http://2014.igem.org/wiki/index.php?title=Special:Contributions/Dbby&feed=atom&limit=50&target=Dbby&year=&month=2014.igem.org - User contributions [en]2024-03-29T06:12:24ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Toulouse/Notebook/project-monitoringTeam:Toulouse/Notebook/project-monitoring2014-10-18T01:22:36Z<p>Dbby: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Project monitoring</p> <br />
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<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
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<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p><br />
<br />
<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">1. Amplification of Binding Module (pEX-K4) into <i>E. coli</i></p><br />
<p class="title3">Transformation of Binding module (pEX-K4) into <I>E. coli</i></p><br />
<p class="texte">Gene "Binding module" synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br><b>Result:</b> We obtained distinct colonies on plates LB + Kanamycin (50 µg/mL)</p><br />
<br />
<p class="title3">Liquid culture of two clones: 1 et 2 (pEX-K4) transformed into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 01/08/2014</p><br />
<br />
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit: two clones of Binding Module (pEX-K4) into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br><b>Result:</b> Binding Module (pEX-K4) obtained</p><br />
<br />
<p class="title3">Digestion of Binding Module (pEX-K4) with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br><b>Result:</b> <br />
<br>- 3 bands : 1500bp (vector with Kanamycin resistance), 1300bp (Module Binding) and 1000bp (vector with pUC Ori)<br />
<br>- The two Binding Module clones are ok</p><br />
<br />
<br />
<p class="title2">2. Cloning Binding Module on pEX-K4 with pSB1C3 (<a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a> without RFP) into <i>E. coli</i></p><br />
<p class="title3">Digestion Binding Module on pEX-K4 and <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a></p><br />
<p class="texte"><b>Date:</b> 23/08/2014<br />
<br><b>Expected bands after digestion:</b><br />
<br>- <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a>: 860 bp for RFP and 2100 bp for vector pSB1C3<br />
<br>- Binding Module: 1400 bp for Binding Module and 1500bp + 1000bp for vector pEX_K4<br />
<br>We decide to do a ligation between pSB1C3 and Binding Module without gel cutting and purification but after a PCR clean-up (Thermo scientific, GeneJET PCR purifiction kit #K0701) to remove the enzymes.</p><br />
<br />
<p class="title3">Ligation Binding Module on pSB1C3</p><br />
<p class="texte"><b>Date:</b> 04/08/2014</p><br />
<br />
<p class="title3">Transformation of Binding Module on pSB1C3 into E. coli</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>Transformation with 10 µL of the ligation mix and plate on Chloremphenicol LA plate<br />
<br><b>Result:</b> many wrong clones</p><br />
<br />
<p class="title2">3. Cloning Binding Module on pEX-K4 with pVeg on pSB1C3 (<a href="http://parts.igem.org/Part:BBa_K823003">BBa_K823003</a>) into <i>E. coli</i></p><br />
<p class="title3">Digestion Binding Module on pEX-K4 and <a href="http://parts.igem.org/Part:BBa_K823003">BBa_K823003</a></p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br><a href="http://parts.igem.org/Part:BBa_K823003">BBa_K823003</a> digested by XbaI and PstI and Binding Module digested by SpeIand PstI<br />
<br>Gel Electrophoresis<br />
<br><b>Result:</b><br />
<br>- Binding Module : 1400 bp for Binding Module and 1500bp + 1000bp for vector pEX_K47<br />
<br>We decide to do a ligation between pVeg and Binding Module without gel cutting and purification but after a PCR clean-up (Thermo scientific, GeneJET PCR purifiction kit #K0701) to remove the enzymes.</p><br />
<br />
<p class="title3">Ligation Binding Module on pVeg with pSB1C3</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br><a href="http://parts.igem.org/Part:BBa_K823003">BBa_K823003</a> digested by XbaI and PstI and Binding Module digested by SpeI and PstI and ligation</p><br />
<br />
<p class="title3">Transformation of Binding Module on pVeg into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br><b>Result:</b> Many good clones (check on 06/08/2014)</p><br />
<br />
<p class="title2">4. Cloning Binding Module with P<sub>veg</sub> (<a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>) on pSB<sub>BS</sub>4S (<a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a>) into <i>E. coli</i></p><br />
<br />
<p class="title3">Digestion Binding Module with P<sub>veg</sub> (<a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>) and pSB<sub>BS</sub>4S (<a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a>)</p><br />
<p class="texte"><b>Date:</b> 07/08/2014<br />
<br><a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a> and Binding Module with P<sub>veg</sub> (<a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>) digested by EcoRI and PstI Gel Electrophoresis<br />
<br><b>Result:</b><br />
<br>- Binding Module with Pveg 1600 bp for Binding Module and 2100bp for vector pSB1C3</p><br />
<br />
<p class="title3">Ligation Binding Module with Pveg on pSBbs4S</p><br />
<p class="texte"><b>Date:</b> 07/08/2014<br />
<br><a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a> and Binding Module with P<sub>veg</sub> (<a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>) digested by EcoRI and PstI<br />
<br>Ligation<br />
<br><b>Result:</b> Ligation between Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S</p><br />
<br />
<p class="title3">Transformation of Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S<br />
<br>Plate on Ampicillin LA plate<br />
<br><b>Result:</b> Many good clones (check on 13/08/2014)</p><br />
<br />
<p class="title3">Transformation of Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S into <i>B. subtilis</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>After 5 hours of incubation and the linearization of 6µl of DNA with 1µl of ScaI, we make the transformation. Plate on Spectinomycin LA plate.<br />
<br><b>Result:</b> One good clone : PCR (check on 09/09/2014) and threonine test (check on 09/09/2014)</p><br />
<br />
<p class="title2">5. Binding Test</p><br />
<p class="texte">See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results#select2">here</a></p><br />
<br />
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</div><br />
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<br />
<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">1. Transformation of chemotaxis (Puc-57) into <i>E. coli</i></p><br />
<p class="texte">Gene chemotaxis synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br><b>Date:</b> 01/08/2014<br />
<br><b>Result:</b> We obtained distinct colonies on LA + Ampicillin (100 µg/mL) plates</p><br />
<br />
<p class="title3">Culture of 4 clones: A, B, C, D of chemotaxis (Puc57) transformed into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014</p><br />
<br />
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit Using a Microcentrifuge: 4 clones of chemotaxis (Puc57) into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 05/08/2014<br />
<br><b>Result:</b> 4*50µL of chemotaxis (Puc57) obtained</p><br />
<br />
<p class="title2">2. Cloning chemotaxis <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> (chemotaxis_Puc57) with digested pSB1C3 with EcoRI and PstI into <i>E. coli</i></p><br />
<br />
<p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> (chemotaxis_Puc57) with EcoRI and PstI - PCR kit Clean up</p><br />
<p class="texte"><b>Date:</b> 07/08/2014<br />
<br><b>Result:</b> Expected band after digestion for <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>: 2300 bp<br />
<br><b>Problem:</b> We can't distinguish the vector band (2500 bp)</p><br />
<br />
<p class="title3">Gel extraction of <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a></p><br />
<p class="texte"><b>Date:</b> 07/08/2014</p><br />
<br />
<p class="title3">Ligation <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> and digested PSB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 08/08/2014</p><br />
<br />
<p class="title3">Transformation <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 08/08/2014<br />
<br><b>Result:</b> We obtained distinct colonies on LA + Cm (15 µg/mL) plates and resuspended 15 colonies in LB+Cm (15 µg/mL)</p><br />
<br />
<p class="title3">Test of sensibility on Ampicillin</p><br />
<p class="texte"><b>Date:</b> 10/08/2014<br />
<br><b>Result:</b> we can determine which colonies are sensible for ampicillin and know which bacterium is carrying the chemotaxis gene.</p><br />
<br />
<p class="title3">PCR</p><br />
<p class="texte"><b>Date:</b> 11/08/2014</p><br />
<br />
<p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> on pSB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 11/08/2014<br />
<br><b>Result:</b> There is one colony which presents the right construction.</p><br />
<br />
<br />
<br />
<p class="title2">3. Cloning chemotaxis <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> (chemotaxis_Puc57) with digested <a href="http://parts.igem.org/Part:BBa_K823003">BBa_823003</a> (P<sub>veg</sub>) on pSB1C3 with SpeI and PstI into <i>E. coli</i></p><br />
<p class="title3">Ligation <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> and digested <a href="http://parts.igem.org/Part:BBa_K823003">BBa_823003</a> on PsB1C3 with SpeI and PstI</p><br />
<p class="texte"><b>Date</b>: 08/08/2014</p><br />
<p class="title3">Transformation <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_1364004</a> in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 8/08/2014</p><br />
<p class="title3">Test of sensibility on Ampicillin</p><br />
<p class="texte"><b>Date:</b> 10/08/2014<br />
<br><b>Result:</b> We can determine which colony is sensible for ampicillin and know which bacterium is carrying the chemotaxis gene. There is one colony which resists on ampicillin.</p><br />
<br />
<p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_1364004</a> on pSBC3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 12/08/2014<br />
<br><b>Result:</b> We did not see any colony with chemotaxis insert.</p><br />
<br />
<p class="title2">4. Cloning chemotaxis <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> (chemotaxis_Puc57) with digested <a href="http://parts.igem.org/Part:BBa_K823003">BBa_823003</a> (P<sub>veg</sub>) on pSB1C3 with SpeI and PstI into <i>E. coli</i></p><br />
<p class="title3">Ligation <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> and digested <a href="http://parts.igem.org/Part:BBa_K823003">BBa_823003</a> on PsB1C3 with SpeI and PstI</p><br />
<p class="texte"><b>Date:</b> 11/08/2014</p><br />
<p class="title3">Transformation <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_1364004</a> in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 11/08/2014</p><br />
<p class="title3">Test of sensibility on Ampicillin</p><br />
<p class="texte"><b>Date:</b> 14/08/2014<br />
<br><b>Result:</b> we obtained 4 colonies sensible at Ampicilline</p><br />
<br />
<p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_1364004</a> on PsB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 18/08/2014</p><br />
<p class="title3">Gel extraction of <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_1364000</a></p><br />
<p class="texte"><b>Date:</b> 19/08/2014</p><br />
<br />
<p class="title2">5. Cloning chemotaxis <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a> with digested pSB<sub>BS</sub>4S with EcorI and PstI into <i>E. coli</i></p><br />
<p class="title3">Ligation <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a> digested EcorI and PstI and digested PsB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 19/08/2014</p><br />
<p class="title3">Transformation <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a> in pSBBS4S in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 19/08/2014 <br />
<br><b>Result:</b> We obtained one colony and resuspended it in LB+ Amp</p><br />
<br />
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</div><br />
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<br />
<div class="technology">Fungicides</div><br />
<div class="thelanguage"><br />
<br />
<div class="technology2">D4E1</div><br />
<div class="thelanguage2"><br />
<br />
<p class="title2">1. Amplification of synthetic gene (D4E1 on pEX-A2)</p><br />
<p class="title3">Transformation of D4E1 (pEX-A2) into <i>E. coli</i></p><br />
<p class="texte">Gene D4E1 synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br>Concentration of D4E1: 115ng/µL<br />
<br><b>Date:</b> 07/21//2014<br />
<br><b>Result:</b> We obtained distinct colonies on LA + Ampicillin (100 µg/mL)</p><br />
<br />
<p class="title3">Culture of 4 clones: A, B, C, D of D4E1 (pEX-A2) transformed into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/22/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of D4E1 (pEX-A2) into E. coli</p><br />
<p class="texte">Buffer EB at 50-55°C<br />
<br><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up</p><br />
<p class="texte"><b>Date:</b> 07/23/2014<br />
<br><b>Result:</b> 4*20µL D4E1 digested with EcoRI and PstI all the clones seem to have the right D4E1 gene.</p><br />
<br />
<p class="title2">2. Cloning D4E1 in pSB1C3</p><br />
<p class="title3">Digestion of D4E1 on pEX-A2 and pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<p class="title3">Ligation of D4E1 in pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<p class="title3">Transformation in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of D4E1 (pSB1C3) transformed into <i>E. coli</i></p><br />
<p class="texte">Processing details: 5mL of LB + chloramphenicol (15µg/mL) , using sterile plastic loop to culture colonies<br />
<br><b>Date:</b> 07/24/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<p class="title3">Digestion of D4E1 (pSB1C3) A, B, C, D, E, F with EcoRI and PstI - PCR kit Clean up + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014<br />
<br><b>Result:</b> clones C, D have the expected construction</p><br />
<p class="title3">PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014<br />
<br><b>Result:</b> clones C, D have the expected construction, and placed in cryopreservation.</p><br />
<br />
<p class="title2">3. Cloning Pveg+D4E1 on Pveg plasmid (pSB1C3)</p><br />
<br />
<p class="title3">Digestion of D4E1 on pEX-A2 and K823003 (Pveg on pSB1C3)</p><br />
<p class="texte"><b>Dated:</b></b> 07/24/2014<br />
<br><b>Result:</b> 20µL digestion of D4E1 on pEX-A2 and of K823003</p><br />
<br />
<p class="title3">Ligation of D4E1 in K823003 (Pveg on pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/24/2014<br />
<br><b>Result:</b> 20µL ligation of D4E1 in K823003</p><br />
<br />
<p class="title3">Transformation of ligation products in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 07/24/2014<br />
<br><b>Result:</b> <i>E.coli</i> transformed by D4E1+K823003</p><br />
<br />
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of transformed <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/26/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Dated:</b> 07/28/2014<br />
<br><b>Result:</b> clones E, F seem to have the expected construction</p><br />
<p class="title3">Digestion of clones E, F of D4E1+K823003 (Pveg on pSB1C3) with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 28/07/2014<br />
<br><b>Result:</b> clones C, D have the expected construction and are placed in cryopreservation.</p><br />
<br />
<p class="title2">4. Cloning P<sub>veg</sub> + D4E1 on pSB<sub>BS</sub>4S (K823022) </p><br />
<p class="texte"><b>Date:</b> 08/13/2014</p><br />
<br />
<p class="title2">5. Cloning P<sub>veg</sub> + D4E1 on pSB<sub>BS</sub>1C lacZ (23)</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology2">GAFP1</div><br />
<div class="thelanguage2"><br />
<br />
<p class="title2">1. Amplification of synthetic gene (GAFP1 on pEX-A2)</p><br />
<br />
<p class="title3">Transformation of GAFP1 (pEX-A2) into <i>E.coli</i></p><br />
<p class="texte">Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br>Concentration of GAFP1 : 145ng/µL<br />
<br><b>Date:</b> 07/21/2014<br />
<br><b>Result:</b> We obtained distinct colonies on plates LB + Ampicillin (100 µg/mL)</p><br />
<br />
<p class="title3">Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/22/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel Electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/23/2014 </p><br />
<br />
<p class="title2">2. Cloning GAFP1 gene on PSB1C3 = K1364002 in E. coli</p><br />
<br />
<p class="title3">Digestion GAFP1_pEX-A2 and <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a> (RFP_pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/23/2014<br />
<br><b>Result:</b> <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a> : 860 bp<br />
<br>We decide to conserve the miniprep B for <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a></p><br />
<br />
<p class="title3">Ligation GAFP1 and <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a></p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Tranformation of ligation products into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Culture of x clones of GAFP1+K606013 in <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/24/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge: 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<br />
<p class="title3">PCR on 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014<br />
<br><b>Result:</b> clones A, C, D, F seem to have the right construction</p><br />
<br />
<p class="title3">Digestion of 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) by EcoR1 and Pst1 + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<br />
<br />
<br />
<p class="title2">3. Cloning GAFP1+terminator(B0015) = K1364007 (pSB1C3)</p><br />
<br />
<p class="title3">Digestion of GAFP1 on pEX-A2 and B0015 (terminator on pSB1C3)<br />
<p class="texte"><b>Date:</b> 07/24/2014<br />
<br />
<p class="title3">Ligation of GAFP1 in B0015 (terminator on pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/24/2014</p><br />
<br />
<p class="title3">Transformation of ligation products in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 07/24/2014</p><br />
<br />
<p class="title3">Culture of 6 clones: A, B, C, D, E, F transformed in <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/26/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">PCR of GAFP1+B0015 = K1364007 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/28/2014<br />
<br><b>Result:</b> clones B, C, D, E, F seem to have the expected construction.</p><br />
<br />
<p class="title3">Digestion of clones E, F of GAFP1+Ter B0015 = K1364007 with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/28/2014<br />
<br><b>Result:</b> clones B, C, D, E, F have the expected construction.</p><br />
<br />
<br />
<br />
<p class="title2">4. Cloning GAFP1+terminator with Pveg on pSB1C3 (K1364008) in <i>E. coli</i></p><br />
<br />
<p class="title3">Digestion of GAFP1+ter = K1364007 on pSB1C3 and K823003 (terminator on pSB1C3) + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/29/2014</p><br />
<br />
<p class="title3">Gel extraction of K1364007 (extraction of GAFP1+ter gene)</p><br />
<p class="texte"><b>Date:</b> 07/29/2014</p><br />
<br />
<p class="title3">Ligation of GAFP1+ter in K823003 (Pveg on pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/29/2014<br />
<br><b>Result:</b> 20µL ligation of GAFP1+ter in K823003</p><br />
<br />
<p class="title3">Transformation of ligation products in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 07/29/2014</p><br />
<br />
<p class="title3">Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli</p><br />
<p class="texte"><b>Date:</b> 07/30/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/31/2014</p><br />
<br />
<p class="title3">PCR of GAFP1+B0015 + K823003 = K1364008 (pSB1C3) A, B, C, D, E, F, G, H + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 31/07/2014<br />
