out of the bag, so the sterility has been conserved over 27 hours.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <div class="title">
| + | |
| − | <h3>Device: biological tests</h3>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | In the end, our objective is to have a bag which contains bacteria to produce alternately butyric acid
| + | |
| − | | + | |
| − | and formic acid during at least ten days in order to be practical for beekeeper.
| + | |
| | <br> | | <br> |
| − | So we faced with some biological questions as:
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <div class="one_quarter">
| |
| − | </div>
| |
| − | <div class="three_quarter">
| |
| − | <ul align="justify" style="font-size:15px;">
| |
| − | <li>Could bacteria live during ten days in micro-aerobic condition?<html></li>
| |
| − | <li>Which carbon source could we have to produce continuously acids?</li>
| |
| − | <li>Would acids be toxic for E.coli?</li>
| |
| − | </ul>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <div class="subtitle">
| |
| − | <h3>Characteristics of <i>E.coli</i> growth</h3>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | In order to know better the <i>E.coli</i> strain we would use for our project, we made a culture in aerobic
| |
| − |
| |
| − | and micro-aerobic conditions. We sampled OD and supernatant as it is explained <a target="_blank" href="https://2015.igem.org/Team:Toulouse/Experiments#erlencult">here</a> to
| |
| − |
| |
| − | see what happen in it.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | Micro-aerobic condition is obtained thanks to cultivation in specific falcons with holes recovered by a
| |
| − |
| |
| − | membrane into the plug which let pass oxygen without opening the falcon. They were incubated at 37 °C
| |
| − |
| |
| − | without agitation to best correspond to our real condition.
| |
| − |
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group">
| |
| − | <p class="text">
| |
| − | Aerobic condition is obtained with classic Erlenmeyer incubated at 37 °C with agitation.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | For the medium, we use a minimal medium M9 because we want to follow acids production by NMR. And
| |
| − | we choose a standard glucose concentration, 15mM.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="subsubtitle">
| |
| − | <h3>Biomass, substrate and products</h3>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | <!-- OD TO CONCENTRATION -->
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | In order to plot biomass concentration it is necessary to convert the OD measured. <br> This equation was used:
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p style="font-size:15px;">
| |
| − | $$ X=OD_{600nm}\times 0,4325 $$
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <p style="font-size:15px;">
| |
| − | Where X is the cell concentration (g.L<SUP>-1</SUP>)
| |
| − | </p>
| |
| − | <!-- OD TO CONCENTRATION -->
| |
| − |
| |
| − |
| |
| | <div class="group center"> | | <div class="group center"> |
| − | <p class="text">
| |
| − | For substrate and products concentration we plotted peak area of each molecule on NMR spectrum.
| |
| | <br> | | <br> |
| − | Then, we calculated concentration with this equation:
| + | <img src="https://static.igem.org/mediawiki/2015/c/c5/TLSE_Attract_fig5.png" /> |
| − | </p>
| + | </div> |
| − | </div>
| + | <div class="group center"> |
| | + | <br> |
| | + | <p class="legend">Figure 5: Reaction catalyzed by acetyl-CoA |
| | + | acetyltransferase </p> |
| | + | </div> |
| | | | |
| − | <!-- NMR TO CONCENTRATION -->
| + | </li> |
| − | <div class="group center">
| + | |
| − | <p style="font-size:15px;">
| + | |
| − | $$[A]=\frac{Area_{molecule}}{Area_TSP} \times [TSP] \times \frac{\textrm{TSP proton number}}{\textrm{A proton number}} \times DF $$
| + | |
| − | </p> | + | |
| − | </div>
| + | |
| − | <!-- NMR TO CONCENTRATION -->
| + | |
| | | | |
| − | <ul style="font-size:15px;">
| + | <li><b><i>hbd</i></b> present in <i>Clostridium acetobutylicum</i> coding for |
| − | <li>[A] = concentration of molecule in our solution in mM</li> | + | 3-hydroxybutyryl-CoA dehydrogenase, an oxidoreductase catalyzing |
| − | <li>Area<SUB>TSP</SUB> = 1</li> | + | the formation of an alcohol function. |
| − | <li>[TSP] = 1.075 mM <br>concentration of Trimethylsilyl propanoic acid in NMR tube, internal reference for
| + | <br> |
| − | | + | |
| − | quantification</li>
| + | |
| − | <li>TSP proton number = 9</li>
| + | |
| − | <li>DF = Dilution Factor = 1.25</li>
| + | |
| − | </ul>
| + | |
| − |
| + | |
| − |
| + | |
| − |
| + | |
| − | <center>
| + | |
| − | <p class="text">
| + | |
| − | Thanks to these calculations we were able to plot biomass, substrate and products depending on
| + | |
| − | time.
