http://2014.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Project/TMAU&feed=atom&action=historyTeam:Paris Bettencourt/Project/TMAU - Revision history2024-03-28T10:20:57ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Project/TMAU&diff=399066&oldid=prevMarguerite at 03:40, 18 October 20142014-10-18T03:40:22Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A simple model was created for the diffusion of trimethylamine into the air using COMSOL Multiphysics (a physics-based interface to solve partial differential equations). The model is shown in Fig.6. From literature, it was found that the odor threshold of trimethylamine in air is approximately 3.2E-5 ppm air (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy"> Nagata, 1993</a>), using the triangle odor bag method. This odor threshold was reached approximately 10 hours in the model, at a distance of 5 cm from the skin surface.</p></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A simple model was created for the diffusion of trimethylamine into the air using COMSOL Multiphysics (a physics-based interface to solve partial differential equations). The model is shown in Fig.6. From literature, it was found that the odor threshold of trimethylamine in air is approximately 3.2E-5 ppm air (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy"> Nagata, 1993</a>), using the triangle odor bag method. This odor threshold was reached approximately 10 hours in the model, at a distance of 5 cm from the skin surface.</p></br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>s<sub>12</sub> is the average collision diameter, which is around 340 Angstroms for gas molecules in air, and w is the dimensionless temperature-dependent collision integral, usually on the order of 1.</br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>s<sub>12</sub> is the average collision diameter, which is around 340 Angstroms for gas molecules in air, and w is the dimensionless temperature-dependent collision integral, usually on the order of 1.</br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Thus, the diffusion coefficient of trimethylamine was tabulated to be 1.9*10<sup>-9</sup> m<sup>2</sup>/s.</br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Thus, the diffusion coefficient of trimethylamine was tabulated to be 1.9*10<sup>-9</sup> m<sup>2</sup>/s.</<ins class="diffchange diffchange-inline">br><br><br><</ins>br></div></td></tr>
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</table>Margueritehttp://2014.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Project/TMAU&diff=397661&oldid=prevPsatin at 03:30, 18 October 20142014-10-18T03:30:20Z<p></p>
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</table>Psatinhttp://2014.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Project/TMAU&diff=397481&oldid=prevPsatin at 03:29, 18 October 20142014-10-18T03:29:11Z<p></p>
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</table>Psatinhttp://2014.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Project/TMAU&diff=397271&oldid=prevRoussetfrancois at 03:27, 18 October 20142014-10-18T03:27:54Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b>GC/MS</b></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>GC/MS <ins class="diffchange diffchange-inline">analysis</ins></b></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the GC/MS analysis, 1µl of the sample was injected into the instrument in split mode. We used a Rtx5MS- 30m column with 0.25-mm ID and 0.25µm df. Data analysis was performed with X Caliber software. Component peaks were quantified by peak area normalized to an internal standard.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the GC/MS analysis, 1µl of the sample was injected into the instrument in split mode. We used a Rtx5MS- 30m column with 0.25-mm ID and 0.25µm df. Data analysis was performed with X Caliber software. Component peaks were quantified by peak area normalized to an internal standard.<br></div></td></tr>
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</table>Roussetfrancoishttp://2014.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Project/TMAU&diff=396966&oldid=prevRoussetfrancois at 03:26, 18 October 20142014-10-18T03:26:08Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>GC/MS</b></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>GC/MS</b></br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>For the GC<del class="diffchange diffchange-inline">-</del>MS analysis, 1µl of the sample was injected into the instrument in split mode. <del class="diffchange diffchange-inline">A </del>Rtx5MS- 30m column with 0.25-mm ID and 0.25µm df were <del class="diffchange diffchange-inline">used</del>. <del class="diffchange diffchange-inline">The GC-MS run uses the following standardized parameters:</del><<del class="diffchange diffchange-inline">/br></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For the GC<ins class="diffchange diffchange-inline">/</ins>MS analysis, 1µl of the sample was injected into the instrument in split mode. <ins class="diffchange diffchange-inline">We used a </ins>Rtx5MS- 30m column with 0.25-mm ID and 0.25µm df<ins class="diffchange diffchange-inline">. Data analysis was performed with X Caliber software. Component peaks </ins>were <ins class="diffchange diffchange-inline">quantified by peak area normalized to an internal standard</ins>.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">- Injection temperature: 300°C</br></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">- Interface temperature: 300°C</br></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">- Ion Source: 250°C</br></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">The temperature program used was 3 min of isothermal heating at 50⁰C followed by heating at 350⁰C for 10 min. Mass spectra were recorded at 2 scan sec-1 with a scanning range of 40 to 850 m/z. </br></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">X Caliber software is used to process and analyze the data from a GC-MS run. Quantify each component based on peak areas and normalization based on the internal standard.</br></del></div></td><td colspan="2"> </td></tr>