<br><b>Result:</b> clones A, B, C, D, E, G, H seem to have the expected construction</p><br />
<br />
<p class="title3">Digestion of clones A, B, C, D, E, G, H of Pveg+K1364007 = K1364008 with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 31/07/2014<br />
<br><b>Result:</b> clones A, B, C, D, E, G, H have the expected construction</p><br />
<br />
<br />
<br />
<p class="title2">5. Cloning Pveg+GAFP1+Ter B0015 (<a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>) on pSBBS4S (<a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a>)</p><br />
<br />
<p class="title3">Digestion of Pveg+GAFP1+ ter B0015 (<a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>) on pSB1C3 and PsBBs4S (<a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a>)</p><br />
<p class="texte"><b>Date:</b> 08/01/2014</p><br />
<br />
<p class="title3">Ligation of K134008 (Pveg+GAFP1+ter fragment) and K823022 (PsBBs4S)</p><br />
<p class="texte"><b>Date:</b> 08/01/2014</p><br />
<br />
<p class="title3">Transformation of ligation products in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 08/01/2014</p><br />
<br />
<p class="title3">Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli</p><br />
<p class="texte"><b>Date:</b> 08/02/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 08/04/2014</p><br />
<br />
<p class="title3">Digestion of clones A, B, C, D, E, G, H of K1364008+K823022 with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 08/04/2014<br />
<br><b>Result:</b> clones A, E, F have the right construction</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology2">EcAMP</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">The EcAMP part was sent by the Utah iGEM team. It was on a pUC plasmid (ampicilin resistance) with BioBrick suffix and prefix.</p><br />
<br />
<p class="title2">1. Transformation of EcAMP in <i>Escherichia coli</i> MC 1061</p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<br />
<p class="title2">2. Spreading of coli cells transformed with EcAMP (plasmid pUC) </p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title2">3. Liquid culture, Miniprep and Test of the miniprep</p><br />
<p class="texte"><b>Date:</b> 07/30/2014</p><br />
<br />
<br />
<br />
<p class="title2">4. Cloning 1: EcAMP + P<sub>veg</sub> + RBS</p><br />
<br />
<p class="title3">Digestion of EcAMP (insert) by XbaI and PstI</p><br />
<p class="texte"><b>Date:</b> 07/31/2014</p><br />
<br />
<p class="title3">Digestion of P<sup>veg</sup> + RBS (VECTOR)</p><br />
<p class="texte"><b>Date:</b> 07/31/2014</p><br />
<br />
<p class="title3">Ligation and transformation</p><br />
<p class="texte"><b>Date:</b> 08/04/2014</p><br />
<br />
<p class="title3">PCR test</p><br />
<p class="texte"><b>Date:</b> 08/05/2014</p><br />
<br />
<p class="title3">Analytical digestion</p><br />
<p class="texte"><b>Date:</b> 08/05/2014<br />
<br />
<br><b>Result:</b> The first cloning show a lack of DNA in the Miniprep EcAMP. The bands are hardly visible on the gel for the linearization of EcAMP + Pveg + RBS. The problem came from the PCR cleanup step: the fragment size of EcAMP is lower than 200bp.</p><br />
<br />
<br />
<br />
<p class="title2">5. Cloning 2: EcAMP + Pveg + RBS </p><br />
<p class="texte"><b>Date:</b> 08/06/2014</p><br />
<p class="title3">Digestion of EcAMP (insert) by XbaI and PstI</p><br />
<p class="texte"><b>Date:</b> 08/06/2014</p><br />
<p class="title3">Digestion of P<sub>veg</sub> + RBS (vector)</p><br />
<p class="texte"><b>Date:</b> 08/06/2014</p><br />
<p class="title3">Heat inactivation of the enzymes</p><br />
<p class="texte"><b>Date:</b> 08/06/2014</p><br />
<p class="title3">Ligation and transformation</p><br />
<p class="texte"><b>Date:</b> 08/06/2014</p><br />
<p class="title3">PCR test</p><br />
<p class="texte"><b>Date:</b> 08/07/2014</p><br />
<br />
<br />
<p class="title3">Striation on a petri dish to purify the clone</p><br />
<p class="texte"><b>Purpose:</b> to isolate a clone with vector+insert<br />
<br><b>Date:</b> 08/072014</p><br />
<br />
<p class="title3">Miniprep of P<sub>veg</sub> + SpoVG + EcAMP and analytic digestion</p><br />
<p class="texte"><b>Date:</b> 08/08/2014</p><br />
<br />
<p class="title3">Ligation of Pveg SpoVG EcAMP with double terminateur B0015 + transformation and liquid culture</p><br />
<p class="texte"><b>Date:</b> 08/11/2014</p><br />
<br />
<p class="title3">Miniprep of P<sub>veg</sub> SpoVG EcAMP + analytic digestion</p><br />
<p class="texte"><b>Date:</b> 08/13/2014</p><br />
<br />
<p class="title3">Cloning K1364011 (EcAMP + P<sub>veg</sub> +SpoVG + B0015) + K823022 (pSB<sub>BS</sub>4S): Digestion, ligation, transformation</p><br />
<p class="texte"><b>Date:</b> 08/19/2014</p><br />
<br />
<p class="title3">Cloning K1364011 + K823023 (pSB<sub>BS</sub>1C) : Digestion, ligation, transformation</p><br />
<p class="texte"><b>Date:</b> 08/18/2014</p><br />
<br />
<p class="title3">Verification of the insertion of K1364011 + K823022 (pSB<sub>BS</sub>4S) into the subtilis genome by threonine test </p><br />
<p class="texte"><b>Date:</b> 08/21/2014</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology2">Assembling the fungicides module: D4E1 + GAFP1</div><br />
<div class="thelanguage2"><br />
<br />
<p class="title2">1. Cloning GAFP1+D4E1 on pSB1C3: <a href="http://parts.igem.org/Part:BBa_K1364012">BBa_K1364012</a></p><br />
<br />
<p class="title3">Digestion of D4E1 on pEX-A2 and GAFP1 on pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/28/2014<br />
<br><b>Result:</b> We obtained 40µL digestion of D4E1 on pEX-A2 and of GAFP1 on pSB1C3.</p><br />
<br />
<p class="title3">Ligation of digestions of D4E1 on pEX-A2 and of GAFP1 on pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">Transformation of ligation of GAFP1 and D4E1 on pSB1C3 into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">PCR of GAFP1 + D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/29/2014<br />
<br><b>Result:</b> clones A, C, F, G seem to have the expected construction.</p><br />
<br />
<p class="title3">Digestion of GAFP1 + D4E1 (pSB1C3) A, C, F, G + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/30/2014<br />
<br><b>Result:</b> clone F (<a href="http://parts.igem.org/Part:BBa_K1364012">BBa_K1364012</a>) has the expected construction and is placed on cryopreservation.</p><br />
<br />
<br />
<br />
<p class="title2">2. Cloning GAFP1 + D4E1 (<a href="http://parts.igem.org/Part:BBa_K1364012">BBa_K1364012</a>) on P<sub>veg</sub> plasmid (<a href="http://parts.igem.org/Part:BBa_K823003">BBa_K823003</a>): <a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a></p><br />
<p class="texte"><b>Date:</b> 08/04/2014<br />
<br />
<p class="title2">3. Cloning P<sub>veg</sub> + GAFP1 + D4E1 (<a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a>) on pSB<sub>BS<sub>4S (<a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a>)</p><br />
<p class="texte"><b>Date:</b> 08/06/2014<br />
<br />
<p class="title2">4. Cloning P<sub>veg</sub> + GAFP1 + D4E1 (<a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a>) on pSB<sub>BS</sub>1C lacZ (<a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>)</p><br />
<p class="texte"><b>Date:</b> 08/11/2014<br />
<br />
<p class="title2">5. Fungicides tests</p><br />
<p class="texte"><b>Date:</b> 08/15/2014<br />
<br />
<p class="title2">Cloning D4E1-GAFP1-EcAMP: <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a></p><br />
<br />
<p class="title2">1. Construction of <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> in pSB1C3, in <i>E. coli</i></p><br />
<br />
<p class="title3">Digestion of <a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a> and <a href="http://parts.igem.org/Part:BBa_K1364012">BBa_K1364012</a></p><br />
<p class="texte">Digestion of <a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a> by SpeI and PstI, and digestion of <a href="http://parts.igem.org/Part:BBa_K1364012">BBa_K1364012</a> by XbaI and PstI.<br />
<br><b>Date:</b> 08/11/2014<br />
<br><b>Result:</b> We obtained digested fragments of <a href="http://parts.igem.org/Part:BBa_K1364012">BBa_K1364012</a> and ~500 bp fragment of <a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a></p><br />
<br />
<p class="title3">Ligation of ~500 bp fragment from <a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a> and <a href="http://parts.igem.org/Part:BBa_K1364012">BBa_K1364012</a></p><br />
<p class="texte"><b>Date:</b> 08/11/2014<br />
<br><b>Result:</b> We obtained ligation of K1364012 and ~500 bp fragment of <a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a></p><br />
<br />
<p class="title3">Transformation of ligation of ~500 bp fragment from <a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a> and <a href="http://parts.igem.org/Part:BBa_K1364012">BBa_K1364012</a> = <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 08/12/2014</p><br />
<br />
<br />
<p class="title3">Testing 8 <i>E.coli</i> + <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> clones</p><br />
<p class="texte"><b>Date:</b> 08/14/2014 </p><br />
<br />
<p class="title3">Testing 15 <i>E.coli</i> + <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> clones</p><br />
<p class="texte"><b>Date:</b> 08/18/2014<br />
<br><b>Result:</b> clones H, J, O, Q, U, V might have the right <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> construction</p><br />
<br />
<br />
<p class="title2">2. <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> in pSB<sub>BS</sub>4S (<a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a>) and pSB<sub>BS</sub>1C (<a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>)</p><br />
<p class="title3">Digestion of <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a>, <a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a> and <a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a></p><br />
<p class="texte"><b>Date:</b> 08/22/2014<br />
<br><b>Result</b>: Digestion of <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a>, <a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a> and <a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a> by EcoRI and PstI were well performed.</p><br />
<br />
<p class="title3">Ligation of <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> with <a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a> and K1364014 with K823023</p><br />
<p class="texte"><b>Date:</b> 08/22/2014<br />
<br><b>Result:</b> We obtained 20µL ligation of K1364014 with <a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a> and of <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> with <a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>.<br />
</p><br />
<p class="title3">Transformation of ligations in <i>E. coli</i></p><br />
<p class="texte"><I>E. coli</I> transformed by <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> + <a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a> spread on LB + Amp 100µg/mL agar plate</p><br />
<p class="texte"><i>E. coli</i> transformed by <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> + <a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a> spread on LB + Amp 100µg/mL agar plate<br />
<br><b>Date:</b> 08/25/2014</p><br />
<br />
<p class="title3">Testing <i>E. coli</i> + <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> + <a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a> and <i>E. coli </i> + <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> + <a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a> clones</p><br />
<p class="texte"><b>Date:</b> 08/27/2014</p><br />
<br />
<br />
<br />
<p class="title2">3. Transformation of <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a>+<a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a> and <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> + <a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a> in <i>B. subtilis</i></p><br />
<p class="texte"><i>B. subtilis</i> transformed by 10µL <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a>+<a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a> spread on LB+Spec 75µg/mL agar plate<br />
<br><i>B. subtilis</i> transformed by 10µL <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a>+<a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a> spread on LB+Cm 15µg/mL agar plate<br />
<br><b>Date:</b> 08/28/2014</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology2">Fungicides tests</div><br />
<div class="thelanguage2"><br />
<br />
<p class="title2">1. D4E1-GAFP1</p><br />
<p class="title3">Transformation of P<sub>veg</sub> - D4E1 - GAFP1 in <i>Bacillus subtilis</i> on pSB<sub>BS</sub>4S </p><br />
<p class="texte"><b>Date:</b> 08/12/2014</p> <br />
<p class="title3">Integration threonine test + fungicide test </p><br />
<p class="texte"><b>Date:</b> 08/13/2014 </p><br />
<br />
<p class="title2"2. D4E1</p><br />
<p class="title3"><br />
Cloning D4E1 into pSB<sub>BS</sub>1C + fungicide test</p><br />
<p class="texte"><b>Date:</b>08/15/2014</p><br />
<p class="title3"><br />
D4E1 on pSB1C3 + fungicide test</p><br />
<p class="texte"><b>Date:</b>08/19/2014</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 1); return false">Collapse</a></p><br />
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{{:Team:Toulouse/template/footer}}</div>Dbbyhttp://2014.igem.org/Team:Toulouse/Team/Fun_factsTeam:Toulouse/Team/Fun facts2014-10-18T01:05:38Z<p>Dbby: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Team&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fun Facts</p> <br />
<br />
</div> <br />
</div><br />
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<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
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<div style="float:left; width:525px;"><br />
<img src="https://static.igem.org/mediawiki/2014/3/30/Kilometers.jpg" style="margin-top:5px; width:470px" /><br />
</div><br />
<br />
<p class="title2">Have you ever tried to estimate the kilometers traveled by your team during this summer?</p><br />
<p class="texte"><br />
According to our calculations, one team member walks approximately 4 kilometers per day all around the <br />
laboratory. This represents 5,544 kilometers for the whole team during the 126 days of this epic <br />
adventure. <br />
What does that mean exactly? Simply that we could walk, cycle, swim or whatever you want to <br />
Kazakhstan, Russia, Kenya… We could even have reached the USA but we decided to stay in the lab <br />
and take the plane to go to Boston!</p><br />
<br />
<br> </br><br />
<br />
<div style="float:right; width:500px;"><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Interview.jpg" style="margin-top:5px; margin-left: 62px; width:450px" /><br />
</div> <br />
<br />
<p class="title2">What do trees lining the “Canal du Midi” think about SubtiTree?</p><br />
<p class="texte"><br />
According to our homemade impartial survey, 94% of the questioned plane trees approve our project and are interested in <br />
serving as guinea pigs for our new bacterial medicine. This percentage represents 41,580 trees which <br />
are also gathered in the association called: “Happy tree friends“.</p><br />
<br />
<br />
<div style="float:left; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Multidisciplinary_yes_we_are.jpg" style="margin-top:5px; width:450px" /><br />
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<br><br />
<br><br />
<br><br />
<br> <br />
<p class="title2">Multidisciplinary… Yes we are!</p><br />
<p class="texte">Housework in our laboratory became necessary when most of people were in vacations except us. <br />
But do you know the novelty this year in our team? Times are changing because now men are <br />
cleaning! ;-)</p><br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
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<br> <br> <br><br />
<br />
<div style="float:right; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/World_cup.jpg" style="margin-top:5px; width:450px; margin-left:62px;" /><br />
</div> <br />
<br> <br> <br> <br> <br> <br><br />
<p class="title2">The “can’t miss it” event of the summer: the 2014 Football (also called Soccer in some third zone countries!!) World Cup!</p><br />
<p class="texte">From 06/12/14 to 07/13/14, the Toulouse iGEM Team was cheering for the French team. During the <br />
games of the French team, our group was juggling with wet lab and large screen projections! Despite <br />
our support, the French team did not win the World Cup. However, we have not said our last world <br />
yet: let’s see what will happen in 2018… ;-)</p><br />
<br />
<div class="clear"></div><br />
<br />
<div style="margin-top:50px; text-align:center;"><br />
<p class="title2">Have you ever...</p><br />
<p class="texte" style="text-align:center;">...forgotten a tube culture or a Petri dish?<br />
<br>Never? Let us show you what happens in that case!</p><br />
</div><br />
<center><br />
<table><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/f/fa/Old_tube1.png" width="100px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/1/1b/Old_tube2.JPG" width="200px"></td></tr><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/d/d4/Old_petri1.png" width="270px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/d/d7/Old_petri2.JPG" width="300px"></td></tr><br />
</table><br />
</center><br><br><br />
<p class="texte" style="text-align:center;">...tried to make a 3% agarose gel?</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/5a/3%25_gel.jpg" width="200px"></center><br />
<br><br />
<br><br />
<br> <br />
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<div style="text-align:center; width:760px; margin:0 auto; margin-top:15px; border-top:1px solid #555; padding-top:60px;"><br />
<p class="title2" style="padding-bottom:15px;">To finish this part, let’s do the official Awards Ceremony of the 2014 Toulouse iGEM Team!</p><br />
<ul><br />
<li class="tree"><p class="texte">The Geek Award goes to... <b>Florie</b> <br />
who spent the longest time in front of her laptop for the modeling part!</p></li><br />
<li class="tree"><p class="texte">The latest-survivor-of-weekly-meetings goes to... <b>Diane</b> who stayed up until 3am because she was skyping from South Korea!<br />
</p></li><br />
<li class="tree"><p class="texte">The worst singer award goes to ...<b>Abdel</b> who <br />
spent the whole day singing badly in the lab!</p></li><br />
<li class="tree"><p class="texte">The dancer award goes to ... <b>Camille</b><br />
who did the famous Plasmid Dance!</p></li><br />
<li class="tree"><p class="texte">The “hello you” award goes to... <b>Pierre</b> who was always saying <br />
“Hello you” each time he meets someone (approximatively 256 times per day)!</p></li><br />
<li class="tree"><p class="texte">The most tired award goes to...<b>Manon</b> but we still do not know why!</p></li><br />
<li class="tree"><p class="texte">The misplaced ideas award goes to... <b>Mathieu</b> but you do not want to know why!</p></li><br />
<li class="tree"><p class="texte">The perseverance award goes to... <b>Emeline</b> who succeeded a cloning after twelve trials!</p></li><br />
<li class="tree"><p class="texte">The drawing award goes to... <b>Fanny</b> <br />
who drew our first SubtiTree logo!</p></li><br />
<li class="tree"><p class="texte">The best phone-caller award goes to...<b>Laureen</b><br />
who was our perfect lab secretary!</p></li><br />
<li class="tree"><p class="texte">The biggest blunder in the lab award goes to ... <b>Aurélie</b> who poured an agarose gel without gel tray!</p></li><br />
<li class="tree"><p class="texte">And last but not least ... The Best Nervous breakdown Award goes to ... <b>Our -20°C freezer</b>! The whole team is grateful for its hard work during a hot summer! Three successive breakdowns during the hot days of summer: thank you freezer, so long chap, we’ll unplug you after iGEM is finished and you’ll retire, hopefully not in the Canal du Midi…</li></p><br />
</ul><br />
</div><br />