| + | |
| − | </p>
| + | |
| − | | + | |
| − |
| + | |
| − | | + | |
| − | <img src="https://static.igem.org/mediawiki/2015/0/08/TLSE_Devicebio_image1.PNG" style="width:60%;"/>
| + | |
| − | <p class="legend">Figure 1: Results of aerobic culture. Culture of BW25113 in M9 medium with [glucose] = 15 mM, in Erlenmeyer at 37 °C </p>
| + | |
| − |
| + | |
| − | | + | |
| − | <img src="https://static.igem.org/mediawiki/2015/2/2b/TLSE_Devicebio_image2.PNG" style="width:60%;"/>
| + | |
| − | <p class="legend">Figure 2: Results of micro-aerobic culture. Culture of <i> E. coli</i> BW25113 in M9 medium with [glucose] = 15 mM, in Falcon at 37 °C </p>
| + | |
| − | </center>
| + | |
| − | | + | |
| − | | + | |
| − |
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | Glucose is consumed approximately at the same rate for both conditions but it is not use for the same thing at all. In aerobic condition biomass reaches 3 g/L whereas in micro-aerobic condition there is six times less biomass. Inversely, there are far less products in aerobic conditions, and bacteria consume them when there is not glucose anymore, than in micro-aerobic condition.
| + | |
| − | | + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | For our objective to produce acids in a microporous bag, it is a really interesting results to have naturally bacteria which have slow growth and fermentation products.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | We can convert formate
| + | |
| − | concentration into formic
| + | |
| − | acid to know how much more
| + | |
| − | we will have to produce to
| + | |
| − | kill varroa. Indeed, the bacteria
| + | |
| − | produce a base but it is the acid that
| + | |
| − | interests us.
| + | |
| − | | + | |
| − | <br>The formula below
| + | |
| − | is used:
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − |
| + | |
| − | <!-- pH equation -->
| + | |
| − | <div class="group center"> | + | |
| − | <p style="font-size:15px;">
| + | |
| − | $$ pH=pKa+log \left(\frac{C_{b}}{C_{a}} \right) $$
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <!-- pH equation -->
| + | |
| − | | + | |
| − | <ul style="font-size:15px;">
| + | |
| − | <li>pH: medium used is buffered with a low concentration in acid. pH = 7.
| + | |
| − | </li>
| + | |
| − | <li>pKa: 3.7 for formic acid and 4.81 for butyric acid</li>
| + | |
| − | <li>C<SUB>b</SUB>: base concentration</li>
| + | |
| − | <li>C<SUB>a</SUB>: acid concentration</li>
| + | |
| − | </ul>
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | As it is said in the “Eradicate” part, our goal is to produce 50 µM of formic acid to kill varroa, thanks to the equation (3) we know it corresponds
| + | |
| − | to 77,7 mM of formate.
| + | |
| − | </p> | + | |
| − | </div>
| + | |
| − |
| + | |
| | <div class="group center"> | | <div class="group center"> |
| − | <p class="text">
| + | <br> |
| − | At the maximum the bacteria
| + | <img src="https://static.igem.org/mediawiki/2015/d/d6/TLSE_Attract_fig6.png" /> |
| − | produces 32mmol/L of formate.
| + | </div> |
| − | It is necessary to add genes
| + | <div class="group center"> |
| − | involved in formate production
| + | <br> |
| − | to regulate production and
| + | <p class="legend">Figure 6: Reaction catalyzed by |
| − | multiply it by 2.4. For a perfect
| + | 3-hydroxybutyryl-CoA dehydrogenase |
| − | regulation it would be necessary to
| + | |
| − | delete pfl-B in <i>E.coli</i> genome not to
| + | |
| − | have formate production during the day.