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</table>Roussetfrancoishttp://2014.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Project/TMAU&diff=396685&oldid=prevRoussetfrancois at 03:24, 18 October 20142014-10-18T03:24:32Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td><b>AIMS</b></br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td><b>AIMS</b></br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class=text1> TMA is also processed by the trimethylamine monooxygenase (TMM) of <i>Ruegeria pomeroyi</i>, an enzyme similar to human <i>FMO3</i>. Expressing this enzyme in human skin bacteria should remove trimethylamine from sweat and reduce its unpleasant odor. TMM-expressing bacteria in a cream or spray could be a cheap and stable way to deliver the therapeutic enzyme to TMAU patients.</p></td></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class=text1> TMA is also processed by the trimethylamine monooxygenase (TMM) of <i>Ruegeria pomeroyi</i>, an enzyme similar to human <i>FMO3</i>. Expressing this enzyme in human skin bacteria should remove trimethylamine from sweat and reduce its unpleasant odor. TMM-expressing bacteria in a cream or spray could be a cheap and stable way to deliver the therapeutic enzyme to TMAU patients.</p></td></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Cloned <i>TMM</i> into <i>E.coli</i> using pSB1C3 and pSEVA315 vectors, creating a new Biobrick: BBa_K1403015.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Cloned <i>TMM</i> into <i>E.coli</i> using pSB1C3 and pSEVA315 vectors, creating a new Biobrick: BBa_K1403015.</li></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We took advangage of this indole production activity to characterize the TMM enzyme. <i>E. coli</i> that were cultured in LB supplemented with tryptophan (2 g/L) produced a deep blue pigment (Fig. 3C) with absorbance properties matching indigo (Fig. 4). Without TMM expression or without tryptophan, indigo production was minimal or absent.</br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We took advangage of this indole production activity to characterize the TMM enzyme. <i>E. coli</i> that were cultured in LB supplemented with tryptophan (2 g/L) produced a deep blue pigment (Fig. 3C) with absorbance properties matching indigo (Fig. 4). Without TMM expression or without tryptophan, indigo production was minimal or absent.</br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b><del class="diffchange diffchange-inline">Gas chromatography-mass spectrometry </del></b></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b><ins class="diffchange diffchange-inline">GC/MS </ins></b></br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Gas chromatography-mass spectrometry (</del>GC/MS<del class="diffchange diffchange-inline">) </del>confirmed the activity of TMM by proving the degradation of trimethylamine (Fig. 5). It was performed on extractions from cultures of TMM-expressing <i>E. coli</i> (TMM) and control expressing an empty vector in a LB medium supplemented with trimethylamine (1 mM). The results show a significant decrease (p-value = 0,0199) of the concentration of TMA in <i>TMM</i>-expressing <i>E.coli</i> (Fig. 5B). This confirms the efficiency of degradation of the fish odor by TMM. </br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>GC/MS confirmed the activity of TMM by proving the degradation of trimethylamine (Fig. 5). It was performed on extractions from cultures of TMM-expressing <i>E. coli</i> (TMM) and control expressing an empty vector in a LB medium supplemented with trimethylamine (1 mM). The results show a significant decrease (p-value = 0,0199) of the concentration of TMA in <i>TMM</i>-expressing <i>E.coli</i> (Fig. 5B). This confirms the efficiency of degradation of the fish odor by TMM. </br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Modeling of TMA diffusion</b></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Modeling of TMA diffusion</b></br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b><del class="diffchange diffchange-inline">Gas chromatography </del>/ <del class="diffchange diffchange-inline">Mass spectrometry</del></b></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b><ins class="diffchange diffchange-inline">GC</ins>/<ins class="diffchange diffchange-inline">MS</ins></b></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the GC-MS analysis, 1µl of the sample was injected into the instrument in split mode. A Rtx5MS- 30m column with 0.25-mm ID and 0.25µm df were used. The GC-MS run uses the following standardized parameters:</br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the GC-MS analysis, 1µl of the sample was injected into the instrument in split mode. A Rtx5MS- 30m column with 0.25-mm ID and 0.25µm df were used. The GC-MS run uses the following standardized parameters:</br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>- Injection temperature: 300°C</br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>- Injection temperature: 300°C</br></div></td></tr>