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{{:Team:Toulouse/template/footer}}</div>Dbbyhttp://2014.igem.org/Team:Toulouse/ethicsTeam:Toulouse/ethics2014-10-18T01:00:29Z<p>Dbby: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
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<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Knowledge without conscience is but the ruin of the soul."</br><br />
"Science sans conscience, n'est que ruine de l'âme", François Rabelais, 16<sup>th</sup> century.<br />
</p><br />
<br />
<p class="texte">The ethical<br />
questioning turned out to be one of the major starting points of our project.<br />
Acting on an established environment and modifying it is by no mean trivial and<br />
our combined technical and philosophical points of view. The actual purpose of our project also leads us to undertake an<br />
ethical questioning about the role of the scientist regarding “useless” things<br />
such as the trees lining along the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="title2">Is it the scientists’ role to protect beauty?<br />
</p><br />
<br />
<p class="texte"> Beauty is a<br />
feeling of satisfaction and is selfless. It is more a feeling than the property<br />
of a thing, this is not a notion we can clearly understand. Indeed, we can find<br />
something beautiful even when we don’t know the purpose of the object...</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/f/fa/Fontaine_Duchamp.jpg" width="400px"><br />
<p class="legend">Figure 1: Fontaine (Marcel Duchamp). Yes this is also art...</p></center><br />
<br />
<p class="texte">There is always<br />
a distinction between natural beauty and artistic beauty according to Hegel,<br />
a German philosopher. The artistic beauty is born from our mind and our<br />
spirit: it is an element of signification of the work of art whereas the<br />
natural beauty of the object is external. In a way, the Canal du Midi combines<br />
both types of beauty: a natural one regarding the Nature, the centenary plane<br />
trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, science judges beauty as a superficial feature not deserving to<br />
undertake any kind of scientific efforts to maintain it. The traditional role<br />
of science is to solve global issues and to elaborate complex strategies in<br />
order to find useful solutions for everyone’s life. Once made this observation,<br />
one may wonder why synthetic biology would be used only to protect the useless<br />
beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial<br />
interrogation leads us to consider science and synthetic biology from a<br />
different point of view. <b>What if the role of scientists was also to make<br />
people rediscovering the beauty of Nature? What if the bases of new scientific<br />
challenges resulted from a more local scale? </b>Science does not have to be it has so much to gain opening itself to these<br />
challenges. First scientifically, as research is never<br />
useless and as we never know the impact and the scope of our results.<br />
Then socially, as we could measure the deep interest raised by our<br />
project within the population and the media. Adopting a new vision of synthetic<br />
biology, we will probably make people change their mind about this innovative<br />
discipline. <br><br />
The traditional cold objectivity of science distances itself from the society.<br />
However, scientists are also being capable of feeling the beauty, sensitive to<br />
the charm of landscapes and <b>able to understand the usefulness of<br />
"useless" trees</b>…<br><br />
The design of a strategy to protect useless beauty may seem senseless but we<br />
believe that it is also the scientist’s duty. We have to remember that thinking<br />
is what distinguish <i>Homo sapiens</i> from other species on earth and this<br />
"thinking" feature allows us to understand the world and be conscious<br />
of our human Nature (Descartes: <i>Cogito ergo sum</i>). The art is an object<br />
of philosophical thought. Consciousness raises humans above all others living<br />
creatures. Thus, it is necessary to respect and protect art. And thus, it<br />
becomes essential to preserve the beauty of the Canal du Midi. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human intervention on Nature</p><br />
<p class="texte">Our main question is to understand the complicated relationship between man and Nature.<br />
Does mankind have the proper right to change the Nature? Is modified Nature considered as artificial?</p><br />
<p class="title2"> Mankind & Nature</p><br />
<br />
<p class="texte"> Nature deserves<br />
to be respected and loved. Mankind has always been linked to Nature as its<br />
survival depends on what comes out of the ground, the trees, the oceans… The<br />
Nature is a source of wealth for mankind. It ensures survival and development<br />
by giving men the wood, the rocks, the soil to build shelters. Being in contact<br />
with Nature can allow men to feel strong emotion, as describe by poets like<br />
Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth<br />
of humanity, man himself understood the importance of studying and mastering<br />
Nature to develop the civilization. Still today the most advanced technologies<br />
often try to mimic natural phenomena. With the development of civilization, men<br />
modified their environment, changing it for their own comfort depending on<br />
their own desire. With the increase of human activity, the natural environment<br />
is modified profundly. With industrialization, the<br />
natural environment suffered from waste discharges, oil slicks, intensive<br />
fishing (and many others...) but also the introduction of devastating species<br />
such as the pathogen,<i> Ceratocystis<br />
platani</i>. However, despite these negative aspects, men<br />
are capable of favorable actions to help the environment and fix their<br />
mistakes. The current trend is to limit the impact of human interventions on<br />
Nature, and hopefully this trend is not transient and will not vanish. A new<br />
desire is born, a wish to protect Nature and wilderness. Humanity can adhere to<br />
this position: human take advantage of the<br />
environment and the environment takes advantage of the reasoned human<br />
interventions. There is an adaptation of mankind to Nature. Moreover, humans<br />
can have empathy: people are capable of understanding emotions and cognitive<br />
states of other organisms. To respond to these feelings, humans have<br />
technological tools allowing them to fight against enemies. This is the case<br />
with our project: fighting <i>Ceratocystis<br />
platani</i>. <br />
</p><br />
<br />
<p class="texte">In conclusion,<br />
by destroying and hammering the Nature, we jeopardize our lives. We need<br />
Nature, we come from Nature and we depend on Nature for survival, food,<br />
discoveries and civilisation. Respecting, loving and<br />
preserving the beauty of it is also a question of<br />
survival.</p><br />
<br />
<p class="title2">Nature and artifice<br />
</p><br />
<br />
<p class="texte">Talking about<br />
the Nature refers to the whole world with an exception: all the transformations<br />
made by mankind. Nature exists regardless of men and his interventions whereas<br />
artificial is everything that exists because to humans.<br />
</p><br />
<br />
<p class="texte">However,<br />
pretending that natural and artificial are opposite does not seem to be true.<br />
Man cannot create without the various elements provided by Nature, he then justs transform Nature. Thus we may wonder if there is a<br />
true difference between natural and artificial. The border between these two<br />
notions is not as obvious as it seems. The landscapes are shaped by the hand of<br />
man, animals are domesticated, and now bacteria are considered as cell<br />
factories. A natural reserve is artificially preserved as a result of human<br />
actions. Is there still something natural since the birth of mankind? Actually,<br />
the artifice is a slight modification of Nature and couldn’t exist by itself.<br />
The distinction between natural and artificial seems sterile and we clearly<br />
understand that these notions are inextricably linked and need each other to<br />
exist. </p><br />
<br />
<p class="texte">In conclusion,<br />
isn't it our duty to use our unique position in the history of life and our human<br />
approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="title2">Back to our project</p><br />
<br />
<br />
<p class="texte">These<br />
inextricable links are obviously the basis of our project. We aim to<br />
artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago.Fighting a<br />
naturally occurring form of life that threatens it maybe just an imitation of<br />
the natural evolution process. What is considered today as ‘non-natural’<br />
may be one day regarded differently. To the extent that everything is done not<br />
to unbalance the ecosystem, our intervention can be judged rightful, even more<br />
than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="title2"> Potential strategies discussed<br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that<br />
SubtiTree will not survive and spread in the<br />
environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the natural environment (outside the tree) thanks to a proline auxotrophy system <br />
<br>- Prevent the sporulation of <i>B. subtilis</i> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild<br />
type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use an integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="title2">Public perception<br />
</p><br />
<br />
<p class="title3">Political and public adhesion</p><br />
<br />
<p class="texte">Due to our<br />
strong implication in preserving this magnificent work of art, our project<br />
interested several governmental services. Indeed some municipalities and<br />
regional councils supported our local engagement. Beyond that, our project<br />
interests the highest level of the “Canal du Midi” administration: the national<br />
navigation authority (VNF) and the Ministry of agriculture. Both of them funded<br />
this project. They are now looking for the continuation of the project after<br />
the iGEM competition. This is clearly a sign that we targeted the right<br />
question. </p><br />
<br />
<p class="texte">This project<br />
also received the attention of the public through several articles in<br />
newspapers, television, radio and internet. First we had just a local coverage,<br />
but days after days there were more and more media interested in SubtiTree. This mediatic coverage<br />
allowed us to contact concerned citizens who participated to the development of<br />
this project. This interaction with the public allowed us to explain and<br />
promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="title3">Safety principle</p><br />
<br />
<p class="texte">One single tree<br />
infected by Canker, and all the trees located in an area of a couple of hundred<br />
meters around are included in the prophylactic cut. We acted to preserve the<br />
surrounding trees. The modification of the endophytic<br />
microbial fauna generated by the introduction of the engineered bacterium has<br />
to be compared to the introduction of chemicals. They contain chlorine atom and<br />
aromatic hydrocarbon, so their remediation is complicated and they represent a<br />
source of pollution. By shortening the lifespan to one season and minimizing<br />
the risks of spreading, we plan a safe and environmental-friendly way to fight<br />
Canker. <br />
<br />
<br />
<p class="title2">Feasability<br />
</p><br />
<br />
<p class="texte">We wonder about<br />
the feasibility of tree’s treatment. As we used endophytic<br />
bacteria, we can count on the natural growth of SubtiTree<br />
inside the sap. So we can inject few bacteria to be sure to have enough<br />
bacteria to protect the tree. Some researchers (Xianling<br />
Ji<sup>1</sup> et al) already injected <i>Bacillus subtilis</i> in plants and<br />
observed an increase of bacteria concentration to a maximum of 10<sup>5</sup><br />
bacteria/mL</p><br />
<br />
<p class="texte">As we aim to<br />
inject a small quantity of bacteria, this treatment remains cheaper than the<br />
injection of several liters of chemical fungicides. In addition, this injection<br />
prevents the preventive tree cutting, which is very expensive. Cutting one tree<br />
cost around € 3000. The administration in charge of the protection of the<br />
“Canal du Midi” already plans to spend 220 million euros to cut and replant all<br />
trees along the Canal. Besides the important cost of cutting trees, it will<br />
destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the <br />
iGEM’s circumstances we could not have the time to go<br />
deeper. First, we can improve the fixation module. Using chitin as fixation<br />
anchor is simple but not enough specific to fix just one fungus type. That’s<br />
why we first think to fix SubtiTree to one protein<br />
included in the <i>Ceratocystis<br />
platani</i>’s<br />
membrane: CP. The bacterial prototype designed this summer can be optimized to<br />
trigger the fungicides production when the binding is completed, and to be more<br />
specific changing the peptides produced.</p><br />
<br />
<p class="title1">References</p><br />
<br />
<li class="tree"><p class="texte"> Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu.<b> Biological control against bacterialwilt and colonization of<br />
mulberry byan endophyticBacillus subtilis strain </b>. FEMS Microbiol Ecol. 65 (2008) 565–573. </p></li><br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Calendar</p> <br />
</div><br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p><br />
<br />
<br />
<div class="technology">December 2013 – January 2014: Team selection</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- iGEM competition presentation and explanations<br/><br />
- Constitution of the <a href="https://2014.igem.org/Team:Toulouse/Team">team</a> by interviewing different students from Université Toulouse III Paul Sabatier and INSA de Toulouse<br />
</p><br />
<p class="texte" style="text-align:center"><B> The adventure begins for the 2014 Toulouse iGEM Team!</B><br />
</p><br />
<br />
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</div><br />
<br />
<br />
<div class="technology">January – June 2014: Projects brainstorming</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- Thanks to weekly meetings, our team was able to discuss about new project ideas.<br />
<br/><br />
- Many ideas were approached and debated regarding the feasibility thanks to the literature and supervisors including teachers and researchers.<br><br />
<br/><br />
This is a list of the main projects: <br />
<br/><br />
- Let's save our trees with SubtiTree: the purpose is to use <i>Bacillus subtilis</i> as an optimized bacterium to target and bind to a specific fungus and secrete fungicides to destroy it.<br />
<br/><br />
- <i>E. coli</i> cancer: the concept was to create a bacterium able to colonize specifically the hypoxic regions of the tumor. The oxygen-sensitive bacterium would not be able to develop in the healthy zones.<br />
<br/><br />
- Reduction of ruminants’ methane production: the goal is to reduce the production of methane by ingesting a microorganism in the animals' rumen. This bacterium would be able to reduce the quantity of metabolic hydrogen and eliminate the methanogenic Archaes population.<br />
</p><br />
<br />
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<br />
<br />
<div class="technology">June 2014: Choice of SubtiTree project</div><br />
<div class="thelanguage"><br />
<p class="texte">The discussion has been long to estimate the practicability of our final project. By the end of May, most of the ideas were eliminated but two of them remained: controlling the cancer or saving the trees. It became easily clear that SubtiTree was successfully completed whether regarding the scientific part or the ethical aspect. Indeed, our knowledge in medicine was not sufficient enough to evaluate the consequences of a bacterium injection in the human body for <i>E. coli</i> cancer.</br><br />
Therefore, the whole team agreed on focusing on a local issue as the preservation of the Canal du Midi plane trees. However, the final goal was based on the idea to allow our optimized bacteria to be efficient for all fungi infections in the environment.</br><br />
By this period, we evaluated our budget to approximately <B>€39,500</B>.<br />
</p> <br />
<br />
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<br />
<div class="technology2">Week 1(16-22 June)</div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
- Bibliography researches about our subject<br/><br />
- Cleaning the whole lab to allow good manipulations and avoid any contamination<br/><br />
- Inventory of necessary lab equipment and biobricks<br/><br />
- Summarize the iGEM protocols<br/><br />
- Prepare all growing media <br/><br />
- Preparing competent cells by optimizing protocols<br/><br />
- Transformation of RFP plasmid to practice<br />
</p><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Increase in our communication strategy to find more sponsors specialized in environment and ecological matters <br/><br />
- Support of Adisseo, Toulouse White Biotechnology, LISBP, INSA Toulouse<br/><br />
- Organization of a weekly meeting each Friday with our supervisors to tackle any problem encountered<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/20/2014)</p><br />
<p class="texte"><br />
- Distribution of the non-scientific tasks regarding the wiki, communication, sponsorship, ethical problems, safety but also the lab work<br/><br />
- Discussion about the different construction parts of the project<br/><br />
- Need to check the absence of stop codon in fungicides sequences<br/><br />
- Ordering the list of necessary products for the laboratory and biobricks<br/><br />
- Checking and validation of the genes sequences<br />
</p> <br />
<br />
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<br />
<br />
<div class="technology2">Week 2 (23-29 June)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- BioSynSyS conference work: powerpoint, logo in order to present our SubtiTree project to a whole audience of scientific and researchers<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- <i>E. coli</i> transformation with BBA_J004450 (pSB1C3)<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/27/2014)</p><br />
<p class="texte"><br />
- Concentration of antibiotics in media must be checked<br/><br />
- The transformation rate for pUC19 must be evaluated<br/><br />
- The transformation efficiency must be calculated regarding the quantity of DNA<br/><br />
- Possibility to contact the cities halls for the sponsorship<br/><br />
- Check the mechanism of action of each fungicide to complete the ethical part<br />
</p><br />
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<br />
<br />
<div class="technology">July 2014</div><br />
<div class="thelanguage"><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Beginning of the laboratory work to create our optimized bacterium <br/><br />
- Research of sponsors and communication thanks to the press<br />
</p> <br />
<br />
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<br />
<div class="technology2">Week 3 (30 June -6 July) and Week 4 (7-13 July)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the BioSynSys conferences: presentation of SubtiTree project<br/><br />
- We have put in place a newsletter system the first friday of each month to keep all our sponsors and people who support us updated about the project (financial, scientific and communication aspects).<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Design of all the constructions needed with cloning and restriction enzymes to save some time and allow a better comprehension of our bacterium system </br><br />