| + | |
| | </p> | | </p> |
| − | </div>
| |
| − |
| |
| − | <div class="subsubtitle">
| |
| − | <h3>Bacteria survival</h3>
| |
| | </div> | | </div> |
| − |
| + | </li> |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | As it is explained <a target="_blank" href="https://2015.igem.org/Team:Toulouse/Experiments#platecult">here</a> we plated bacteria on Petri dish to know if they were alive or not because OD measure cannot discriminate alive bacteria from dead. This test show us that wild type bacteria can easily survive during at least 15 days. So if we bring them a carbon source during this period they should survive even better.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/5/56/TLSE_Devicebio_image3.PNG" style="width:60%;"/></center>
| + | |
| − | <div class="group center">
| + | |
| − | <p class="legend">Figure 3: Bacteria survival results from culture
| + | |
| − | test with BW25113 on M9 with 15mM of glucose during 15 days to mime
| + | |
| − | real
| + | |
| − | survival condition. </p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | <!---partie bio2--->
| + | |
| − | | + | |
| − | <div class="subtitle">
| + | |
| − | <h3>Choice of carbon source to produce acids during 10 days<h3>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="subsubtitle">
| + | |
| − | <h3>Characteristics of Biosilta kit</h3>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | En Presso B is a technology which permits to produce a lot of
| + | |
| − | recombinant proteins thanks to a low substrate delivering during
| + | |
| − | 24 hours. This technology is based on polymer degradation by an enzyme
| + | |
| − | which permits to have the right quantity of substrate at each moment.
| + | |
| − | We would like to use this technology to cultivate our cells during one
| + | |
| − | or two weeks in good conditions in order to produce butyrate and formate.
| + | |
| − | The medium with the polymer is solid and contained in separate bags.
| + | |
| − | To know which quantity of butyrate and formate we can produce, we have
| + | |
| − | to know the quantity of substrate we could obtain with the polymer so
| + | |
| − | we made a kinetic test with a high enzyme concentration (50 U/L).
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <center>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/8/8c/TLSE_Devicebio_image4.PNG" style="width:60%;"/>
| + | |
| − | </center> | + | |
| | | | |
| | + | <li><b><i>crt</i></b> present in <i>C.acetobutylicum</i> |
| | + | coding for 3-hydroxybutyryl-CoA dehydratase, |
| | + | a lyase cleaving carbon-oxygen bond. |
| | + | <br> |
| | <div class="group center"> | | <div class="group center"> |
| − | <p class="legend">
| |
| − | Figure 4: Kinetic test of enzyme which degrades polymer from Biosilta
| |
| − | kit. [Enzyme] = 50 U/L in order to have a complete degradation of
| |
| − | polymer.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group">
| |
| − | <p class="text">
| |
| − | In order to have a global idea of the rate
| |
| − | of glucose releasing we calculate an average speed.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p style="font-size:15px;">
| |
| − | $$ v_{glucose1}=\frac{[glucose]}{time}=\frac{11.1}{3.97}=2.80 g.L^{-1}.h^{-1}
| |
| − | , for [E]_{1}=50 U.L^{-1} (4) $$
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group">
| |
| − | <p class="text">
| |
| − | With a final glucose concentration of 13 g/L for one bag of polymer,
| |
| − | a rate of glucose releasing can be calculate in order to have glucose
| |
| − | during 13 days.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p style="font-size:15px;">
| |
| − | $$ v_{glucose2}=\frac{13}{13 days}=\frac{13}{322 hours}=0.0403
| |
| − | g.L^{-1}.h^{-1} (5) $$
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group">
| |
| − | <p class="text">
| |
| − | The reduction factor is calculated:
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p style="font-size:15px;">
| |
| − | $$ RF=\frac{v_{glucose1}}{v_{glucose2}}=\frac{2.8}{0.0403}=69.44 (6)$$
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group">
| |
| − | <p class="text">
| |
| − | So, the concentration of enzyme that we have to use is:
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p style="font-size:15px;">
| |
| − | $$ [E]_{2}=\frac{[E]_{1}}{RF}=\frac{50}{69.44}=0.72 U.L^{-1} (7)$$
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="subsubtitle">
| |
| − | <h3>Growth culture with Biosilta kit<h3>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | As we do not know any growth characteristic with Biosilta
| |
| − | medium we tested different enzyme concentrations and not
| |
| − | only the one which allow growth during 13 days. We made acquisition in two times because of software constraints,
| |
| − | this is why there is a break at 5 days:
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <center>
| |
| − | <img src="https://static.igem.org/mediawiki/2015/3/37/TLSE_Devicebio_image5.PNG"
| |
| − | style="width:60%;"/>
| |
| − | <p class="legend">
| |
| − | Figure 5: Bacteria growth as a function of different enzyme
| |
| − | concentrations in Biosilta medium. Test was made in 48 wells
| |
| − | plate with OD reader.
