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| | <!-- ################################ ICI CONTENEUR OU TU PEUX T'AMUSER A METTRE LES DIVS ############################### --> | | <!-- ################################ ICI CONTENEUR OU TU PEUX T'AMUSER A METTRE LES DIVS ############################### --> |
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| | + | |
| | | | |
| | <div class="title"> | | <div class="title"> |
| − | <h3>Device : physical tests</h3>
| |
| − | </div>
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| − |
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| − | <div class="subtitle">
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| | <h3>Trap Construction</h3> | | <h3>Trap Construction</h3> |
| | </div> | | </div> |
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| | </div> | | </div> |
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| − | <div class="subtitle"> | + | <div class="title"> |
| | <h3>TPX Bag</h3> | | <h3>TPX Bag</h3> |
| | </div> | | </div> |
| | | | |
| − | <div class="subsubtitle"> | + | <div class="subtitle"> |
| | <h3>Safety tests</h3> | | <h3>Safety tests</h3> |
| | </div> | | </div> |
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| | </p> | | </p> |
| | </div> | | </div> |
| − |
| |
| − | <div class="subsubtitle">
| |
| − | <h3>Gas diffusion tests</h3>
| |
| − | </div>
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| − |
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| − | <div class="group center">
| |
| − | <p class="text">
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| − | 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>
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| − | </div>
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| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
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| − | For butyric acid we did not detect gas butyric acid by NMR,
| |
| − | the solution we used to solubilize gas should be not basic enough.
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| − | 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
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| − | solution of 4% (V/V) of butyric acid.
| |
| − | </p>
| |
| − | </div>
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| − |
| |
| − | <center>
| |
| − | <img src="https://static.igem.org/mediawiki/2015/a/a0/TLSE_Device_Physical_tests_image1.png" style="width:60%;"/>
| |
| − | </center>
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| − |
| |
| − | <div class="group center">
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| − | <p class="legend">
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| − |
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| − | Figure 1: NMR Spectrum of butyric acid liquid control
| |
| − | in red and butyric acid liquid which passed through
| |
| − | TPX bag in blue. Red curve is zoomed 1340 times more
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| − | than blue curve. Each condition was tested in two replicates.
| |
| − | </p></div>
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| − |
| |
| − |
| |
| − | <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">
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| − |
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| − | Table 1: Concentrations of butyric acid corresponding to
| |
| − | NMR spectrum
| |
| − | </p></div>
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| − |
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | Thanks to these results, TPX® overlook butyric acid
| |
| − | 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>
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| − | For formic acid we were able to detect it
| |
| − | in gas, probably because its pka is lower than butyric acid.
| |
| − | </p>
| |
| − | </div>
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| − |
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| − |
| |
| − |
| |
| − | <center>
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| − | <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">
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| − |
| |
| − | 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.
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| − | Each condition was tested in two replicates.
| |
| − | </p></div>
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| − |
| |
| − |
| |
| − |
| |
| − | <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">
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| − |
| |
| − | Table 2: Concentrations of formic acid corresponding to NMR spectrum
| |
| − | </p></div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
| |
| − | According to these results, TPX® overlook 56 % of formic acid
| |
| − | 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>
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| − |
| |
| − |
| |
| − | <div class="title">
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| − | <h3>Device: biological tests</h3>
| |
| − | </div>
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| − |
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| − | <div class="group center">
| |
| − | <p class="text">
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| − | In the end, our objective is to have a bag which contains bacteria to produce alternately butyric acid
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| − |
| |
| − | and formic acid during at least ten days in order to be practical for beekeeper.
| |
| − | <br>
| |
| − | So we faced with some biological questions as:
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="group center">
| |
| − | <div class="one_quarter">
| |
| − | </div>
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| − | <div class="three_quarter">
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| − | <ul align="justify" style="font-size:15px;">
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| − | <li>Could bacteria live during ten days in micro-aerobic condition?<html></li>
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| − | <li>Which carbon source could we have to produce continuously acids?</li>
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| − | <li>Would acids be toxic for E.coli?</li>
| |
| − | </ul>
| |
| − | </div>
| |
| − | </div>
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| − |
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| − |
| |
| − | <div class="subtitle">
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| − | <h3>Characteristics of <i>E.coli</i> growth</h3>
| |
| − | </div>
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| − |
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| − | <div class="group center">
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| − | <p class="text">
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| − | In order to know better the <i>E.coli</i> strain we would use for our project, we made a culture in aerobic
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| − |
| |
| − | 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
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| − |
| |
| − | see what happen in it.
| |
| − | </p>
| |
| − | </div>
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| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
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| − | Micro-aerobic condition is obtained thanks to cultivation in specific falcons with holes recovered by a
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| − |
| |
| − | membrane into the plug which let pass oxygen without opening the falcon. They were incubated at 37 °C
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| − |
| |
| − | without agitation to best correspond to our real condition.
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| − |
| |
| − | </p>
| |
| − | </div>
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| − |
| |
| − | <div class="group">
| |
| − | <p class="text">
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| − | Aerobic condition is obtained with classic Erlenmeyer incubated at 37 °C with agitation.
| |
| − | </p>
| |
| − | </div>
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| − |
| |
| − | <div class="group center">
| |
| − | <p class="text">
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| − | 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>
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| − |
| |
| − | <div class="subsubtitle">
| |
| − | <h3>Biomass, substrate and products</h3>
| |
| − | </div>
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| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | <!-- 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">
| |
| − | <p class="text">
| |
| − | For substrate and products concentration we plotted peak area of each molecule on NMR spectrum.
| |
| − | <br>
| |
| − | Then, we calculated concentration with this equation:
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <!-- NMR TO CONCENTRATION -->
| |
| − | <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>[A] = concentration of molecule in our solution in mM</li>
| |
| − | <li>Area<SUB>TSP</SUB> = 1</li>
| |
| − | <li>[TSP] = 1.075 mM <br>concentration of Trimethylsilyl propanoic acid in NMR tube, internal reference for
| |
| − |
| |
| − | 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">
| |
| − | <p class="text">
| |
| − | At the maximum the bacteria
| |
| − | produces 32mmol/L of formate.
| |
| − | It is necessary to add genes
| |
| − | involved in formate production
| |
| − | to regulate production and
| |
| − | multiply it by 2.4. For a perfect
| |
| − | regulation it would be necessary to
| |
| − | delete pfl-B in <i>E.coli</i> genome not to
| |
| − | have formate production during the day.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="subsubtitle">
| |
| − | <h3>Bacteria survival</h3>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <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 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>
| |
| − |
| |
| − | <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 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>
| |
| | | | |
| | </main> | | </main> |