Problem: A problem was reported in the use of the CYP. Indeed, the BioBrick is not expressed properly in <i>Bacillus subtilis</i> strain. Therefore we had to look at the literature to find a new fungicide called GAFP-1.<br/><br />
- A bioreactor was conceived to reproduce the composition of the sap in the plane trees. The main goal was to identify the behavior of <i>Bacillus</i> strain in a medium composed of many different carbon sources.<br/><br />
- Competent cells and transformation practice using GFP and RFP.<br />
</p><br />
<br />
<p class="title3">Weekly meetings with the instructors (07/04/2014) and (07/11/2014)</p><br />
<p class="texte"><br />
- iGEM efficiency kit does not work, we shall not use it anymore<br/><br />
- Organization of a timetable to check the stored and sterile equipment everyday</br><br />
- Try a transformation in <i>Bacillus subtilis</i><br />
</p> <br />
<br />
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<br />
<div class="technology2">Week 5 (14-20 July)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Our team was interviewed by many local newspapers such as 20minutes, Metronews, La Voix du Midi, La Dépêche but also by the local television channel France3 Midi-Pyrénées to present our draft to the community. Moreover, SubtiTree was approached in some radio programs like Virgin Radio Toulouse and France info.</br><br />
- Support of ThermoFisher, New Englands BioLabs, Qiagen, Eurofins but also GenoToul, Labex Tulip, L’Université Paul Sabatier, CROUS.</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- GFP and RFP digestion amplified by miniprep and gel electrophoresis <br/><br />
Problem: the digested GFP doesn’t appear on the gel contrary to RFP plasmid. We assumed a problem with the GFP miniprep. We tried the miniprep from the INSA Toulouse team from 2013 and the GFP plasmid was fully digested.<br/><br />
- Transformation and cryopreservation of BioBricks BBa_K823002 (P<sub>lep</sub>A), BBa_K823003 (P<sub>veg</sub>), BBa_K606061 (RBS SpoVG) and BBa_B0015 (double terminator), BBa_K823023 (pSB<sub>BS</sub>1C), BBa_K733013 (P<sub>veg</sub> + RBS) in <i>E. coli</i>.<br/><br />
- Transformation of the Munich <i>B. subtilis</i> backbones<br/><br />
- Cloning of BBa_K606061 (RBS SpoVG) with BBa_K823003 (P<sub>veg</sub>) and BBa_K823002 (P<sub>lepA</sub>)<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (07/18/2014)</p><br />
<p class="texte"><br />
- Transformation of the Eurofins genes<br/><br />
- Start assembling the BioBricks for the fungicides<br/><br />
- Check all the cloning<br />
</p><br />
<br />
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<br />
<div class="technology2">Week 6 (21 July-27 July)</div><br />
<div class="thelanguage2"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Description of SubtiTree project and determination of the goodies for a crowdfunding campaign on Ulule website</p><br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Transformation of BBA_1364002 (GAFP1) and BBA_1364003 (D4E1) fungicides genes<br/><br />
- Subculture of the clones for each gene<br/><br />
- PCR and migration on electrophoresis gel<br/><br />
- Transformation BBA_1364003 (D4E1) on pSB1C3 and BBA_1364002 (GAFP1) on pSB1C3<br/><br />
- Cloning BBA_1364002 (GAFP1) with BBa_B0015 (double terminator) and BBA_1364003 (D4E1) with BBa_K823003 (P<sub>veg</sub>)<br />
</p><br />
<br />
<p class="title3">Others:</p><br />
<p class="texte"><br />
- Research for plane tickets and hotels in Boston for the Giant Jamboree<br/><br />
- New idea: analyze the plane tree sap to determine its composition<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology">August 2014</div><br />
<div class="thelanguage"><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finishing the cloning for the different parts of the bacterium<br/><br />
- Putting in place the fungicides, binding and chemotaxis tests<br />
</p> <br />
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<br />
<div class="technology2">Week 7 (28 July-3 August)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Financial supports of the Ministery (Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt) and Voies Navigables de France<br/><br />
- Interview with Johan Langot from Sciences Animation for a possible presentation at the Toulouse Festival of Science<br/><br />
- Launch of the crowdfunding campaign on Ulule<br />
</p><br />
<br />
<p class="title3">Lab work: </p><br />
<p class="texte"><br />
- Cloning BBa_K823003 (P<sub>veg</sub>) + RFP and BBa_K823002 (P<sub>lepA</sub>) + RFP in pSB<sub>Bs</sub>1C<br/><br />
- Test of every pSB<sub>BS</sub> vector: BBa_K823021 (pSB<sub>BS</sub>1C-lacZ), BBa_K823022 (pSB<sub>BS</sub>4S), BBa_K823023 (pSB<sub>BS</sub>1C), minipreps and cryopreservation of the best clones<br/><br />
- Checking of BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) transformants (BioBrick BBa_K1364007) and BBA_1364003 (D4E1) + BBa_K823003 (P<sub>veg</sub>) BioBrick BBa_K1364009<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) + pSB<sub>BS</sub>4S (BBa_K823022) and subculture of the colonies<br/><br />
- Cloning of BBa_K823003 (Pveg) + BBA_1364003 (D4E1) with BBa_B0015 (double terminator)<br/><br />
- Cloning of BBa_K823003 (Pveg) + RFP (BBa_K1364017) in pSB<sub>BS</sub>4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_K823002 (PlepA) + RFP (BBa_K1364016) in pSB<sub>BS</sub>4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_1364018 (Strong Promotor + RFP)<br/><br />
- Cloning of BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) + Promotor BBa_K823003 (P<sub>veg</sub>) BioBrick BBa_K1364012 with gel extraction for BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) and a PCR cleanup for BBa_K823003 (P<sub>veg</sub>), subculture<br/><br />
- Cloning BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBS4S in <i>E. coli</i><br/><br />
- Transformation of binding gene with <i>E. coli</i> competent cells<br/><br />
- Transformation of BBa_K1364000, the chemotaxis gene in <i>E. coli</i><br/><br />
Problem: the transformation worked but a cell layer appeared. A new subculture of the colonies was made as well as a new spreading on petri dishes.<br/><br />
- Spreading of BBa_K1162001 (EcAMP), miniprep and digestion<br/><br />
Problem: the digestion did not work => Issue with the quantity of DNA in the miniprep is assumed<br/><br />
- First experience of chemotaxis with a glucose chemo-attractant<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (08/01/2014)</p><br />
<p class="texte"><br />
- Possible problem with the restriction enzymes regarding the digestion: new recommendations<br />
<br/><br />
- Discussion about EcAMP cloning which presents some issues<br />
<br/><br />
- Discussion about the transfer of pSB1C3 from <i>E. coli</i> to <i>B. subtilis</i> strain<br />
</p> <br />
<br />
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<br />
<div class="technology2">Week 8 (04-10 August)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the “Passion Jeune” competition organized by a French bank foundation named Fondation Crédit Agricole Toulouse<br />
</p><br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Check the cloning of BBa_K823003 (P<sub>veg</sub>)+BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSB<sub>BS</sub>4S and cryopreservation<br />
<br/><br />
- Transformation of BBa_K823003 (P<sub>veg</sub>) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) (BioBrick BBa_ K1364008) in BBa_K823022 (pSB<sub>BS</sub>4S) <br />
<br/><br />
- Cloning of BBa_K1364001, the binding gene + BBa_K823003 (P<sub>veg</sub>) on pSB1C3 and pSB<sub>BS</sub>4S<br />
<br/><br />
Problem: the band is at 1500bp instead of 1300bp on the gel<br />
<br/><br />
- Cloning of BBa_K1162001 (EcAMP) + BBa_K823003 (P<sub>veg</sub>) + BBa_K606061 (RBS SpoVG), miniprep, PCR and digestion <br />
<br/><br />
Problem: the fragment of DNA is too small (149bp) to be seen on the gel and was probably lost during the PCR cleanup. Instead of that, we used the heat enzymes inactivation at 95°C for 15 minutes. <br />
<br/><br />
- Cloning of Promotor + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) in pSB1C3 and pSB<sub>BS</sub>4S and cryopreservation<br />
<br/><br />
- Elaboration of an efficient fungicide test protocol <br />
<br/><br />
- Test of the fungus growth on PDA medium + test of growth for <i>B. subtilis</i> liquid culture on PDA<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/08/2014)</p><br />
<p class="texte"><br />
- Ask iGEM headquarters if all the biobricks must be on pSB1C3 plasmid<br />
<br/><br />
- Use another strain of <i>E. coli</i> (DH5-1 instead of DH5 alpha) to have a faster growth<br />
<br/><br />
- Check which quantity of <i>B. subtilis</i> is necessary to naturally destroy the fungus<br />
</p><br />
<br />
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<br />
<div class="technology2">Week 9 (11-17 August)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Publication of more articles about our project in Labiotech, Développement durable (Networkvisio), Midi Libre, l’Indépendant, DigiSchool, La voix du Midi Lauragais, LURIO Addl.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Evaluation of the bacterial decrease rate from 10°C to 4°C with a sporuling strain of <i>B. subtilis</i>.<br />
<br/><br />
- Chemotaxis test with different concentrations of glucose on a 0.3% agar.<br />
<br/><br />
- Miniprep of pSB<sub>B</sub>4S with binding gene and transformation in <i>B. subtilis</i>.<br />
<br/><br />
Problem: no digestion was visible on the gel => the cloning failed<br />
<br/><br />
- Transformation of BBa_K1374010 (P<sub>veg</sub> + RBS SpoVG + EcAMP) + BBa_K1364012 (RBS + GAFP1 + D4E1 + double terminator), subculture<br />
<br/><br />
- Subculture of <i>B. subtilis</i> clones transformed by BBa_K823003 (P<sub>veg</sub>) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) in pSB<sub>B</sub>4S, cryopreservation<br />
<br/><br />
- Fungicide test<br />
</p><br />
<br />
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<br />
<div class="technology2">Week 10 (18-24 August)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Ethics: Interview with a specialist in ethical sciences, Vincent Grégoire Delory to complete our reflexion about the ethical issues in SubtiTree project.<br />
<br/><br />
- Wiki: hiring a web designer, Adrien Nicod, to improve the aesthetic effect and the logo.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Subculture and transformation in <i> B. subtilis </i> of BBa_K1364011 in pSB<sub>BS</sub>4S (BBa_K823022) and cloning of BBa_K1364011 on pSB<sub>BS</sub>1C (BBa_K823023).<br />
<br/><br />
Problem: no clone had the insert on pSB<sub>BS</sub>1C => the cloning had to be done again<br />
<br>- Miniprep of BBa_K1364011 in BBa_K823023 (pSB<sub>BS</sub>1C) + Transformation in <i>B. subtilis</i>.<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) , BBA_1364002 (GAFP1) , BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) on BBa_K823023 (pSB<sub>BS</sub>1C) <br />
<br/><br />
- Transformation BBa_1364004 (in pSB<sub>BS</sub>4S in <i>E. coli</i> )<br />
<br/><br />
- Cloning of BBa_K1364014 (P<sub>veg</sub> + RBS SpoVG + EcAMP + GAFP1 + D4E1 + double terminator) in K823022 (pSB<sub>BS</sub>4S) and in BBa_K823023 (pSB<sub>BS</sub>1C), miniprep and digestion by restriction enzymes to see the size of the insert on a 1% gel<br />
<br/><br />
Problem: no clone had the insert on pSB<sub>BS</sub>1C => the cloning had to be done again<br />
</p><br />
<br />
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<br />
<div class="technology2">Week 11 (25- 31 August)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Beginning of the creation and redaction of the different parts of the wiki by the Toulouse iGEM Toulouse<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Cloning of BBa_K1364014 in BBa_K823022 and in BBa_K823023 <br />
<br/><br />
- Cloning GAFP1 + BBa_K823023 (pSB<sub>BS</sub>1C) in <i>B. subtilis</i> with a change in the protocol: pre-induction of the culture with 0.125 µg/ml of chloramphenicol one hour after the addition of DNA, and let grow for another hour.<br />
<br/><br />
Problem: no colony <br />
<br/><br />
- Liquid culture of <i>Bacillus subtilis</i> + BBA_1364002 (GAFP1); <i>Bacillus subtilis</i> + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) ; <i>Bacillus subtilis</i> + BBA_1364003 (D4E1) for future fungicide tests<br />
<br/><br />
- Miniprep of BBa_ K1364011 in BBa_ K823023 (pSB<sub>BS</sub>1C) and transformation in <i>Bacillus subtilis</i> strain. <br />
<br/><br />
- Fungicide test of BBa_K1364011 in pSB<sub>BS</sub>4S, Promotor + BBA_1364002 (GAFP1) + Terminator (BioBrick BBa_K1364008) in pSB<sub>BS</sub>4S, Promotor + BBA_1364003 (D4E1) + Terminator (BioBrick BBa_K1364009) in pSB<sub>BS</sub>4S, Promotor + BBA_1364002 (GAFP1) + BA_1364003 (D4E1) + Terminator (BioBrick BBa_K1364013) in pSB<sub>BS</sub>4S<br />
<br/><br />
- Transformation of a new vector PKL 190 (threonine integrative) in <i>Bacillus</i> strain <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/29/2014)</p><br />
<p class="texte"><br />
- Boston accommodation must be booked<br />
<br/><br />
- Discussion about the contamination seen on the fungicide tests : the issue seems to come from the sterile water which contained contaminants<br />
<br/><br />
- Objectives : chemotaxis, binding and fungicides tests have to be performed again<br />
</p> <br />
<br />
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<br />
<div class="technology">September 2014</div><br />
<div class ="thelanguage"><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finish the tests of the different modules<br />
<br/><br />
- Establish the final editorial parts for the wiki<br />
<br/><br />
- Design of the wiki<br />
<br/><br />
- Sequencing the different assembled parts of our bacterium<br />
</p> <br />
<br />
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<br />
<div class="technology2">Week 12 (1-7 September)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Different parts of the wiki by the Toulouse iGEM Toulouse<br/><br />
- Reservation of Boston accommodation<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Chemotaxis test and modeling<br />
<br/><br />
- Fungicides tests on EcAMP-A, EcAMP-B, EcAMP-C, EcAMP-D, MaxiOpA, MaxiOpB, MaxiOpC, MaxiOpD from 2ml of overnight cultures. Only with 25000 conidia with both fungi.<br />
<br/><br />
- PCR on pSB<sub>BS</sub>4S + BBa_K1162001 (EcAMP) <br />
<br/><br />
Problem: failed on both diluted plasmid and colony<br />
<br/><br />
- Spreading of BBa_K1364014 + BBa_ K823022 in threonine medium and in medium without threonine <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/05/2014)</p> <br />
<p class="texte"><br />
- The first sequencing was not successful => new sequencing with one primer/tube<br/><br />
- Chemotaxis test: increase the concentration in bacterium, try a new protocol by capillarity<br/><br />
- End of the binding test: IT WORKS PERFECTLY! SubtiTree has the good phenotype and presents a better binding property than the wild type <i>Bacillus subtilis</i> strain.<br/><br />
- Fungicides test: contaminants with the fungus. Need to ask for another culture of our fungi.<br />
</p><br />
<br />
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<br />
<div class="technology2">Week 13 (8- 14 September)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Telephone interview for an article in a French national review named Fluvial which will be published at the end of September.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- PCR on D4E1 colony with different primers<br />
<br/>- Culture and miniprep of EcAMP to get more DNA + analytic digestion <br />
<br/>- Transformation of K1364014 + K1364006 in <i>E. coli</i> <br />
<br/>- Transformation of GAFP1 + D4E1, GAFP1 and EcAMP + GAFP1 + D4E1 on <i>B. subtilis</i> + colony PCR + threonine test<br />
<br/><i>NB: good results for the maxi operon and GAFP1 but not for GAFP1 + D4E1</i><br />
<br/>- Cloning of K606013 + pEX_K4, 823022 + P<sub>lepA</sub>_RFP, 823022 + P<sub>veg</sub>_RFP<br />
<br/>- Chemotaxis tests: different times of LB + agar addition in the LB medium (after 3 minutes, 5 minutes, 8 minutes, 10 minutes…) are performed => No difference of the results after spreading on the plates.<br />
<br/>- New test of chemotaxis: capillary between two Eppendorf tubes<br />
<br/>- Spreading of the fungi on a medium which mimics the sap<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/11/2014)</p> <br />
<p class="texte"><br />
- Try to use the GFP or RFP to follow the chemotaxis test.<br />
<br/>- Make a new glass process for the chemotaxis capillary test.<br />
<br/>- Possibility of contacting an INRA laboratory to get pictures of the binding test with confocal microscope.<br />
<br/>- Sequencing the binding construction.<br />
<br/>- Try different temperatures for the fungicide tests: 20°C, 25°C, and 37°C to reduce the fungus growth.<br />
</p><br />
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<br />
<div class="technology2">Week 14 (15 - 21 September)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Support of two French city halls: Mairie de Montréal and Mairie de Ventenac en Minervois.<br />
<br/>- First interview by the French national radio France Inter which will be aired on October 19<sup>th</sup>.<br />
<br/>- Beginning of the organization of the Toulouse iGEM team acknowledgement day: the purpose is to present the project to the students and give a feedback on the competition to the sponsors.<br />
<br/>- Final design of the wiki<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Threonine tests for the BBa_K1364014 maxi operon (EcAMP1 – GAFP1 – D4E1) and mini operon BBa_K1364013 (GAFP1 - D4E1)<br />
<br/>- Colony PCR on the mini operon BBa_K1364013 (GAFP1 - D4E1)<br />
<br/>- Colony PCR on BBa_K1364011 on BBa_K823022 (pSBBS4S)<br />
<br/>- Chemotaxis test with wild type <i>B. subtilis</i> strain using Imperial College protocol<br />
<br/>- Tansformation of BBa_K1364011 + BBa_K823022 (pSB<sub>BS</sub>4S) in <i>B. subtilis</i><br />
<br/>- Culture of fungi with fungicides<br />
<br/>- Sequencing of plasmids containing the fungicides<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (09/18/2014)</p> <br />
<p class="texte"><br />
- Sequencing worked for D4E1 and GAFP1 and the mini operon BBa_K1364013 (GAFP1-D4E1).<br />
<br/>- The next step is to sequence the maxi operon (EcAMP1 – GAFP1 – D4E1).<br />
<br/>- Try different approaches for the chemotaxis:<br />
<br/>New protocol with the capillary assay with a new system made by a glassblower.<br />
<br/>Make the Imperial College test again by testing 3 different solutions: chitin, N-acetylglucosamine and another carbon source.<br />
<br/>- Decrease the number of conidia and temperature for the fungicide test.<br />
</p><br />
<br />
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<br />
<div class="technology2">Week 15 (22 - 28 September)</div><br />
<div class="thelanguage2"><br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- New support of Sallèles d’Aude and Labastide d'Anjou city halls but also the Département de l'Hérault.<br />
<br/>- Payment of the plane tickets.<br />
<br/>- Acknowledgement day (4<sup>th</sup> December): contact with catering service, reservation of the amphitheater, list of guests…<br />
<br/>- Order of the T shirt for the Jamboree! We look awesome with them!<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Last experiments regarding chemotaxis tests.<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/26/2014)</p> <br />
<p class="texte"><br />
- Focus on the wiki design and writing.<br />
<br/>- Start making the power point presentation for the Giant Jamboree and the poster.<br />
<br/>- Division of responsibilities for the non-scientific work.<br />
</p><br />
<br />
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<div class="technology">October 2014</div><br />
<div class="thelanguage"><br />
<p class="title2">Main activites</p><br />