| |
| − | </p>
| |
| − | </center>
| |
| − |
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | Except for 1.5 U/L enzyme, OD increase during 12 days so glucose
| |
| − | releasing seems to function well. At the beginning there is
| |
| − | exponential growth because some glucose is directly available on
| |
| − | medium. Since 2 days until the end growth is linear, only the slope
| |
| − | change. It is higher between 2 and 4 days than after probably because
| |
| − | bacteria were in worse condition after few days so they are not able
| |
| − | anymore to consume all glucose available.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | Thanks to those results we know it is possible to have continuous
| |
| − | growth during at least 12 days. The only problem is that our control,
| |
| − | without enzyme, grows also so either another substrate is available or
| |
| − | bacteria could able to degrade polymer that could be a problem.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | In order to answer these questions we did culture in falcon in order
| |
| − | to analyze products and to see evolution of polymer quantity. We made
| |
| − | culture without enzyme and we test concentration of enzyme of 0.72 U/L
| |
| − | because it this one which allows to reach the highest OD in figure 5 and
| |
| − | it is the one we calculated above to have glucose releasing during 13
| |
| − | days.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <center> <img src="https://static.igem.org/mediawiki/2015/2/29/TLSE_Devicebio_image6.PNG"
| |
| − | style="width:60%;"/>
| |
| − |
| |
| − | <p class="legend">
| |
| − | Figure 6: Results of BW25113 culture on Biosilta medium without enzyme.
| |
| − | </p>
| |
| − | </center>
| |
| − |
| |
| − | <center> <img src="https://static.igem.org/mediawiki/2015/c/cf/TLSE_Devicebio_image7.PNG "
| |
| − | style="width:60%;"/>
| |
| − |
| |
| − | <p class="legend">
| |
| − | Figure 7: Results of BW25113 culture on Biosilta medium with [Enzyme] = 0.72 U/L.
| |
| − | </p>
| |
| − | </center>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | Figure 6 shows that polymer is not degraded, so it is only enzyme of Biosilta kit which releases glucose. Enzyme concentration could be correlate to glucose rate releasing for further modelling. So bacteria find another carbon source in Biosilta medium but we were not able to determine which one. Products concentrations are nearly identical to those in M9 medium.
| |
| − | <br><br>
| |
| − |
| |
| − | In figure 7, polymer area decreases, so enzyme degrades it well. At the beginning, glucose concentration is almost constant so bacteria consumed it directly when enzyme releases it. At the end glucose concentration increases a bit, bacteria either did not consume it as fast as the beginning or they consumed formate because its concentration decreases. This could be a problem for us because we look for produce more formate so we would have to think about it.
| |
| − | <br><br>
| |
| − | Fermentation products have high concentrations in comparison to culture in M9 with 15mM of glucose, around 20 times more for lactate, 3 times more for acetate and 2 times more for ethanol. So if we delete production ways for lactate, acetate and ethanol and degradation way of formate we would able to produce enough formate and butyrate.
| |
| − | <br><br>
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="subsubtitle">
| |
| − | <h3>Acids production modelling</h3>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | With the rate of glucose calculated above, an FBA and FVA simulation were launched as explained in “Modelling” part. Some conversion between the model and the real condition are necessary and they are explain in “Modelling” part.
| |
| | <br> | | <br> |
| | + | <img src="https://static.igem.org/mediawiki/2015/6/67/TLSE_Attract_fig7.png" /> |
| | + | </div> |
| | + | <div class="group center"> |
| | <br> | | <br> |
| − | In order to model production in the most similar conditions to fit real experiment we chose a glucose rate of 0.0403 g.L-1.h-1 that correspond to 0.72 U/L of enzyme.
| + | <p class="legend">Figure 7: |
| − | <br> | + | Reaction catalyzed by 3-hydroxybutyryl-CoA deshydratase |
| − | <br>
| + | |
| − | To convert formate production into formic acid concentration we use equation (3).