</table>Roussetfrancoishttp://2014.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Project/TMAU&diff=396519&oldid=prevRoussetfrancois at 03:23, 18 October 20142014-10-18T03:23:35Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td><b>AIMS</b></br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td><b>AIMS</b></br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class=text1> TMA is also processed by the trimethylamine monooxygenase (TMM) of <i>Ruegeria pomeroyi</i>, an enzyme similar to human <i>FMO3</i>. Expressing this enzyme in human skin bacteria should remove trimethylamine from sweat and reduce its unpleasant odor. TMM-expressing bacteria in a cream or spray could be a cheap and stable way to deliver the therapeutic enzyme to TMAU patients.</p></td></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class=text1> TMA is also processed by the trimethylamine monooxygenase (TMM) of <i>Ruegeria pomeroyi</i>, an enzyme similar to human <i>FMO3</i>. Expressing this enzyme in human skin bacteria should remove trimethylamine from sweat and reduce its unpleasant odor. TMM-expressing bacteria in a cream or spray could be a cheap and stable way to deliver the therapeutic enzyme to TMAU patients.</p></td></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <td><b><del class="diffchange diffchange-inline">RESULTS</del></b></br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <td><b><ins class="diffchange diffchange-inline">ACHIEVEMENT</ins></b></br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class=text1><ul id=alignliste></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class=text1><ul id=alignliste></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Cloned <i>TMM</i> into <i>E.coli</i> using pSB1C3 and pSEVA315 vectors, creating a new Biobrick: BBa_K1403015.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Cloned <i>TMM</i> into <i>E.coli</i> using pSB1C3 and pSEVA315 vectors, creating a new Biobrick: BBa_K1403015.</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Characterized and quantified the activity of TMM by a colorimetric assay.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Characterized and quantified the activity of TMM by a colorimetric assay.</li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li>Confirmed the degradation of trimethylamine by <del class="diffchange diffchange-inline">gas chromatography - mass spectrometry (</del>GC/MS<del class="diffchange diffchange-inline">)</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li>Confirmed the degradation of trimethylamine by GC/MS.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></ul></td></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></ul></td></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </tr></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </tr></div></td></tr>
</table>Roussetfrancoishttp://2014.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Project/TMAU&diff=392974&oldid=prevPsatin at 02:58, 18 October 20142014-10-18T02:58:51Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Modeling of TMA diffusion</b></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Modeling of TMA diffusion</b></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A simple model was created for the diffusion of trimethylamine into the air using COMSOL Multiphysics (a physics-based interface to solve partial differential equations). The model is shown in Fig.6. From literature, it was found that the odor threshold of trimethylamine in air is approximately 3.2E-5 ppm air (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy"> Nagata, 1993</a>), using the triangle odor bag method. This odor threshold was reached approximately 10 hours in the model, at a distance of 5 cm from the skin surface.</p></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A simple model was created for the diffusion of trimethylamine into the air using COMSOL Multiphysics (a physics-based interface to solve partial differential equations). The model is shown in Fig.6. From literature, it was found that the odor threshold of trimethylamine in air is approximately 3.2E-5 ppm air (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy"> Nagata, 1993</a>), using the triangle odor bag method. This odor threshold was reached approximately 10 hours in the model, at a distance of 5 cm from the skin surface.</p></br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></br></br></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </br><h6>Methods</h6><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </br><h6>Methods</h6><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p class=text1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p class=text1></div></td></tr>