<p class="texte"><br />
- Preparation of the wiki every day... all day long... so hard to work on it, especially at night!<br />
<br/>- Preparation of the poster and the presentation for the Giant Jamboree with our instructors <br />
<br/><br />
- Daily presentations in front of the laboratory teachers to repeat again and again and again… let’s get ready for the Jamboree!<br />
</p> <br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Calendar</p> <br />
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<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p><br />
<br />
<br />
<div class="technology">December 2013 – January 2014: Team selection</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- iGEM competition presentation and explanations<br/><br />
- Constitution of the <a href="https://2014.igem.org/Team:Toulouse/Team">team</a> by interviewing different students from Université Toulouse III Paul Sabatier and INSA de Toulouse<br />
</p><br />
<p class="texte" style="text-align:center"><B> The adventure begins for the 2014 Toulouse iGEM Team!</B><br />
</p><br />
<br />
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<br />
<br />
<div class="technology">January – June 2014: Projects brainstorming</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- Thanks to weekly meetings, our team was able to discuss about new project ideas.<br />
<br/><br />
- Many ideas were approached and debated regarding the feasibility thanks to the literature and supervisors including teachers and researchers.<br><br />
<br/><br />
This is a list of the main projects: <br />
<br/><br />
- Let's save our trees with SubtiTree: the purpose is to use <i>Bacillus subtilis</i> as an optimized bacterium to target and bind to a specific fungus and secrete fungicides to destroy it.<br />
<br/><br />
- <i>E. coli</i> cancer: the concept was to create a bacterium able to colonize specifically the hypoxic regions of the tumor. The oxygen-sensitive bacterium would not be able to develop in the healthy zones.<br />
<br/><br />
- Reduction of ruminants’ methane production: the goal is to reduce the production of methane by ingesting a microorganism in the animals' rumen. This bacterium would be able to reduce the quantity of metabolic hydrogen and eliminate the methanogenic Archaes population.<br />
</p><br />
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<br />
<br />
<div class="technology">June 2014: Choice of SubtiTree project</div><br />
<div class="thelanguage"><br />
<p class="texte">The discussion has been long to estimate the practicability of our final project. By the end of May, most of the ideas were eliminated but two of them remained: controlling the cancer or saving the trees. It became easily clear that SubtiTree was successfully completed whether regarding the scientific part or the ethical aspect. Indeed, our knowledge in medicine was not sufficient enough to evaluate the consequences of a bacterium injection in the human body for <i>E. coli</i> cancer.</br><br />
Therefore, the whole team agreed on focusing on a local issue as the preservation of the Canal du Midi plane trees. However, the final goal was based on the idea to allow our optimized bacteria to be efficient for all fungi infections in the environment.</br><br />
By this period, we evaluated our budget to approximately <B>€39,500</B>.<br />
</p> <br />
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<br />
<div class="technology2">Week 1(16-22 June)</div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
- Bibliography researches about our subject<br/><br />
- Cleaning the whole lab to allow good manipulations and avoid any contamination<br/><br />
- Inventory of necessary lab equipment and biobricks<br/><br />
- Summarize the iGEM protocols<br/><br />
- Prepare all growing media <br/><br />
- Preparing competent cells by optimizing protocols<br/><br />
- Transformation of RFP plasmid to practice<br />
</p><br />
<br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Increase in our communication strategy to find more sponsors specialized in environment and ecological matters <br/><br />
- Support of Adisseo, Toulouse White Biotechnology, LISBP, INSA Toulouse<br/><br />
- Organization of a weekly meeting each Friday with our supervisors to tackle any problem encountered<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors (06/20/2014)</p><br />
<p class="texte"><br />
- Distribution of the non-scientific tasks regarding the wiki, communication, sponsorship, ethical problems, safety but also the lab work<br/><br />
- Discussion about the different construction parts of the project<br/><br />
- Need to check the absence of stop codon in fungicides sequences<br/><br />
- Ordering the list of necessary products for the laboratory and biobricks<br/><br />
- Checking and validation of the genes sequences<br />
</p> <br />
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<br />
<div class="technology2">Week 2 (23-29 June)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- BioSynSyS conference work: powerpoint, logo in order to present our SubtiTree project to a whole audience of scientific and researchers<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- <i>E. coli</i> transformation with BBA_J004450 (pSB1C3)<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors (06/27/2014)</p><br />
<p class="texte"><br />
- Concentration of antibiotics in media must be checked<br/><br />
- The transformation rate for pUC19 must be evaluated<br/><br />
- The transformation efficiency must be calculated regarding the quantity of DNA<br/><br />
- Possibility to contact the cities halls for the sponsorship<br/><br />
- Check the mechanism of action of each fungicide to complete the ethical part<br />
</p><br />
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<br />
<div class="technology">July 2014</div><br />
<div class="thelanguage"><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Beginning of the laboratory work to create our optimized bacterium <br/><br />
- Research of sponsors and communication thanks to the press<br />
</p> <br />
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<br />
<br />
<div class="technology2">Week 3 (30 June -6 July) and Week 4 (7-13 July)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the BioSynSys conferences: presentation of SubtiTree project<br/><br />
- We have put in place a newsletter system the first friday of each month to keep all our sponsors and people who support us updated about the project (financial, scientific and communication aspects).<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Design of all the constructions needed with cloning and restriction enzymes to save some time and allow a better comprehension of our bacterium system </br><br />
Problem: A problem was reported in the use of the CYP. Indeed, the BioBrick is not expressed properly in <i>Bacillus subtilis</i> strain. Therefore we had to look at the literature to find a new fungicide called GAFP-1.<br/><br />
- A bioreactor was conceived to reproduce the composition of the sap in the plane trees. The main goal was to identify the behavior of <i>Bacillus</i> strain in a medium composed of many different carbon sources.<br/><br />
- Competent cells and transformation practice using GFP and RFP.<br />
</p><br />
<br />
<p class="title4">Weekly meetings with the instructors (07/04/2014) and (07/11/2014)</p><br />
<p class="texte"><br />
- iGEM efficiency kit does not work, we shall not use it anymore<br/><br />
- Organization of a timetable to check the stored and sterile equipment everyday</br><br />
- Try a transformation in <i>Bacillus subtilis</i><br />
</p> <br />
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<br />
<div class="technology2">Week 5 (14-20 July)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Our team was interviewed by many local newspapers such as 20minutes, Metronews, La Voix du Midi, La Dépêche but also by the local television channel France3 Midi-Pyrénées to present our draft to the community. Moreover, SubtiTree was approached in some radio programs like Virgin Radio Toulouse and France info.</br><br />
- Support of ThermoFisher, New Englands BioLabs, Qiagen, Eurofins but also GenoToul, Labex Tulip, L’Université Paul Sabatier, CROUS.</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- GFP and RFP digestion amplified by miniprep and gel electrophoresis <br/><br />
Problem: the digested GFP doesn’t appear on the gel contrary to RFP plasmid. We assumed a problem with the GFP miniprep. We tried the miniprep from the INSA Toulouse team from 2013 and the GFP plasmid was fully digested.<br/><br />
- Transformation and cryopreservation of BioBricks BBa_K823002 (P<sub>lep</sub>A), BBa_K823003 (P<sub>veg</sub>), BBa_K606061 (RBS SpoVG) and BBa_B0015 (double terminator), BBa_K823023 (pSB<sub>BS</sub>1C), BBa_K733013 (P<sub>veg</sub> + RBS) in <i>E. coli</i>.<br/><br />
- Transformation of the Munich <i>B. subtilis</i> backbones<br/><br />
- Cloning of BBa_K606061 (RBS SpoVG) with BBa_K823003 (P<sub>veg</sub>) and BBa_K823002 (P<sub>lepA</sub>)<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors (07/18/2014)</p><br />
<p class="texte"><br />
- Transformation of the Eurofins genes<br/><br />
- Start assembling the BioBricks for the fungicides<br/><br />
- Check all the cloning<br />
</p><br />
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<br />
<div class="technology2">Week 6 (21 July-27 July)</div><br />
<div class="thelanguage2"><br />
<br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Description of SubtiTree project and determination of the goodies for a crowdfunding campaign on Ulule website</p><br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Transformation of BBA_1364002 (GAFP1) and BBA_1364003 (D4E1) fungicides genes<br/><br />
- Subculture of the clones for each gene<br/><br />
- PCR and migration on electrophoresis gel<br/><br />
- Transformation BBA_1364003 (D4E1) on pSB1C3 and BBA_1364002 (GAFP1) on pSB1C3<br/><br />
- Cloning BBA_1364002 (GAFP1) with BBa_B0015 (double terminator) and BBA_1364003 (D4E1) with BBa_K823003 (P<sub>veg</sub>)<br />
</p><br />
<br />
<p class="title4">Others:</p><br />
<p class="texte"><br />
- Research for plane tickets and hotels in Boston for the Giant Jamboree<br/><br />
- New idea: analyze the plane tree sap to determine its composition<br />
</p><br />
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<br />
<div class="technology">August 2014</div><br />
<div class="thelanguage"><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finishing the cloning for the different parts of the bacterium<br/><br />
- Putting in place the fungicides, binding and chemotaxis tests<br />
</p> <br />
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<br />
<div class="technology2">Week 7 (28 July-3 August)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Financial supports of the Ministery (Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt) and Voies Navigables de France<br/><br />
- Interview with Johan Langot from Sciences Animation for a possible presentation at the Toulouse Festival of Science<br/><br />
- Launch of the crowdfunding campaign on Ulule<br />
</p><br />
<br />
<p class="title4">Lab work: </p><br />
<p class="texte"><br />
- Cloning BBa_K823003 (P<sub>veg</sub>) + RFP and BBa_K823002 (P<sub>lepA</sub>) + RFP in pSB<sub>Bs</sub>1C<br/><br />
- Test of every pSB<sub>BS</sub> vector: BBa_K823021 (pSB<sub>BS</sub>1C-lacZ), BBa_K823022 (pSB<sub>BS</sub>4S), BBa_K823023 (pSB<sub>BS</sub>1C), minipreps and cryopreservation of the best clones<br/><br />
- Checking of BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) transformants (BioBrick BBa_K1364007) and BBA_1364003 (D4E1) + BBa_K823003 (P<sub>veg</sub>) BioBrick BBa_K1364009<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) + pSB<sub>BS</sub>4S (BBa_K823022) and subculture of the colonies<br/><br />
- Cloning of BBa_K823003 (Pveg) + BBA_1364003 (D4E1) with BBa_B0015 (double terminator)<br/><br />
- Cloning of BBa_K823003 (Pveg) + RFP (BBa_K1364017) in pSB<sub>BS</sub>4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_K823002 (PlepA) + RFP (BBa_K1364016) in pSB<sub>BS</sub>4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_1364018 (Strong Promotor + RFP)<br/><br />
- Cloning of BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) + Promotor BBa_K823003 (P<sub>veg</sub>) BioBrick BBa_K1364012 with gel extraction for BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) and a PCR cleanup for BBa_K823003 (P<sub>veg</sub>), subculture<br/><br />
- Cloning BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBS4S in <i>E. coli</i><br/><br />
- Transformation of binding gene with <i>E. coli</i> competent cells<br/><br />
- Transformation of BBa_K1364000, the chemotaxis gene in <i>E. coli</i><br/><br />
Problem: the transformation worked but a cell layer appeared. A new subculture of the colonies was made as well as a new spreading on petri dishes.<br/><br />
- Spreading of BBa_K1162001 (EcAMP), miniprep and digestion<br/><br />
Problem: the digestion did not work => Issue with the quantity of DNA in the miniprep is assumed<br/><br />
- First experience of chemotaxis with a glucose chemo-attractant<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors (08/01/2014)</p><br />
<p class="texte"><br />
- Possible problem with the restriction enzymes regarding the digestion: new recommendations<br />
<br/><br />
- Discussion about EcAMP cloning which presents some issues<br />
<br/><br />
- Discussion about the transfer of pSB1C3 from <i>E. coli</i> to <i>B. subtilis</i> strain<br />
</p> <br />
<br />
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<div class="technology2">Week 8 (04-10 August)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the “Passion Jeune” competition organized by a French bank foundation named Fondation Crédit Agricole Toulouse<br />
</p><br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Check the cloning of BBa_K823003 (P<sub>veg</sub>)+BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSB<sub>BS</sub>4S and cryopreservation<br />
<br/><br />
- Transformation of BBa_K823003 (P<sub>veg</sub>) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) (BioBrick BBa_ K1364008) in BBa_K823022 (pSB<sub>BS</sub>4S) <br />
<br/><br />
- Cloning of BBa_K1364001, the binding gene + BBa_K823003 (P<sub>veg</sub>) on pSB1C3 and pSB<sub>BS</sub>4S<br />
<br/><br />
Problem: the band is at 1500bp instead of 1300bp on the gel<br />
<br/><br />
- Cloning of BBa_K1162001 (EcAMP) + BBa_K823003 (P<sub>veg</sub>) + BBa_K606061 (RBS SpoVG), miniprep, PCR and digestion <br />
<br/><br />
Problem: the fragment of DNA is too small (149bp) to be seen on the gel and was probably lost during the PCR cleanup. Instead of that, we used the heat enzymes inactivation at 95°C for 15 minutes. <br />
<br/><br />
- Cloning of Promotor + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) in pSB1C3 and pSB<sub>BS</sub>4S and cryopreservation<br />
<br/><br />
- Elaboration of an efficient fungicide test protocol <br />
<br/><br />
- Test of the fungus growth on PDA medium + test of growth for <i>B. subtilis</i> liquid culture on PDA<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors(08/08/2014)</p><br />
<p class="texte"><br />
- Ask iGEM headquarters if all the biobricks must be on pSB1C3 plasmid<br />
<br/><br />
- Use another strain of <i>E. coli</i> (DH5-1 instead of DH5 alpha) to have a faster growth<br />
<br/><br />
- Check which quantity of <i>B. subtilis</i> is necessary to naturally destroy the fungus<br />
</p><br />
<br />
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<br />
<div class="technology2">Week 9 (11-17 August)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Publication of more articles about our project in Labiotech, Développement durable (Networkvisio), Midi Libre, l’Indépendant, DigiSchool, La voix du Midi Lauragais, LURIO Addl.<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Evaluation of the bacterial decrease rate from 10°C to 4°C with a sporuling strain of <i>B. subtilis</i>.<br />
<br/><br />
- Chemotaxis test with different concentrations of glucose on a 0.3% agar.<br />
<br/><br />
- Miniprep of pSB<sub>B</sub>4S with binding gene and transformation in <i>B. subtilis</i>.<br />
<br/><br />
Problem: no digestion was visible on the gel => the cloning failed<br />
<br/><br />
- Transformation of BBa_K1374010 (P<sub>veg</sub> + RBS SpoVG + EcAMP) + BBa_K1364012 (RBS + GAFP1 + D4E1 + double terminator), subculture<br />
<br/><br />
- Subculture of <i>B. subtilis</i> clones transformed by BBa_K823003 (P<sub>veg</sub>) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) in pSB<sub>B</sub>4S, cryopreservation<br />
<br/><br />
- Fungicide test<br />
</p><br />
<br />
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<br />
<div class="technology2">Week 10 (18-24 August)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Ethics: Interview with a specialist in ethical sciences, Vincent Grégoire Delory to complete our reflexion about the ethical issues in SubtiTree project.<br />
<br/><br />
- Wiki: hiring a web designer, Adrien Nicod, to improve the aesthetic effect and the logo.<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Subculture and transformation in <i> B. subtilis </i> of BBa_K1364011 in pSB<sub>BS</sub>4S (BBa_K823022) and cloning of BBa_K1364011 on pSB<sub>BS</sub>1C (BBa_K823023).<br />
<br/><br />
Problem: no clone had the insert on pSB<sub>BS</sub>1C => the cloning had to be done again<br />
<br>- Miniprep of BBa_K1364011 in BBa_K823023 (pSB<sub>BS</sub>1C) + Transformation in <i>B. subtilis</i>.<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) , BBA_1364002 (GAFP1) , BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) on BBa_K823023 (pSB<sub>BS</sub>1C) <br />
<br/><br />
- Transformation BBa_1364004 (in pSB<sub>BS</sub>4S in <i>E. coli</i> )<br />
<br/><br />
- Cloning of BBa_K1364014 (P<sub>veg</sub> + RBS SpoVG + EcAMP + GAFP1 + D4E1 + double terminator) in K823022 (pSB<sub>BS</sub>4S) and in BBa_K823023 (pSB<sub>BS</sub>1C), miniprep and digestion by restriction enzymes to see the size of the insert on a 1% gel<br />
<br/><br />
Problem: no clone had the insert on pSB<sub>BS</sub>1C => the cloning had to be done again<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology2">Week 11 (25- 31 August)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Beginning of the creation and redaction of the different parts of the wiki by the Toulouse iGEM Toulouse<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Cloning of BBa_K1364014 in BBa_K823022 and in BBa_K823023 <br />
<br/><br />
- Cloning GAFP1 + BBa_K823023 (pSB<sub>BS</sub>1C) in <i>B. subtilis</i> with a change in the protocol: pre-induction of the culture with 0.125 µg/ml of chloramphenicol one hour after the addition of DNA, and let grow for another hour.<br />
<br/><br />
Problem: no colony <br />
<br/><br />
- Liquid culture of <i>Bacillus subtilis</i> + BBA_1364002 (GAFP1); <i>Bacillus subtilis</i> + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) ; <i>Bacillus subtilis</i> + BBA_1364003 (D4E1) for future fungicide tests<br />
<br/><br />
- Miniprep of BBa_ K1364011 in BBa_ K823023 (pSB<sub>BS</sub>1C) and transformation in <i>Bacillus subtilis</i> strain. <br />
<br/><br />
- Fungicide test of BBa_K1364011 in pSB<sub>BS</sub>4S, Promotor + BBA_1364002 (GAFP1) + Terminator (BioBrick BBa_K1364008) in pSB<sub>BS</sub>4S, Promotor + BBA_1364003 (D4E1) + Terminator (BioBrick BBa_K1364009) in pSB<sub>BS</sub>4S, Promotor + BBA_1364002 (GAFP1) + BA_1364003 (D4E1) + Terminator (BioBrick BBa_K1364013) in pSB<sub>BS</sub>4S<br />
<br/><br />
- Transformation of a new vector PKL 190 (threonine integrative) in <i>Bacillus</i> strain <br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors(08/29/2014)</p><br />
<p class="texte"><br />
- Boston accommodation must be booked<br />
<br/><br />
- Discussion about the contamination seen on the fungicide tests : the issue seems to come from the sterile water which contained contaminants<br />