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | | | |
| − | <center> <img src="https://static.igem.org/mediawiki/2015/7/7a/TLSE_Devicebio_image8.PNG "
| |
| − | style="width:50%;"/>
| |
| − |
| |
| − | <p class="legend">
| |
| − | Figure 8: Modelling of formic acid production as a function of different growth rates for a glucose rate of 0.0403 g.L<sup>-1</sup>.h<sup>-1</sup>.
| |
| − | </p>
| |
| − | </center>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | Our goal is to produce at least 50µmol/L and with this graph the maximum production could be 6µmol/L. So we have to produce nearly 10 times more formic acid. In order to reach our goal we could see which rate of glucose we needed with a reverse thought.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <center> <img src="https://static.igem.org/mediawiki/2015/3/33/TLSE_Devicebio_image9.PNG "
| |
| − | style="width:60%;"/>
| |
| − |
| |
| − | <p class="legend">
| |
| − | Figure 9 : Modelling of formic acid production as a function of glucose rate for different growth rates (in h<sup>-1</sup>).
| |
| − | </p>
| |
| − | </center>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | To produce 50 µmol/L of formic acid different strategies are available. Either a low growth rate is chosen so a low glucose rate would be necessary or a high growth rate is chosen and a high glucose rate would be necessary. As bacteria have to live during at least ten days it will be better to have a continuous slow growth rate. Moreover, it will consume less glucose per hour so we would need a lower polymer concentration in our bag at the beginning. So, we chose a growth rate of 0.2h-1, and we can determine rate of glucose needed.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p style="font-size:15px;">
| |
| − | [Formic acid] (μmol.L<sup>-1</sup> )=166.88 ×[Glucose] (g.L<sup>-1</sup>.h<sup>-1</sup> ) (9)
| |
| − |
| |
| − | $$ [Glucose] = \frac{50}{166.88}=0.3 g.L^{-1}.h^{-1} (10) $$
| |
| | </p> | | </p> |
| − | </div>
| |
| − |
| |
| − | <div class="group">
| |
| − | <p class="text">
| |
| − | Now, we will see which butyric acid concentration we could produce.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <center> <img src="https://static.igem.org/mediawiki/2015/7/7c/TLSE_Devicebio_image10.PNG "
| |
| − | style="width:60%;"/>
| |
| − |
| |
| − | <p class="legend">
| |
| − | Figure 10: Modelling of butyric acid production as a function of glucose rate for different growth rates.
| |
| − | </p>
| |
| − | </center>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | According to modelling results in figure 10, we would be able to produce around 100µmol/L of butyric acid that correspond to 0.0092% (V/V). As we explained in “Results” part, <a target="_blank" href="https://2015.igem.org/Team:Toulouse/Description/Attract">"attract"</a> section, our objective is to produce at least 0.00001%, so with modelling we reach it.
| |
| − | <br><br>
| |
| − | Nevertheless, in order to have this right glucose rate it is necessary to calculate how much polymer is needed at the beginning and which enzyme concentration.
| |
| − | <br><br>
| |
| − | With the same equations as we used in “Characteristics of Biosilta kit” we can determine which quantity of glucose is needed in total during a fortnight.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p style="font-size:15px;">
| |
| − | $$[Glucose]=v_{glucose}\times time=0.3\times 322=96.6 g.L^{-1} (11)$$
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | Knowing that one Biosilta kit contains the equivalence of 13 g/L of glucose, we have to concentrate the medium 7 times.
| |
| − |
| |
| − | Concerning the glucose rate, the 0.3 g.L-1.h-1 value correspond to 5 U/L of enzyme.