</table>Psatinhttp://2014.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Project/TMAU&diff=391995&oldid=prevRoussetfrancois at 02:51, 18 October 20142014-10-18T02:51:35Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td><b>RESULTS</b></br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td><b>RESULTS</b></br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class=text1><ul id=alignliste></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class=text1><ul id=alignliste></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li>Cloned <i><del class="diffchange diffchange-inline">tmm</del></i> into <i>E.coli</i> using pSB1C3 and pSEVA315 vectors, creating a new Biobrick: BBa_K1403015.</li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li>Cloned <i><ins class="diffchange diffchange-inline">TMM</ins></i> into <i>E.coli</i> using pSB1C3 and pSEVA315 vectors, creating a new Biobrick: BBa_K1403015.</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Characterized and quantified the activity of TMM by a colorimetric assay.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Characterized and quantified the activity of TMM by a colorimetric assay.</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Confirmed the degradation of trimethylamine by gas chromatography - mass spectrometry (GC/MS).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Confirmed the degradation of trimethylamine by gas chromatography - mass spectrometry (GC/MS).</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p class=text1>Trimethylamine (TMA) is a volatile compound smelling strongly of spoiled fish. It is produced during human digestion when choline, a B-complex vitamin, is fermented by gut bacteria (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Craciun, S. <i>et al.</i>, 2012</a>) (Fig. 1A). Normally it proceeds to the liver where it is oxidized by FMO3, a flavin-containing monooxygenase (Fig. 1B). The product of FMO3 is trimethylamine oxide (TMAO), which is odorless.</br></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p class=text1>Trimethylamine (TMA) is a volatile compound smelling strongly of spoiled fish. It is produced during human digestion when choline, a B-complex vitamin, is fermented by gut bacteria (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Craciun, S. <i>et al.</i>, 2012</a>) (Fig. 1A). Normally it proceeds to the liver where it is oxidized by FMO3, a flavin-containing monooxygenase (Fig. 1B). The product of FMO3 is trimethylamine oxide (TMAO), which is odorless.</br></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Trimethylaminuria (TMAU), or fish odor syndrome, is a rare genetic disorder in which patients are not able to fully oxidize TMA. TMAO is an autosomal recessive disorder, requiring two nonfunctional alleles of the FMO3 gene. More than 30 known mutations can inactivate FMO3, and inactive alleles have an estimated 0.1-1% global frequency (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Mitchell, S.C <i>et al.</i>, 2001</a>). TMAU currently has no cure.</br></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Trimethylaminuria (TMAU), or fish odor syndrome, is a rare genetic disorder in which patients are not able to fully oxidize TMA. TMAO is an autosomal recessive disorder, requiring two nonfunctional alleles of the FMO3 gene. More than 30 known mutations can inactivate FMO3, and inactive alleles have an estimated 0.1-1% global frequency (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Mitchell, S.C <i>et al.</i>, 2001</a>). TMAU currently has no cure.</br></br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We propose that TMAU could be treated with a genetically modified skin bacteria. The trimethylamine monooxygenase (<del class="diffchange diffchange-inline">Tmm</del>) of <i>Ruegeria pomeroyi</i> also oxdizes TMA to TMAO (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Chen, Y. <i>et al.</i>, 2011</a>). Applying this enzyme to the skin should eliminate TMA in sweat and, therefore, unpleasant odor. Expression in a bacterium of the skin microbiome could be a cheap and stable way to deliver the therapeutic enzyme. Here we present the expression and characterization of <del class="diffchange diffchange-inline">Tmm </del>in <i>E. coli</i> and <i>Corynebacterium striatum</i>, a skin-native bacterium.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We propose that TMAU could be treated with a genetically modified skin bacteria. The trimethylamine monooxygenase (<ins class="diffchange diffchange-inline">TMM</ins>) of <i>Ruegeria pomeroyi</i> also oxdizes TMA to TMAO (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Chen, Y. <i>et al.</i>, 2011</a>). Applying this enzyme to the skin should eliminate TMA in sweat and, therefore, unpleasant odor. Expression in a bacterium of the skin microbiome could be a cheap and stable way to deliver the therapeutic enzyme. Here we present the expression and characterization of <ins class="diffchange diffchange-inline">TMM </ins>in <i>E. coli</i> and <i>Corynebacterium striatum</i>, a skin-native bacterium.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div id=part3 class=project></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div id=part3 class=project></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Figure 3. Characterisation of TMM using indigo production.</b></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Figure 3. Characterisation of TMM using indigo production.</b></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>(A) TMM allows the degradation of trimethylamine into trimethylamine-N-oxide.</br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>(A) TMM allows the degradation of trimethylamine into trimethylamine-N-oxide.</br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>(B) TMM activity can be detected by the presence of indigo, a blue dye. Indole is naturally produced from tryptophan by tryptophanase in <i>E. coli</i> and can be converted into indigo by <del class="diffchange diffchange-inline">Tmm</del>.