<br/><br />
- Objectives : chemotaxis, binding and fungicides tests have to be performed again<br />
</p> <br />
<br />
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<br />
<br />
<div class="technology">September 2014</div><br />
<div class ="thelanguage"><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finish the tests of the different modules<br />
<br/><br />
- Establish the final editorial parts for the wiki<br />
<br/><br />
- Design of the wiki<br />
<br/><br />
- Sequencing the different assembled parts of our bacterium<br />
</p> <br />
<br />
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<br />
<div class="technology2">Week 12 (1-7 September)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Different parts of the wiki by the Toulouse iGEM Toulouse<br/><br />
- Reservation of Boston accommodation<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Chemotaxis test and modeling<br />
<br/><br />
- Fungicides tests on EcAMP-A, EcAMP-B, EcAMP-C, EcAMP-D, MaxiOpA, MaxiOpB, MaxiOpC, MaxiOpD from 2ml of overnight cultures. Only with 25000 conidia with both fungi.<br />
<br/><br />
- PCR on pSB<sub>BS</sub>4S + BBa_K1162001 (EcAMP) <br />
<br/><br />
Problem: failed on both diluted plasmid and colony<br />
<br/><br />
- Spreading of BBa_K1364014 + BBa_ K823022 in threonine medium and in medium without threonine <br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors(09/05/2014)</p> <br />
<p class="texte"><br />
- The first sequencing was not successful => new sequencing with one primer/tube<br/><br />
- Chemotaxis test: increase the concentration in bacterium, try a new protocol by capillarity<br/><br />
- End of the binding test: IT WORKS PERFECTLY! SubtiTree has the good phenotype and presents a better binding property than the wild type <i>Bacillus subtilis</i> strain.<br/><br />
- Fungicides test: contaminants with the fungus. Need to ask for another culture of our fungi.<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology2">Week 13 (8- 14 September)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Telephone interview for an article in a French national review named Fluvial which will be published at the end of September.<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- PCR on D4E1 colony with different primers<br />
<br/>- Culture and miniprep of EcAMP to get more DNA + analytic digestion <br />
<br/>- Transformation of K1364014 + K1364006 in <i>E. coli</i> <br />
<br/>- Transformation of GAFP1 + D4E1, GAFP1 and EcAMP + GAFP1 + D4E1 on <i>B. subtilis</i> + colony PCR + threonine test<br />
<br/><i>NB: good results for the maxi operon and GAFP1 but not for GAFP1 + D4E1</i><br />
<br/>- Cloning of K606013 + pEX_K4, 823022 + P<sub>lepA</sub>_RFP, 823022 + P<sub>veg</sub>_RFP<br />
<br/>- Chemotaxis tests: different times of LB + agar addition in the LB medium (after 3 minutes, 5 minutes, 8 minutes, 10 minutes…) are performed => No difference of the results after spreading on the plates.<br />
<br/>- New test of chemotaxis: capillary between two Eppendorf tubes<br />
<br/>- Spreading of the fungi on a medium which mimics the sap<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors(09/11/2014)</p> <br />
<p class="texte"><br />
- Try to use the GFP or RFP to follow the chemotaxis test.<br />
<br/>- Make a new glass process for the chemotaxis capillary test.<br />
<br/>- Possibility of contacting an INRA laboratory to get pictures of the binding test with confocal microscope.<br />
<br/>- Sequencing the binding construction.<br />
<br/>- Try different temperatures for the fungicide tests: 20°C, 25°C, and 37°C to reduce the fungus growth.<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology2">Week 14 (15 - 21 September)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Support of two French city halls: Mairie de Montréal and Mairie de Ventenac en Minervois.<br />
<br/>- First interview by the French national radio France Inter which will be aired on October 19<sup>th</sup>.<br />
<br/>- Beginning of the organization of the Toulouse iGEM team acknowledgement day: the purpose is to present the project to the students and give a feedback on the competition to the sponsors.<br />
<br/>- Final design of the wiki<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Threonine tests for the BBa_K1364014 maxi operon (EcAMP1 – GAFP1 – D4E1) and mini operon BBa_K1364013 (GAFP1 - D4E1)<br />
<br/>- Colony PCR on the mini operon BBa_K1364013 (GAFP1 - D4E1)<br />
<br/>- Colony PCR on BBa_K1364011 on BBa_K823022 (pSBBS4S)<br />
<br/>- Chemotaxis test with wild type <i>B. subtilis</i> strain using Imperial College protocol<br />
<br/>- Tansformation of BBa_K1364011 + BBa_K823022 (pSB<sub>BS</sub>4S) in <i>B. subtilis</i><br />
<br/>- Culture of fungi with fungicides<br />
<br/>- Sequencing of plasmids containing the fungicides<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (09/18/2014)</p> <br />
<p class="texte"><br />
- Sequencing worked for D4E1 and GAFP1 and the mini operon BBa_K1364013 (GAFP1-D4E1).<br />
<br/>- The next step is to sequence the maxi operon (EcAMP1 – GAFP1 – D4E1).<br />
<br/>- Try different approaches for the chemotaxis:<br />
<br/>New protocol with the capillary assay with a new system made by a glassblower.<br />
<br/>Make the Imperial College test again by testing 3 different solutions: chitin, N-acetylglucosamine and another carbon source.<br />
<br/>- Decrease the number of conidia and temperature for the fungicide test.<br />
</p><br />
<br />
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<br />
<div class="technology2">Week 15 (22 - 28 September)</div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- New support of Sallèles d’Aude and Labastide d'Anjou city halls but also the Département de l'Hérault.<br />
<br/>- Payment of the plane tickets.<br />
<br/>- Acknowledgement day (4<sup>th</sup> December): contact with catering service, reservation of the amphitheater, list of guests…<br />
<br/>- Order of the T shirt for the Jamboree! We look awesome with them!<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Last experiments regarding chemotaxis tests.<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors(09/26/2014)</p> <br />
<p class="texte"><br />
- Focus on the wiki design and writing.<br />
<br/>- Start making the power point presentation for the Giant Jamboree and the poster.<br />
<br/>- Division of responsibilities for the non-scientific work.<br />
</p><br />
<br />
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<br />
<div class="technology">October 2014</div><br />
<div class="thelanguage"><br />
<p class="title2">Main activites</p><br />
<p class="texte"><br />
- Preparation of the wiki every day... all day long... so hard to work on it, especially at night!<br />
<br/>- Preparation of the poster and the presentation for the Giant Jamboree with our instructors <br />
<br/><br />
- Daily presentations in front of the laboratory teachers to repeat again and again and again… let’s get ready for the Jamboree!<br />
</p> <br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Calendar</p> <br />
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<br />
<div class="technology">December 2013 – January 2014: Team selection</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- iGEM competition presentation and explanations<br/><br />
- Constitution of the <a href="https://2014.igem.org/Team:Toulouse/Team">team</a> by interviewing different students from Université Toulouse III Paul Sabatier and INSA de Toulouse<br />
</p><br />
<p class="texte" style="text-align:center"><B> The adventure begins for the 2014 Toulouse iGEM Team!</B><br />
</p><br />
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<div class="technology"><p class="title2"; "line-height:6px"; "padding:10px";>January – June 2014: Projects brainstorming</p></div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- Thanks to weekly meetings, our team was able to discuss about new project ideas.<br />
<br/><br />
- Many ideas were approached and debated regarding the feasibility thanks to the literature and supervisors including teachers and researchers.<br><br />
<br/><br />
This is a list of the main projects: <br />
<br/><br />
- Let's save our trees with SubtiTree: the purpose is to use <i>Bacillus subtilis</i> as an optimized bacterium to target and bind to a specific fungus and secrete fungicides to destroy it.<br />
<br/><br />
- <i>E. coli</i> cancer: the concept was to create a bacterium able to colonize specifically the hypoxic regions of the tumor. The oxygen-sensitive bacterium would not be able to develop in the healthy zones.<br />
<br/><br />
- Reduction of ruminants’ methane production: the goal is to reduce the production of methane by ingesting a microorganism in the animals' rumen. This bacterium would be able to reduce the quantity of metabolic hydrogen and eliminate the methanogenic Archaes population.<br />
</p><br />
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<div class="technology"><p class="title2"; "line-height:6px"; "padding:10px";>June 2014: Choice of SubtiTree project</p></div><br />
<div class="thelanguage"><br />
<p class="texte">The discussion has been long to estimate the practicability of our final project. By the end of May, most of the ideas were eliminated but two of them remained: controlling the cancer or saving the trees. It became easily clear that SubtiTree was successfully completed whether regarding the scientific part or the ethical aspect. Indeed, our knowledge in medicine was not sufficient enough to evaluate the consequences of a bacterium injection in the human body for <i>E. coli</i> cancer.</br><br />
Therefore, the whole team agreed on focusing on a local issue as the preservation of the Canal du Midi plane trees. However, the final goal was based on the idea to allow our optimized bacteria to be efficient for all fungi infections in the environment.</br><br />
By this period, we evaluated our budget to approximately <B> €39,500 </B>.<br />
</p> <br />
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<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 1(16-22 June)</p></div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
- Bibliography researches about our subject<br/><br />
- Cleaning the whole lab to allow good manipulations and avoid any contamination<br/><br />
- Inventory of necessary lab equipment and biobricks<br/><br />
- Summarize the iGEM protocols<br/><br />
- Prepare all growing media <br/><br />
- Preparing competent cells by optimizing protocols<br/><br />
- Transformation of RFP plasmid to practice<br />
</p><br />
<br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Increase in our communication strategy to find more sponsors specialized in environment and ecological matters <br/><br />
- Support of Adisseo, Toulouse White Biotechnology, LISBP, INSA Toulouse<br/><br />
- Organization of a weekly meeting each Friday with our supervisors to tackle any problem encountered<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors (06/20/2014)</p><br />
<p class="texte"><br />
- Distribution of the non-scientific tasks regarding the wiki, communication, sponsorship, ethical problems, safety but also the lab work<br/><br />
- Discussion about the different construction parts of the project<br/><br />
- Need to check the absence of stop codon in fungicides sequences<br/><br />
- Ordering the list of necessary products for the laboratory and biobricks<br/><br />
- Checking and validation of the genes sequences<br />
</p> <br />
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<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 2 (23-29 June)</p></div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- BioSynSyS conference work: powerpoint, logo in order to present our SubtiTree project to a whole audience of scientific and researchers<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- <i>E. coli</i> transformation with BBA_J004450 (pSB1C3)<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors (06/27/2014)</p><br />
<p class="texte"><br />
- Concentration of antibiotics in media must be checked<br/><br />
- The transformation rate for pUC19 must be evaluated<br/><br />
- The transformation efficiency must be calculated regarding the quantity of DNA<br/><br />
- Possibility to contact the cities halls for the sponsorship<br/><br />
- Check the mechanism of action of each fungicide to complete the ethical part<br />
</p><br />
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<div class="technology"><p class="title2"; "line-height:6px"; "padding:10px";>July 2014</p></div><br />
<div class="thelanguage"><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Beginning of the laboratory work to create our optimized bacterium <br/><br />
- Research of sponsors and communication thanks to the press<br />
</p> <br />
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<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 3 (30 June -6 July) and Week 4 (7-13 July)</p></div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the BioSynSys conferences: presentation of SubtiTree project<br/><br />
- We have put in place a newsletter system the first friday of each month to keep all our sponsors and people who support us updated about the project (financial, scientific and communication aspects).<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Design of all the constructions needed with cloning and restriction enzymes to save some time and allow a better comprehension of our bacterium system </br><br />
Problem: A problem was reported in the use of the CYP. Indeed, the BioBrick is not expressed properly in <i>Bacillus subtilis</i> strain. Therefore we had to look at the literature to find a new fungicide called GAFP-1.<br/><br />
- A bioreactor was conceived to reproduce the composition of the sap in the plane trees. The main goal was to identify the behavior of <i>Bacillus</i> strain in a medium composed of many different carbon sources.<br/><br />
- Competent cells and transformation practice using GFP and RFP.<br />
</p><br />
<br />
<p class="title4">Weekly meetings with the instructors (07/04/2014) and (07/11/2014)</p><br />
<p class="texte"><br />
- iGEM efficiency kit does not work, we shall not use it anymore<br/><br />
- Organization of a timetable to check the stored and sterile equipment everyday</br><br />
- Try a transformation in <i>Bacillus subtilis</i><br />
</p> <br />
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<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 5 (14-20 July)</p></div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Our team was interviewed by many local newspapers such as 20minutes, Metronews, La Voix du Midi, La Dépêche but also by the local television channel France3 Midi-Pyrénées to present our draft to the community. Moreover, SubtiTree was approached in some radio programs like Virgin Radio Toulouse and France info.</br><br />
- Support of ThermoFisher, New Englands BioLabs, Qiagen, Eurofins but also GenoToul, Labex Tulip, L’Université Paul Sabatier, CROUS.</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- GFP and RFP digestion amplified by miniprep and gel electrophoresis <br/><br />
Problem: the digested GFP doesn’t appear on the gel contrary to RFP plasmid. We assumed a problem with the GFP miniprep. We tried the miniprep from the INSA Toulouse team from 2013 and the GFP plasmid was fully digested.<br/><br />
- Transformation and cryopreservation of BioBricks BBa_K823002 (P<sub>lep</sub>A), BBa_K823003 (P<sub>veg</sub>), BBa_K606061 (RBS SpoVG) and BBa_B0015 (double terminator), BBa_K823023 (pSB<sub>BS</sub>1C), BBa_K733013 (P<sub>veg</sub> + RBS) in <i>E. coli</i>.<br/><br />
- Transformation of the Munich <i>B. subtilis</i> backbones<br/><br />
- Cloning of BBa_K606061 (RBS SpoVG) with BBa_K823003 (P<sub>veg</sub>) and BBa_K823002 (P<sub>lepA</sub>)<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors (07/18/2014)</p><br />
<p class="texte"><br />
- Transformation of the Eurofins genes<br/><br />
- Start assembling the BioBricks for the fungicides<br/><br />
- Check all the cloning<br />
</p><br />
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<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 6 (21 July-27 July)</p></div><br />
<div class="thelanguage2"><br />
<br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Description of SubtiTree project and determination of the goodies for a crowdfunding campaign on Ulule website</p><br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Transformation of BBA_1364002 (GAFP1) and BBA_1364003 (D4E1) fungicides genes<br/><br />
- Subculture of the clones for each gene<br/><br />
- PCR and migration on electrophoresis gel<br/><br />
- Transformation BBA_1364003 (D4E1) on pSB1C3 and BBA_1364002 (GAFP1) on pSB1C3<br/><br />
- Cloning BBA_1364002 (GAFP1) with BBa_B0015 (double terminator) and BBA_1364003 (D4E1) with BBa_K823003 (P<sub>veg</sub>)<br />
</p><br />
<br />
<p class="title4">Others:</p><br />
<p class="texte"><br />
- Research for plane tickets and hotels in Boston for the Giant Jamboree<br/><br />
- New idea: analyze the plane tree sap to determine its composition<br />
</p><br />
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<div class="technology"><p class="title2"; "line-height:6px"; "padding:10px";>August 2014</p> </div><br />
<div class="thelanguage"><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finishing the cloning for the different parts of the bacterium<br/><br />
- Putting in place the fungicides, binding and chemotaxis tests<br />
</p> <br />
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<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 7 (28 July-3 August)</p></div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Financial supports of the Ministery (Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt) and Voies Navigables de France<br/><br />
- Interview with Johan Langot from Sciences Animation for a possible presentation at the Toulouse Festival of Science<br/><br />
- Launch of the crowdfunding campaign on Ulule<br />
</p><br />
<br />
<p class="title4">Lab work: </p><br />
<p class="texte"><br />
- Cloning BBa_K823003 (P<sub>veg</sub>) + RFP and BBa_K823002 (P<sub>lepA</sub>) + RFP in pSB<sub>Bs</sub>1C<br/><br />
- Test of every pSB<sub>BS</sub> vector: BBa_K823021 (pSB<sub>BS</sub>1C-lacZ), BBa_K823022 (pSB<sub>BS</sub>4S), BBa_K823023 (pSB<sub>BS</sub>1C), minipreps and cryopreservation of the best clones<br/><br />
- Checking of BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) transformants (BioBrick BBa_K1364007) and BBA_1364003 (D4E1) + BBa_K823003 (P<sub>veg</sub>) BioBrick BBa_K1364009<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) + pSB<sub>BS</sub>4S (BBa_K823022) and subculture of the colonies<br/><br />
- Cloning of BBa_K823003 (Pveg) + BBA_1364003 (D4E1) with BBa_B0015 (double terminator)<br/><br />
- Cloning of BBa_K823003 (Pveg) + RFP (BBa_K1364017) in pSB<sub>BS</sub>4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_K823002 (PlepA) + RFP (BBa_K1364016) in pSB<sub>BS</sub>4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_1364018 (Strong Promotor + RFP)<br/><br />
- Cloning of BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) + Promotor BBa_K823003 (P<sub>veg</sub>) BioBrick BBa_K1364012 with gel extraction for BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) and a PCR cleanup for BBa_K823003 (P<sub>veg</sub>), subculture<br/><br />
- Cloning BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBS4S in <i>E. coli</i><br/><br />
- Transformation of binding gene with <i>E. coli</i> competent cells<br/><br />
- Transformation of BBa_K1364000, the chemotaxis gene in <i>E. coli</i><br/><br />
Problem: the transformation worked but a cell layer appeared. A new subculture of the colonies was made as well as a new spreading on petri dishes.<br/><br />
- Spreading of BBa_K1162001 (EcAMP), miniprep and digestion<br/><br />