| |
| − |
| |
| − | Thus, we tested different concentrations of Biosilta medium with different enzyme concentrations.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="subsubtitle">
| |
| − | <h3>Testing different concentrations of Biosilta kit<h3>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | As we do not know the exact composition of Biosilta medium,
| |
| − | we are not able to say if there is a molecule which could be
| |
| − | toxic at high concentrations. We could only have a global analysis
| |
| − | on our results :
| |
| − |
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <center> <img src="https://static.igem.org/mediawiki/2015/a/a3/TLSE_Devicebio_image11.PNG "
| |
| − | style="width:60%;"/>
| |
| − |
| |
| − | <p class="legend">
| |
| − | Figure 11: Bacteria growth as a function of time on Biosilta medium concentrated 6 times. Culture with BW25113 on 48 wells plate and optical reader
| |
| − | </p>
| |
| − | </center>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | There is hardly any growth during three first days, bacteria probably adapt themselves to the medium. Since 3 days until the end, OD increases up to 1 for 1.5 U/L enzyme but it is still slow. Moreover, bacteria grow better with 0.72 and 1.5 U/L than with 3 or 4 U/L. It could be explained by an excess of glucose that inhibits bacteria growth. Indeed, enzyme could release too much glucose that bacteria would not consume as fast, then glucose accumulated itself in medium. We tested with a less concentrated medium in order to see if latency period could be reduce.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <center> <img src="https://static.igem.org/mediawiki/2015/b/b6/TLSE_Devicebio_image12.PNG "
| |
| − | style="width:60%;"/>
| |
| − |
| |
| − | <p class="legend">
| |
| − | Figure 12: Bacteria growth as a function of time on Biosilta medium concentrated 4 times. Culture with BW25113 on 48 wells plate and optical reader
| |
| − | </p>
| |
| − | </center>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class ="text">
| |
| − | With a 4 times concentrated medium, there is no latency period anymore but enzyme concentration seem not to affect bacteria growth. Bacteria probably consume all free glucose in medium and then enzyme does not have enough time to degrade polymer. A longer period test would be necessary to know if bacteria were able to consume glucose as fast as enzyme released it. Maybe by testing a twice concentrated medium, we would be able to answer it.
| |
| − | </p>
| |
| | </div> | | </div> |
| − |
| + | </li> |
| − | <center> <img src="https://static.igem.org/mediawiki/2015/5/50/TLSE_Devicebio_image13.PNG "
| + | |
| − | style="width:60%;"/>
| + | |
| | | | |
| − | <p class="legend"> | + | <li><b><i>ccr</i></b> present in |
| − | Figure 13: Bacteria growth as a function of time on Biosilta medium concentrated twice. Culture with BW25113 on 48 wells plate and optical reader
| + | <i>Streptomyces collinus</i> coding |
| − | </p>
| + | for crotonyl-CoA reductase, |
| − | </center>
| + | an oxidoreductase acting on |
| − |
| + | CH=CH double bond. This enzyme |
| − | <div class="group center">
| + | is also in <i>C.acetobutylicum</i> with |
| − | <p class="text">
| + | <b>bcd</b> gene coding for butyryl-CoA dehydrogenase, |
| − | Decline of curve for 0.72U/L was not expected because in “normal” Biosilta medium, figure 5, bacteria grew during 12 days. We cannot explain this result, but it shows that it is complicated to work with a medium with an unknown composition.
| + | with the disadvantage |
| | + | to run with Electron Transfer |
| | + | Flavoprotein (ETF) which complicates the reaction [6]. |
| | <br> | | <br> |
| | + | <div class="group center"> |
| | <br> | | <br> |
| − | Curves for 3 and 4 U/L enzyme or very similar so it seems that bacteria are not able to consume all glucose released by enzyme. As we only measured OD we do not know if bacteria would assimilate glucose for another way that growth metabolism.
| + | <img src="https://static.igem.org/mediawiki/2015/5/57/TLSE_Attract_fig8.png" /> |
| | + | </div> |
| | + | <br> |
| | + | <div class="group center"> |
| | <br> | | <br> |
| − | <br> | + | <p class="legend">Figure 8: Reaction |
| − | Thanks to figure 11, 12 and 13 we know that it would not be possible to have enough polymer in our medium. As a solution we think to use a dialysis system: in one side there will be bacteria and in the other side there will be the polymer with enzyme. Membrane which separates them will allow only small molecules to pass like glucose. Thanks to this system our device will have enough substrate for two weeks.