</br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>(B) TMM activity can be detected by the presence of indigo, a blue dye. Indole is naturally produced from tryptophan by tryptophanase in <i>E. coli</i> and can be converted into indigo by <ins class="diffchange diffchange-inline">TMM</ins>.</br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>(C) Indigo can be detected in TMM-expressing <i>E.coli</i> (left) but not in the control (right) after 14h of culture in LB medium supplemented with 2g/l of tryptophan.</br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>(C) Indigo can be detected in TMM-expressing <i>E.coli</i> (left) but not in the control (right) after 14h of culture in LB medium supplemented with 2g/l of tryptophan.</br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src='https://static.igem.org/mediawiki/2014/f/f0/Spectrum_indigoPB.png' width='800px'></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src='https://static.igem.org/mediawiki/2014/f/f0/Spectrum_indigoPB.png' width='800px'></br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b>Figure 4. Average absorbances of DMSO extractions from bacterial lysates shows <del class="diffchange diffchange-inline">Tmm </del>activity.</b></br> This graph shows the absorbance spectra of DMSO extractions from TMM-expressing <i>E. coli</i> (+TMM) or control (-TMM) in LB medium (-trp) or in LB supplemented with tryptophan (+trp). The grey area shows the expected absorbance peak of indigo.</br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>Figure 4. Average absorbances of DMSO extractions from bacterial lysates shows <ins class="diffchange diffchange-inline">TMM </ins>activity.</b></br> This graph shows the absorbance spectra of DMSO extractions from TMM-expressing <i>E. coli</i> (+TMM) or control (-TMM) in LB medium (-trp) or in LB supplemented with tryptophan (+trp). The grey area shows the expected absorbance peak of indigo.</br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src='https://static.igem.org/mediawiki/2014/8/88/TMMGC_PB.png' width='800px'></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src='https://static.igem.org/mediawiki/2014/8/88/TMMGC_PB.png' width='800px'></br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Cloning</b></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Cloning</b></br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We synthesized <del class="diffchange diffchange-inline">Tmm </del>from <i>R. pomeroyi</i> and expressed it in <i>E. coli</i>. The construct (BBa_K1403015) included a constitutive promoter and was cloned in the standard high-copy BioBrick vector pSB1C3. Following 24 hours of incubation at 37 C, a dark pigment was visible in the transformed colonies.</br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We synthesized <ins class="diffchange diffchange-inline">TMM </ins>from <i>R. pomeroyi</i> and expressed it in <i>E. coli</i>. The construct (BBa_K1403015) included a constitutive promoter and was cloned in the standard high-copy BioBrick vector pSB1C3. Following 24 hours of incubation at 37 C, a dark pigment was visible in the transformed colonies.</br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Characterisation of TMM activity in <i>E.coli</i></b></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Characterisation of TMM activity in <i>E.coli</i></b></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The <del class="diffchange diffchange-inline">Tmm </del>enzyme is not specific to TMA as a substrate. It is also known to oxidize indole to indoxyl, which dimerizes into the well known blue pigment indigo (Fig 3B). Indole is a natural product of tryptophan metabolism in <i>E. coli</i>.</br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The <ins class="diffchange diffchange-inline">TMM </ins>enzyme is not specific to TMA as a substrate. It is also known to oxidize indole to indoxyl, which dimerizes into the well known blue pigment indigo (Fig 3B). Indole is a natural product of tryptophan metabolism in <i>E. coli</i>.</br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We took advangage of this indole production activity to characterize the <del class="diffchange diffchange-inline">Tmm </del>enzyme. <i>E. coli</i> that were cultured in LB supplemented with tryptophan (2 g/L) produced a deep blue pigment (Fig. 3C) with absorbance properties matching indigo (Fig. 4). Without <del class="diffchange diffchange-inline">Tmm </del>expression or without tryptophan, indigo production was minimal or absent.</br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We took advangage of this indole production activity to characterize the <ins class="diffchange diffchange-inline">TMM </ins>enzyme. <i>E. coli</i> that were cultured in LB supplemented with tryptophan (2 g/L) produced a deep blue pigment (Fig. 3C) with absorbance properties matching indigo (Fig. 4). Without <ins class="diffchange diffchange-inline">TMM </ins>expression or without tryptophan, indigo production was minimal or absent.