Problem: the digestion did not work => Issue with the quantity of DNA in the miniprep is assumed<br/><br />
- First experience of chemotaxis with a glucose chemo-attractant<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors (08/01/2014)</p><br />
<p class="texte"><br />
- Possible problem with the restriction enzymes regarding the digestion: new recommendations<br />
<br/><br />
- Discussion about EcAMP cloning which presents some issues<br />
<br/><br />
- Discussion about the transfer of pSB1C3 from <i>E. coli</i> to <i>B. subtilis</i> strain<br />
</p> <br />
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<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 8 (04-10 August)</p></div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the “Passion Jeune” competition organized by a French bank foundation named Fondation Crédit Agricole Toulouse<br />
</p><br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Check the cloning of BBa_K823003 (P<sub>veg</sub>)+BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSB<sub>BS</sub>4S and cryopreservation<br />
<br/><br />
- Transformation of BBa_K823003 (P<sub>veg</sub>) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) (BioBrick BBa_ K1364008) in BBa_K823022 (pSB<sub>BS</sub>4S) <br />
<br/><br />
- Cloning of BBa_K1364001, the binding gene + BBa_K823003 (P<sub>veg</sub>) on pSB1C3 and pSB<sub>BS</sub>4S<br />
<br/><br />
Problem: the band is at 1500bp instead of 1300bp on the gel<br />
<br/><br />
- Cloning of BBa_K1162001 (EcAMP) + BBa_K823003 (P<sub>veg</sub>) + BBa_K606061 (RBS SpoVG), miniprep, PCR and digestion <br />
<br/><br />
Problem: the fragment of DNA is too small (149bp) to be seen on the gel and was probably lost during the PCR cleanup. Instead of that, we used the heat enzymes inactivation at 95°C for 15 minutes. <br />
<br/><br />
- Cloning of Promotor + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) in pSB1C3 and pSB<sub>BS</sub>4S and cryopreservation<br />
<br/><br />
- Elaboration of an efficient fungicide test protocol <br />
<br/><br />
- Test of the fungus growth on PDA medium + test of growth for <i>B. subtilis</i> liquid culture on PDA<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors(08/08/2014)</p><br />
<p class="texte"><br />
- Ask iGEM headquarters if all the biobricks must be on pSB1C3 plasmid<br />
<br/><br />
- Use another strain of <i>E. coli</i> (DH5-1 instead of DH5 alpha) to have a faster growth<br />
<br/><br />
- Check which quantity of <i>B. subtilis</i> is necessary to naturally destroy the fungus<br />
</p><br />
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<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 9 (11-17 August)</p></div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Publication of more articles about our project in Labiotech, Développement durable (Networkvisio), Midi Libre, l’Indépendant, DigiSchool, La voix du Midi Lauragais, LURIO Addl.<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Evaluation of the bacterial decrease rate from 10°C to 4°C with a sporuling strain of <i>B. subtilis</i>.<br />
<br/><br />
- Chemotaxis test with different concentrations of glucose on a 0.3% agar.<br />
<br/><br />
- Miniprep of pSB<sub>B</sub>4S with binding gene and transformation in <i>B. subtilis</i>.<br />
<br/><br />
Problem: no digestion was visible on the gel => the cloning failed<br />
<br/><br />
- Transformation of BBa_K1374010 (P<sub>veg</sub> + RBS SpoVG + EcAMP) + BBa_K1364012 (RBS + GAFP1 + D4E1 + double terminator), subculture<br />
<br/><br />
- Subculture of <i>B. subtilis</i> clones transformed by BBa_K823003 (P<sub>veg</sub>) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) in pSB<sub>B</sub>4S, cryopreservation<br />
<br/><br />
- Fungicide test<br />
</p><br />
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<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 10 (18-24 August)</p></div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Ethics: Interview with a specialist in ethical sciences, Vincent Grégoire Delory to complete our reflexion about the ethical issues in SubtiTree project.<br />
<br/><br />
- Wiki: hiring a web designer, Adrien Nicod, to improve the aesthetic effect and the logo.<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Subculture and transformation in <i> B. subtilis </i> of BBa_K1364011 in pSB<sub>BS</sub>4S (BBa_K823022) and cloning of BBa_K1364011 on pSB<sub>BS</sub>1C (BBa_K823023).<br />
<br/><br />
Problem: no clone had the insert on pSB<sub>BS</sub>1C => the cloning had to be done again<br />
<br>- Miniprep of BBa_K1364011 in BBa_K823023 (pSB<sub>BS</sub>1C) + Transformation in <i>B. subtilis</i>.<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) , BBA_1364002 (GAFP1) , BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) on BBa_K823023 (pSB<sub>BS</sub>1C) <br />
<br/><br />
- Transformation BBa_1364004 (in pSB<sub>BS</sub>4S in <i>E. coli</i> )<br />
<br/><br />
- Cloning of BBa_K1364014 (P<sub>veg</sub> + RBS SpoVG + EcAMP + GAFP1 + D4E1 + double terminator) in K823022 (pSB<sub>BS</sub>4S) and in BBa_K823023 (pSB<sub>BS</sub>1C), miniprep and digestion by restriction enzymes to see the size of the insert on a 1% gel<br />
<br/><br />
Problem: no clone had the insert on pSB<sub>BS</sub>1C => the cloning had to be done again<br />
</p><br />
<br />
<br />
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<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 11 (25- 31 August)</p></div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Beginning of the creation and redaction of the different parts of the wiki by the Toulouse iGEM Toulouse<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Cloning of BBa_K1364014 in BBa_K823022 and in BBa_K823023 <br />
<br/><br />
- Cloning GAFP1 + BBa_K823023 (pSB<sub>BS</sub>1C) in <i>B. subtilis</i> with a change in the protocol: pre-induction of the culture with 0.125 µg/ml of chloramphenicol one hour after the addition of DNA, and let grow for another hour.<br />
<br/><br />
Problem: no colony <br />
<br/><br />
- Liquid culture of <i>Bacillus subtilis</i> + BBA_1364002 (GAFP1); <i>Bacillus subtilis</i> + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) ; <i>Bacillus subtilis</i> + BBA_1364003 (D4E1) for future fungicide tests<br />
<br/><br />
- Miniprep of BBa_ K1364011 in BBa_ K823023 (pSB<sub>BS</sub>1C) and transformation in <i>Bacillus subtilis</i> strain. <br />
<br/><br />
- Fungicide test of BBa_K1364011 in pSB<sub>BS</sub>4S, Promotor + BBA_1364002 (GAFP1) + Terminator (BioBrick BBa_K1364008) in pSB<sub>BS</sub>4S, Promotor + BBA_1364003 (D4E1) + Terminator (BioBrick BBa_K1364009) in pSB<sub>BS</sub>4S, Promotor + BBA_1364002 (GAFP1) + BA_1364003 (D4E1) + Terminator (BioBrick BBa_K1364013) in pSB<sub>BS</sub>4S<br />
<br/><br />
- Transformation of a new vector PKL 190 (threonine integrative) in <i>Bacillus</i> strain <br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors(08/29/2014)</p><br />
<p class="texte"><br />
- Boston accommodation must be booked<br />
<br/><br />
- Discussion about the contamination seen on the fungicide tests : the issue seems to come from the sterile water which contained contaminants<br />
<br/><br />
- Objectives : chemotaxis, binding and fungicides tests have to be performed again<br />
</p> <br />
<br />
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<br />
<div class="technology"><p class="title2"; "line-height:6px"; "padding:10px";>September 2014</p></div><br />
<div class ="thelanguage"><p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finish the tests of the different modules<br />
<br/><br />
- Establish the final editorial parts for the wiki<br />
<br/><br />
- Design of the wiki<br />
<br/><br />
- Sequencing the different assembled parts of our bacterium<br />
</p> <br />
<br />
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<br />
<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 12 (1-7 September)</p></div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Different parts of the wiki by the Toulouse iGEM Toulouse<br/><br />
- Reservation of Boston accommodation<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Chemotaxis test and modeling<br />
<br/><br />
- Fungicides tests on EcAMP-A, EcAMP-B, EcAMP-C, EcAMP-D, MaxiOpA, MaxiOpB, MaxiOpC, MaxiOpD from 2ml of overnight cultures. Only with 25000 conidia with both fungi.<br />
<br/><br />
- PCR on pSB<sub>BS</sub>4S + BBa_K1162001 (EcAMP) <br />
<br/><br />
Problem: failed on both diluted plasmid and colony<br />
<br/><br />
- Spreading of BBa_K1364014 + BBa_ K823022 in threonine medium and in medium without threonine <br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors(09/05/2014)</p> <br />
<p class="texte"><br />
- The first sequencing was not successful => new sequencing with one primer/tube<br/><br />
- Chemotaxis test: increase the concentration in bacterium, try a new protocol by capillarity<br/><br />
- End of the binding test: IT WORKS PERFECTLY! SubtiTree has the good phenotype and presents a better binding property than the wild type <i>Bacillus subtilis</i> strain.<br/><br />
- Fungicides test: contaminants with the fungus. Need to ask for another culture of our fungi.<br />
</p><br />
<br />
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<br />
<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 13 (8- 14 September)</p></div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Telephone interview for an article in a French national review named Fluvial which will be published at the end of September.<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- PCR on D4E1 colony with different primers<br />
<br/>- Culture and miniprep of EcAMP to get more DNA + analytic digestion <br />
<br/>- Transformation of K1364014 + K1364006 in <i>E. coli</i> <br />
<br/>- Transformation of GAFP1 + D4E1, GAFP1 and EcAMP + GAFP1 + D4E1 on <i>B. subtilis</i> + colony PCR + threonine test<br />
<br/><i>NB: good results for the maxi operon and GAFP1 but not for GAFP1 + D4E1</i><br />
<br/>- Cloning of K606013 + pEX_K4, 823022 + P<sub>lepA</sub>_RFP, 823022 + P<sub>veg</sub>_RFP<br />
<br/>- Chemotaxis tests: different times of LB + agar addition in the LB medium (after 3 minutes, 5 minutes, 8 minutes, 10 minutes…) are performed => No difference of the results after spreading on the plates.<br />
<br/>- New test of chemotaxis: capillary between two Eppendorf tubes<br />
<br/>- Spreading of the fungi on a medium which mimics the sap<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors(09/11/2014)</p> <br />
<p class="texte"><br />
- Try to use the GFP or RFP to follow the chemotaxis test.<br />
<br/>- Make a new glass process for the chemotaxis capillary test.<br />
<br/>- Possibility of contacting an INRA laboratory to get pictures of the binding test with confocal microscope.<br />
<br/>- Sequencing the binding construction.<br />
<br/>- Try different temperatures for the fungicide tests: 20°C, 25°C, and 37°C to reduce the fungus growth.<br />
</p><br />
<br />
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<br />
<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 14 (15 - 21 September)</p></div><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- Support of two French city halls: Mairie de Montréal and Mairie de Ventenac en Minervois.<br />
<br/>- First interview by the French national radio France Inter which will be aired on October 19<sup>th</sup>.<br />
<br/>- Beginning of the organization of the Toulouse iGEM team acknowledgement day: the purpose is to present the project to the students and give a feedback on the competition to the sponsors.<br />
<br/>- Final design of the wiki<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Threonine tests for the BBa_K1364014 maxi operon (EcAMP1 – GAFP1 – D4E1) and mini operon BBa_K1364013 (GAFP1 - D4E1)<br />
<br/>- Colony PCR on the mini operon BBa_K1364013 (GAFP1 - D4E1)<br />
<br/>- Colony PCR on BBa_K1364011 on BBa_K823022 (pSBBS4S)<br />
<br/>- Chemotaxis test with wild type <i>B. subtilis</i> strain using Imperial College protocol<br />
<br/>- Tansformation of BBa_K1364011 + BBa_K823022 (pSB<sub>BS</sub>4S) in <i>B. subtilis</i><br />
<br/>- Culture of fungi with fungicides<br />
<br/>- Sequencing of plasmids containing the fungicides<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (09/18/2014)</p> <br />
<p class="texte"><br />
- Sequencing worked for D4E1 and GAFP1 and the mini operon BBa_K1364013 (GAFP1-D4E1).<br />
<br/>- The next step is to sequence the maxi operon (EcAMP1 – GAFP1 – D4E1).<br />
<br/>- Try different approaches for the chemotaxis:<br />
<br/>New protocol with the capillary assay with a new system made by a glassblower.<br />
<br/>Make the Imperial College test again by testing 3 different solutions: chitin, N-acetylglucosamine and another carbon source.<br />
<br/>- Decrease the number of conidia and temperature for the fungicide test.<br />
</p><br />
<br />
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<br />
<div class="technology2"><p class="title3"; "line-height:6px"; "padding:10px";>Week 15 (22 - 28 September)</div></p><br />
<div class="thelanguage2"><br />
<p class="title4">Communication and financial support:</p><br />
<p class="texte"><br />
- New support of Sallèles d’Aude and Labastide d'Anjou city halls but also the Département de l'Hérault.<br />
<br/>- Payment of the plane tickets.<br />
<br/>- Acknowledgement day (4<sup>th</sup> December): contact with catering service, reservation of the amphitheater, list of guests…<br />
<br/>- Order of the T shirt for the Jamboree! We look awesome with them!<br />
</p><br />
<br />
<p class="title4">Lab work:</p><br />
<p class="texte"><br />
- Last experiments regarding chemotaxis tests.<br />
</p><br />
<br />
<p class="title4">Weekly meeting with the instructors(09/26/2014)</p> <br />
<p class="texte"><br />
- Focus on the wiki design and writing.<br />
<br/>- Start making the power point presentation for the Giant Jamboree and the poster.<br />
<br/>- Division of responsibilities for the non-scientific work.<br />
</p><br />
<br />
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<br />
<div class="technology"><p class="title2"; "line-height:6px"; "padding:10px";>October 2014</p></div><br />
<div class="thelanguage"><br />
<p class="title2">Main activites</p><br />
<p class="texte"><br />
- Preparation of the wiki every day... all day long... so hard to work on it, especially at night!<br />
<br/>- Preparation of the poster and the presentation for the Giant Jamboree with our instructors <br />
<br/><br />
- Daily presentations in front of the laboratory teachers to repeat again and again and again… let’s get ready for the Jamboree!<br />
</p> <br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
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<div id="innercontenthome"><br />
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<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Knowledge without conscience is but the ruin of the soul."</br><br />
"Science sans conscience, n'est que ruine de l'âme", François Rabelais, 16<sup>th</sup> century.<br />
</p><br />
<br />
<p class="texte">The ethical<br />
questioning turned out to be one of the major starting points of our project.<br />
Acting on an established environment and modifying it is by no mean trivial and<br />
our combined technical and philosophical points of view. The actual purpose of our project also leads us to undertake an<br />
ethical questioning about the role of the scientist regarding “useless” things<br />
such as the trees lining along the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="title2">Is it the scientists’ role to protect beauty?<br />
</p><br />
<br />
<p class="texte"> Beauty is a<br />
feeling of satisfaction and is selfless. It is more a feeling than the property<br />
of a thing, this is not a notion we can clearly understand. Indeed, we can find<br />
something beautiful even when we don’t know the purpose of the object...</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/f/fa/Fontaine_Duchamp.jpg" width="400px"><br />
<p class="legend">Figure 1: Fontaine (Marcel Duchamp). Yes this is also art...</p></center><br />
<br />
<p class="texte">There is always<br />
a distinction between natural beauty and artistic beauty according to Hegel,<br />
the famous philosopher. The artistic beauty is born from our mind and our<br />
spirit: it is an element of signification of the work of art whereas the<br />
natural beauty of the object is external. In a way, the Canal du Midi combines<br />
both types of beauty: a natural one regarding the Nature, the centenary plane<br />
trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, science judges beauty as a superficial feature not deserving to<br />
undertake any kind of scientific efforts to maintain it. The traditional role<br />
of science is to solve global issues and to elaborate complex strategies in<br />
order to find useful solutions for everyone’s life. Once made this observation,<br />
one may wonder why synthetic biology would be used only to protect the useless<br />
beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial<br />
interrogation leads us to consider science and synthetic biology from a<br />
different point of view. <b>What if the role of scientists was also to make<br />
people rediscovering the beauty of Nature? What if the bases of new scientific<br />
challenges resulted from a more local scale? </b>Science does not have to be it has so much to gain opening itself to these<br />
challenges. First scientifically, as research is never<br />
useless and as we never know the impact and the scope of our results.<br />
Then socially, as we could measure the deep interest raised by our<br />
project within the population and the media. Adopting a new vision of synthetic<br />
biology, we will probably make people change their mind about this innovative<br />
discipline. <br><br />
The traditional cold objectivity of science distances itself from the society.<br />
However, scientists are also being capable of feeling the beauty, sensitive to<br />
the charm of landscapes and <b>able to understand the usefulness of<br />
"useless" trees</b>…<br><br />
The design of a strategy to protect useless beauty may seem senseless but we<br />
believe that it is also the scientist’s duty. We have to remember that thinking<br />
is what distinguish <i>Homo sapiens</i> from other species on earth and this<br />
"thinking" feature allows us to understand the world and be conscious<br />
of our human Nature (Descartes: <i>Cogito ergo sum</i>). The art is an object<br />
of philosophical thought. Consciousness raises humans above all others living<br />
creatures. Thus, it is necessary to respect and protect art. And thus, it<br />
becomes essential to preserve the beauty of the Canal du Midi. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human intervention on Nature</p><br />
<p class="texte">Our main question is to understand the complicated relationship between man and Nature.<br />
Does mankind have the proper right to change the Nature? Is modified Nature considered as artificial?</p><br />
<p class="title2"> Mankind & Nature</p><br />
<br />
<p class="texte"> Nature deserves<br />
to be respected and loved. Mankind has always been linked to Nature as its<br />
survival depends on what comes out of the ground, the trees, the oceans… The<br />
Nature is a source of wealth for mankind. It ensures survival and development<br />
by giving men the wood, the rocks, the soil to build shelters. Being in contact<br />
with Nature can allow men to feel strong emotion, as describe by poets like<br />
Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth<br />
of humanity, man himself understood the importance of studying and mastering<br />
Nature to develop the civilization. Still today the most advanced technologies<br />
often try to mimic natural phenomena. With the development of civilization, men<br />
modified their environment, changing it for their own comfort depending on<br />