| + | catalyzed by crotonyl-CoA reductase |
| − | <br>
| + | |
| − | <br>Regarding the rate of glucose assimilation we could do additional tests where we would measure glucose in medium to determine maximum rate of assimilation. An optimization of this assimilation could be essential.
| + | |
| | </p> | | </p> |
| − | </div> | + | </div> |
| − | | + | |
| − | <div class="subtitle">
| + | |
| − | <h3>Testing acids toxicity<h3>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="subsubtitle">
| + | |
| − | <h3>Effects of medium</h3>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | In order to optimize resistance of BW25113 to different acids concentrations we tested two medium: LB and M9 with 15mM of glucose.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <center> <img src="https://static.igem.org/mediawiki/2015/0/0d/TLSE_Devicebio_image14.PNG "
| + | |
| − | style="width:60%;"/>
| + | |
| | | | |
| − | <p class="legend"> | + | </li> |
| − | Figure 14: Optic density in function of time for different formic acid concentrations and two medium. LB medium is represented with green curves and M9 medium with blue curves. Each condition is tested in three replicates so standard deviation is represented in orange.
| + | |
| − | </p>
| + | |
| − |
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/a/ab/TLSE_Devicebio_image15.PNG "
| + | |
| − | style="width:60%;"/>
| + | |
| | | | |
| − | <p class="legend">
| + | <li><b><i>tesB</i></b> present in <i>E.coli</i> |
| − | Figure 15: Optic density in function of time for different butyric acid concentrations and two medium. LB medium is represented with green curves and M9 medium with blue curves. Each condition is tested in three replicates so standard deviation is represented in orange.
| + | coding for acyl-CoA transferase 2, |
| − | </p>
| + | a thiolase which enables coenzyme A transfer. |
| − | </center>
| + | <br> |
| − | | + | <div class="group center"> |
| − | <div class="group center"> | + | |
| − | <p class="text">
| + | |
| − | In M9 medium, growth is slower at the beginning in both figures but OD max is almost the same for both medium.
| + | |
| | <br> | | <br> |
| | + | <img src="https://static.igem.org/mediawiki/2015/3/34/TLSE_Attract_fig9.png" /> |
| | + | </div> |
| | + | <div class="group center"> |
| | <br> | | <br> |
| − | For formic acid, the only significant difference is for 10mM with a slower growth in LB than in M9. For butyric acid the difference is stronger because in LB bacteria do not grow anymore with 109mM whereas in M9 there is growth.
| + | <p class="legend">Figure 9: Reaction |
| − | <br>
| + | catalyzed by acyl-CoA transferase 2 |
| − | <br> | + | |
| − | In fact, M9 is buffered and not LB so we measured pH in both medium with different acids concentrations in order to see if there were a correlation.
| + | |
| | </p> | | </p> |
| − | </div>
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| − |
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| − |
| |
| − | <center>
| |
| − | <img src="https://static.igem.org/mediawiki/2015/b/b2/TLSE_Devicebio_image16.PNG "
| |
| − | style="width:60%;"/>
| |
| − |
| |
| − | <p class="legend">
| |
| − | Figure 16: pH in function of concentration in mM for formic acid and butyric acid. LB medium is represented with green curves and M9 medium with blue curves. pH was measured with pH paper because only order of magnitude interested us. Each condition was tested three times and give us the exactly the same results.
| |
| − | </p>
| |
| − | </center>
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | It is clear that in M9 medium pH stay at pH 7 for higher acids concentrations than LB medium. Moreover, thanks to previous figures, it is possible to see that bacteria do not grow anymore when pH is around 5. This results show that bacteria are sensitive to acid pH, but they may resist to higher acids concentrations if the medium was better buffered.
| |
| − | We will now see if it would be interesting or not to buffer better our medium.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="subtitle">
| |
| − | <h3>Formic acid toxicity</h3>
| |
| | </div> | | </div> |
| − |
| |
| − | <center>
| |
| − | <img src="https://static.igem.org/mediawiki/2015/c/c6/TLSE_Devicebio_image17.PNG "
| |
| − | style="width:60%;"/>
| |
| | | | |
| − | <p class="legend"> | + | </li> |
| − | Figure 17: Toxicity test of formic acid, OD of BW on M9 15mM glucose.