</br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Gas chromatography-mass spectrometry </b></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Gas chromatography-mass spectrometry </b></br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Cloning</b></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Cloning</b></br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The <del class="diffchange diffchange-inline">Tmm </del>gene was produced by gene synthesis (IDT). The final construct was codon-optimized for <i>E. coli</i> expression and included a strong constitutive promoter and ribosome binding site.</br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The <ins class="diffchange diffchange-inline">TMM </ins>gene was produced by gene synthesis (IDT). The final construct was codon-optimized for <i>E. coli</i> expression and included a strong constitutive promoter and ribosome binding site.</br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>For <i>E. coli</i> expression, we cloned the <del class="diffchange diffchange-inline">Tmm </del>construct into the standard high-copy BioBrick vector pSB1C3. For expression in <i>C. striatum</i> we used the vector pSEVA351, a universal vector with a high-copy replication origin (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Standard European Vector Architecture</a>).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For <i>E. coli</i> expression, we cloned the <ins class="diffchange diffchange-inline">TMM </ins>construct into the standard high-copy BioBrick vector pSB1C3. For expression in <i>C. striatum</i> we used the vector pSEVA351, a universal vector with a high-copy replication origin (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Standard European Vector Architecture</a>).</div></td></tr>
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</table>Roussetfrancoishttp://2014.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Project/TMAU&diff=391911&oldid=prevRoussetfrancois at 02:50, 18 October 20142014-10-18T02:50:55Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h6>Introduction</h6><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h6>Introduction</h6><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p class=text1>Trimethylamine (TMA) is a volatile compound smelling strongly of spoiled fish. It is produced during human digestion when choline, a B-complex vitamin, is fermented by gut bacteria (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Craciun, S. <i>et al.</i>, 2012</a>) (Fig. 1A). Normally it proceeds to the liver where it is oxidized by FMO3, a flavin-containing monooxygenase (Fig. 1B). The product of FMO3 is trimethylamine oxide (TMAO), which is odorless.</br></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p class=text1>Trimethylamine (TMA) is a volatile compound smelling strongly of spoiled fish. It is produced during human digestion when choline, a B-complex vitamin, is fermented by gut bacteria (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Craciun, S. <i>et al.</i>, 2012</a>) (Fig. 1A). Normally it proceeds to the liver where it is oxidized by FMO3, a flavin-containing monooxygenase (Fig. 1B). The product of FMO3 is trimethylamine oxide (TMAO), which is odorless.</br></br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Trimethylaminuria (TMAU), or fish odor syndrome, is a rare genetic disorder in which patients are not able to fully oxidize TMA. TMAO is an autosomal recessive disorder, requiring two nonfunctional alleles of the FMO3 gene. More than 30 known mutations can inactivate FMO3, and inactive alleles have an estimated 0.1-1% global frequency (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Mitchell, S.C <i>et al.</i>, 2001</a>). TMAU currently has no cure.</br</br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Trimethylaminuria (TMAU), or fish odor syndrome, is a rare genetic disorder in which patients are not able to fully oxidize TMA. TMAO is an autosomal recessive disorder, requiring two nonfunctional alleles of the FMO3 gene. More than 30 known mutations can inactivate FMO3, and inactive alleles have an estimated 0.1-1% global frequency (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Mitchell, S.C <i>et al.</i>, 2001</a>). TMAU currently has no cure.</br<ins class="diffchange diffchange-inline">></ins></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We propose that TMAU could be treated with a genetically modified skin bacteria. The trimethylamine monooxygenase (Tmm) of <i>Ruegeria pomeroyi</i> also oxdizes TMA to TMAO (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Chen, Y. <i>et al.</i>, 2011</a>). Applying this enzyme to the skin should eliminate TMA in sweat and, therefore, unpleasant odor. Expression in a bacterium of the skin microbiome could be a cheap and stable way to deliver the therapeutic enzyme. Here we present the expression and characterization of Tmm in <i>E. coli</i> and <i>Corynebacterium striatum</i>, a skin-native bacterium.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We propose that TMAU could be treated with a genetically modified skin bacteria. The trimethylamine monooxygenase (Tmm) of <i>Ruegeria pomeroyi</i> also oxdizes TMA to TMAO (<a href="https://2014.igem.org/Team:Paris_Bettencourt/Bibliograpy">Chen, Y. <i>et al.</i>, 2011</a>). Applying this enzyme to the skin should eliminate TMA in sweat and, therefore, unpleasant odor. Expression in a bacterium of the skin microbiome could be a cheap and stable way to deliver the therapeutic enzyme. Here we present the expression and characterization of Tmm in <i>E. coli</i> and <i>Corynebacterium striatum</i>, a skin-native bacterium.</p></div></td></tr>
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</table>Roussetfrancois