| + | |
| − | </p>
| + | |
| − | </center>
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | Figure 17 shows a dose/response relationship between formic acid concentration and bacteria growth. We would like to produce at least 50µmol/L of formic acid in order to kill varroas and bacteria growth normally up to 1mM. So we should not have toxicity problem during the treatment.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <div class="subtitle">
| + | |
| − | <h3>Butyric acid toxicity</h3>
| + | |
| − | </div>
| + | |
| − | <center>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/5/50/TLSE_Devicebio_image18.PNG "
| + | |
| − | style="width:60%;"/>
| + | |
| | | | |
| − | <p class="legend">
| + | </ul> |
| − | Figure 18: Toxicity test of butyric acid, OD of BW on M9 15mM glucose.
| + | |
| − | </p>
| + | |
| − | </center>
| + | |
| − |
| + | |
| − | <div class="group center">
| + | |
| − | <p class="text">
| + | |
| − | The higher is butyric concentration, the less is bacteria growth, as our previous results with formic acid. However we have an intermediate result with 109mM of butyric acid. We do not have a specific butyric acid concentration to produce and modelling show us that we could produce around 15mM with all optimizations so we would not have any butyric acid toxicity during our treatment.
| + | |
| − | <br><br>
| + | |
| − | <i>Nota Bene</i>: For our conclusions about acids toxicity, we consider that acids evaporate during day for formic acid and night for butyric acid, so there would not be a lot of acid accumulation.
| + | |
| − | | + | |
| − | </p>
| + | |
| | </div> | | </div> |
| | + | <div class="group center"> |
| | + | <p align="justify" style="font-size:15px;">Concerning heterologous genes (hbd, crt and ccr), a codon optimization has been performed in order to enable a good expression of these genes in <i>E. coli</i>. |
| | + | The genetic construction is then done by assembling the five genes presented earlier, which are placed under the control of P(Bla) constitutive promoter (BBa_I14018). In between the genes are placed ribosome binding sites (RBS) (BBa_B0030) to improve protein expression, and a strong terminator (BBa_B1006) is used to end this construction, which is to be cloned into a pSB1C3 vector (<a href="https://static.igem.org/mediawiki/2015/9/9e/PSB1C3_ccr_butyrate.xdna.png">here</a>).</p> |
| | + | </div> |
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| − | | + | <center><img src="https://static.igem.org/mediawiki/2015/2/20/TLSE_Attract_fig10.png" style="width:65%;"/></center> |
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| | + | [1] Boecking O, Genersch E. 2008. Varroosis – the Ongoing Crisis in Bee Keeping. J. Verbr. Lebensm. 3:221–228.</li> |
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| | <li> | | <li> |
| − | [1] REFERENCE 1 Mukesh Saini, Zei Wen Wang, Chung-Jen Chiang, and Yun-Peng Chao, Metabolic Engineering of Escherichia coli for Production of Butyric Acid | + | [2] Le Conte Y, Arnold G, Trouiller J, Masson C, Chappe B, Ourisson G. 1989. Attraction of the parasitic mite varroa to the drone larvae of honey bees by simple aliphatic esters. Science 245:638–639.</li> |
| − | </li> | + | |
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| | <li> | | <li> |
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| + | [3] Methods for attracting honey bee parasitic mites. [accessed 2015 Jul 24]. |
| − | [2] REFERENCE 2 AVEC UN LIEN <a href="http://www.google.com/patents/US8647615">See more</a> | + | |
| | </li> | | </li> |
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| − | [3] REFERENCE 2 AVEC UN LIEN qui ouvre dans une nouvelle fenêtre <a href="http://www.google.com/patents/US8647615">See more</a> | + | [4] Louis P, Flint HJ. 2009. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol. Lett. 294:1–8. |
| − | </a> | + | </li> |
| | + | [5] Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T, Liao JC. 2008. Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic Engineering 10:305–311. |
| | + | <li> |
| | + | [6] Wallace KK, Bao Z-Y, Dai H, Digate R, Schuler G, Speedie MK, Reynolds KA. 1995. Purification of Crotonyl-CoA Reductase from Streptomyces collinus and Cloning, Sequencing and Expression of the Corresponding Gene in Escherichia coli. European Journal of Biochemistry 233:954–962. |
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