their own desire. With the increase of human activity, the natural environment<br />
is modified profundly. With industrialization, the<br />
natural environment suffered from waste discharges, oil slicks, intensive<br />
fishing (and many others...) but also the introduction of devastating species<br />
such as the pathogen,<i> Ceratocystis<br />
platani</i>. However, despite these negative aspects, men<br />
are capable of favorable actions to help the environment and fix their<br />
mistakes. The current trend is to limit the impact of human interventions on<br />
Nature, and hopefully this trend is not transient and will not vanish. A new<br />
desire is born, a wish to protect Nature and wilderness. Humanity can adhere to<br />
this position: human take advantage of the<br />
environment and the environment takes advantage of the reasoned human<br />
interventions. There is an adaptation of mankind to Nature. Moreover, humans<br />
can have empathy: people are capable of understanding emotions and cognitive<br />
states of other organisms. To respond to these feelings, humans have<br />
technological tools allowing them to fight against enemies. This is the case<br />
with our project: fighting <i>Ceratocystis<br />
platani</i>. <br />
</p><br />
<br />
<p class="texte">In conclusion,<br />
by destroying and hammering the Nature, we jeopardize our lives. We need<br />
Nature, we come from Nature and we depend on Nature for survival, food,<br />
discoveries and civilisation. Respecting, loving and<br />
preserving the beauty of it is also a question of<br />
survival.</p><br />
<br />
<p class="title2">Nature and artifice<br />
</p><br />
<br />
<p class="texte">Talking about<br />
the Nature refers to the whole world with an exception: all the transformations<br />
made by mankind. Nature exists regardless of men and his interventions whereas<br />
artificial is everything that exists because to humans.<br />
</p><br />
<br />
<p class="texte">However,<br />
pretending that natural and artificial are opposite does not seem to be true.<br />
Man cannot create without the various elements provided by Nature, he then justs transform Nature. Thus we may wonder if there is a<br />
true difference between natural and artificial. The border between these two<br />
notions is not as obvious as it seems. The landscapes are shaped by the hand of<br />
man, animals are domesticated, and now bacteria are considered as cell<br />
factories. A natural reserve is artificially preserved as a result of human<br />
actions. Is there still something natural since the birth of mankind? Actually,<br />
the artifice is a slight modification of Nature and couldn’t exist by itself.<br />
The distinction between natural and artificial seems sterile and we clearly<br />
understand that these notions are inextricably linked and need each other to<br />
exist. </p><br />
<br />
<p class="texte">In conclusion,<br />
isn't it our duty to use our unique position in the history of life and our human<br />
approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="title2">Back to our project</p><br />
<br />
<br />
<p class="texte">These<br />
inextricable links are obviously the basis of our project. We aim to<br />
artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago.Fighting a<br />
naturally occurring form of life that threatens it maybe just an imitation of<br />
the natural evolution process. What is considered today as ‘non-natural’<br />
may be one day regarded differently. To the extent that everything is done not<br />
to unbalance the ecosystem, our intervention can be judged rightful, even more<br />
than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="title2"> Potential strategies discussed<br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that<br />
SubtiTree will not survive and spread in the<br />
environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the natural environment (outside the tree) thanks to a proline auxotrophy system <br />
<br>- Prevent the sporulation of <i>B. subtilis</i> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild<br />
type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use an integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="title2">Public perception<br />
</p><br />
<br />
<p class="title3">Political and public adhesion</p><br />
<br />
<p class="texte">Due to our<br />
strong implication in preserving this magnificent work of art, our project<br />
interested several governmental services. Indeed some municipalities and<br />
regional councils supported our local engagement. Beyond that, our project<br />
interests the highest level of the “Canal du Midi” administration: the national<br />
navigation authority (VNF) and the Ministry of agriculture. Both of them funded<br />
this project. They are now looking for the continuation of the project after<br />
the iGEM competition. This is clearly a sign that we targeted the right<br />
question. </p><br />
<br />
<p class="texte">This project<br />
also received the attention of the public through several articles in<br />
newspapers, television, radio and internet. First we had just a local coverage,<br />
but days after days there were more and more media interested in SubtiTree. This mediatic coverage<br />
allowed us to contact concerned citizens who participated to the development of<br />
this project. This interaction with the public allowed us to explain and<br />
promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="title3">Safety principle</p><br />
<br />
<p class="texte">One single tree<br />
infected by Canker, and all the trees located in an area of a couple of hundred<br />
meters around are included in the prophylactic cut. We acted to preserve the<br />
surrounding trees. The modification of the endophytic<br />
microbial fauna generated by the introduction of the engineered bacterium has<br />
to be compared to the introduction of chemicals. They contain chlorine atom and<br />
aromatic hydrocarbon, so their remediation is complicated and they represent a<br />
source of pollution. By shortening the lifespan to one season and minimizing<br />
the risks of spreading, we plan a safe and environmental-friendly way to fight<br />
Canker. <br />
<br />
<br />
<p class="title2">Feasability<br />
</p><br />
<br />
<p class="texte">We wonder about<br />
the feasibility of tree’s treatment. As we used endophytic<br />
bacteria, we can count on the natural growth of SubtiTree<br />
inside the sap. So we can inject few bacteria to be sure to have enough<br />
bacteria to protect the tree. Some researchers (Xianling<br />
Ji<sup>1</sup> et al) already injected <i>Bacillus subtilis</i> in plants and<br />
observed an increase of bacteria concentration to a maximum of 10<sup>5</sup><br />
bacteria/mL</p><br />
<br />
<p class="texte">As we aim to<br />
inject a small quantity of bacteria, this treatment remains cheaper than the<br />
injection of several liters of chemical fungicides. In addition, this injection<br />
prevents the preventive tree cutting, which is very expensive. Cutting one tree<br />
cost around € 3000. The administration in charge of the protection of the<br />
“Canal du Midi” already plans to spend 220 million euros to cut and replant all<br />
trees along the Canal. Besides the important cost of cutting trees, it will<br />
destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the <br />
iGEM’s circumstances we could not have the time to go<br />
deeper. First, we can improve the fixation module. Using chitin as fixation<br />
anchor is simple but not enough specific to fix just one fungus type. That’s<br />
why we first think to fix SubtiTree to one protein<br />
included in the <i>Ceratocystis<br />
platani</i>’s<br />
membrane: CP. The bacterial prototype designed this summer can be optimized to<br />
trigger the fungicides production when the binding is completed, and to be more<br />
specific changing the peptides produced.</p><br />
<br />
<p class="title1">References</p><br />
<br />
<li class="tree"><p class="texte"> Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu.<b> Biological control against bacterialwilt and colonization of<br />
mulberry byan endophyticBacillus subtilis strain </b>. FEMS Microbiol Ecol. 65 (2008) 565–573. </p></li><br />
</div><br />
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
</div><br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
<br />
<p class="texte">How did we validate the three modules and improve our new protocols? Click below to find out…</p><br />
<br />
<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p><br />
<br />
<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">We performed several assays to demonstrate the chemotaxis ability of our engineered <i>Bacillus subtilis</i> strain to move towards N-acetylglucosamine (NAG), the base unit of chitin. The ability of the <i>wild-type</i> strain to move towards glucose was the positive control. <br />
</p><br />
<br />
<p class="title2">1. Petri dishes Test</p><br />
<p class="texte"><br />
The first chemotaxis assay was done in Petri dishes filled with a growth medium containing 0,3% agar. This semi-solid medium was supposed to favor bacterial motility. A paper disk soaked with the attractive compound was placed in the middle of the dish, then cells were loaded into the medium (see Figure 1). This protocol was from the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Petri dishes chemotaxis assay. (A) pipetman was used to inject cells into the semi-solid medium. (B) Bacteria would move toward the attractive compound diffusing from a paper disk.<br />
</p><br />
<br />
<p class="texte">With this assay, we however failed to see any chemotaxis of the <i>wild-type</i> control toward glucose (Figure 2-A).As <i>B. subtilis</i> is attracted by many other glucides and amino-acids that can be found in rich medium such as in LB medium. Therefore, with the hope of improving the experimental conditions, we have challenged the cells with paper discs soaked in LB medium containing glucose (Figure 2-B).<br />
</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with paper discs soaked in either a Glucose solution (A) or in Glucose containing LB medium (B) as attractive compound. Paper discs soaked with water were used as negative controls.<br />
</p><br />
<br />
<p class="texte">No difference was observed between the Petri dishes with or without glucose. With glucose containing LB medium, a large halo around the paper disk was observed. This halo might be due to cells attracted by the solution, as it was not observed when cells were not inoculated in the Petri dish (data not shown). However this result was barely reproducible. Moreover with the addition of LB medium, it was hard to make the distinction between attractive effects and cell growth resulting from random diffusion. We have therefore given up this protocol and tested alternative protocols.<br />
</p><br />
<br />
<p class="title2">2. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol was from a PhD work (see references [1]). <i>B.subtilis</i> was grown overnight to a density of 8.10<sup>8</sup> cells/mL. 10mL of the culture was mixed with 15mL of LB medium containing 1.5% agar kept at 45°C. The final agar concentration was 0.9%. Tetracycline (25 µg/ml) was added to inhibit growth in order to only observe the chemotaxis phenomenon. Plates were cooled down and dried, before digging wells with either a punch or 1mL tips. The wells were then filled with the attractive compound (Figure 3). After one hour at room temperature, pictures of the plates were taken and the results analyzed.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Plug-in-pond test design.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
After one hour, no tangible results were obtained. After 12h we observed halos around the 1M glucose containing wells in the plates without tetracycline but not in the plates with tetracycline. Again, because cells could use glucose for growth, we could not distinguish between growth and chemotaxis. Making the hypothesis that the concentration of tetracycline could have been too high and inhibited any bacterial activity, we thereafter lowered the tetracycline concentration to 15µg/mL. We repeated this protocol with this new experimental condition. We made two wells per plate (Figure 5), one with either Glucose or n-acetyl-glucosamine and one with LB medium. As previously, no results were achieved after 1h, but after 12h we could notice halos.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>B. subtilis</i> WT. The upper well contains attractive compound and the lower well contains medium without any attractive compound.<br />
</p><br />
<br />
<p class="texte"><br />
Results were not as clear as in the previous assay (Figure 4), but halos around the wells with glucose at 250mM with and without tetracycline were observed. With N-acetyl-glucosamine (NAG) as the attractive compound, halos were observed for a concentration of 25mM with tetracycline and for a concentration of 250mM without tetracycline, thus suggesting that our <i>B. subtilis</i> 168 strain is attracted toward NAG and uses it to grow.<br />
</p><br />
<br />
<br />
<p class="texte"><br />
<b>References:</b></br><br />
[1]: Claudine Baraquet. <b>Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez <i>Escherichia coli</i> et <i>Shewanella oneidensis</i></b>. 2008, Université de la Méditerranée Aix-Marseille II.<br />
</p><br />
<br />
<br />
<br />
<br />
<p class="title2">4. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin dye towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse 2014 iGEM team asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte"><br />
This new system was tested with the fuchsin dye and the assay was made with WT <i>B. subtilis</i> and N-Acetylglucosamine as the attractive compound.<br />
<br><br><br />
<i>NB: We could not see any diffusion of the fushin dye from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to prevent the presence of any air bubbles which could have impeded diffusion.<br><br />
- Then, the tube 2 was plugged and maintained with the thumb while another iGEM mate was adding the bacterial suspention of WT <i>B. subtilis</i> into the tube 1.<br><br />
- The tube 1 was also plugged and only then the thumb could be removed from the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2.<br><br />
- The same process was made with xylose as a positive control.<br><br />
<br><br />
<i>NB: According to the paper "Chemotaxis towards sugars by </i>Bacillus subtilis", (Ordal et al., 1979),<i> glucose and xylose have the same attractant power. We have privileged a positive control instead of a negative one as we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2 hours. Every 40 minutes, samples from each were removed and streaked on solid medium (dilution 1/1,000) in order to estimate the bacterial concentration.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement. As we did not find any information in the litterature and did not have enough time to optimize this protocol, we dicided to test again the first protocol from the Imperial college 2011 iGEM team : the tips capillary test.<br />
</p><br />
<br />
<p class="title2"> 5. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol from Imperial College 2011 iGEM team was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br />
<br><br />
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br><br />
- 15µL of each chemo-attractant was pipetted.<br><br />
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tips.<br><br />
- The tip was then sealed with a piece of parafilm in order to keep the liquid sterile and inside the tip.<br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- When all the chemo-attractants were added, the were fixed on a green support. The whole process can be seen on Figure 10.<br><br />
- Each tip was then immersed into 300 µL of a bacterial suspension in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and kept it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria suspensions and the bacteria content of the tips was monitored with a Thoma cell under the microscope.<br><br />
<br><br />
We experienced several problems with this system:<br><br />
- The liquid level decreasing so much during the course of the experiment that we did not have enough liquid to fill the Thoma cell for counting.<br><br />
- The bacteria were moving, therefore preventing us from accurately counting them. Taking into account this probelem, we decided to estimate the bacterial concentration by streaking the tips content on agar plate instead of using Thoma cell and microscopy.<br />
<br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte">And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>B. subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than that of the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
At the beginning, the experiment was conducted with only one negative control, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis</i> 168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. In contrast, a cell layer was observed for the NAG plates with every concentration, indicating that a large number of cells have been inoculated in the plates.<br><br />
<br><br />
Thus, we assumed that WT <i>B. subtilis</i> was more attracted by NAG than by fuchsin. In addition we could neglect the bacterial growth because the test only lasted one hour. We also neglected diffusion and osmolality phenomena for the reasons explained above. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells and thus make the fuschin dye an appropriate negative control.<br><br />
<br><br />
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
Hopefully, we managed to find a negative control: galactose (25mM). The article "Chemotaxis towards sugars by <i>B. subtilis</i>" (<i>Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<p class="texte"> The miracle arrived! We managed to prove that our WT <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p><br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
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<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2">1. Preliminary experiments</p><br />
<p class="texte">For the first experiment we wanted to check if the buffer of the binding assay was compatible with <i>B. subtilis</i> survival. To do that, we tested four bacterial concentrations (from OD 0.1 to OD 0.01). These <i>B. subtilis</i> suspensions were incubated 1 hour at 4°C with 500µL of either Chitin Binding Buffer (CBB) or water. 100 µL cell suspension were plated on LB medium in order to count surviving cells.</br><br />
Cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025.<br />
</br>We observed similar survival rates between cells treated with CBB or water (data not shown).<br />
Thus, the experimental conditions of the chitin binding assay are compatible with the bacterial life.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 16</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein allowing the bacterium to bind chitin. It is composed of the Cell Wall Binding Domain of a <i>B. subtilis</i> protein LycT, and of the GbpA domain 4 of <i>Vibrio cholerae</i> able to bind chitin. The capacity to bind chitin was assessed by bringing together either the WT strain or the engineered bacterium with chitin beads. After several washes, bacteria still bound to the beads were counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We measured the quantity of bacteria before the binding assay (Direct), in the eluted fraction of the first (Wash A) and the second (Wash B) washing steps, and associated with the beads.</br> <br />
<br />
Both bacterial solutions of WT and engineered bacterium used for the binding assay had the initial same concentration before the assay (Direct on Figure 17).<br />
There is also no significant difference between both strains after the first wash (Wash A on Figure 17). However, the concentration of cells quantified in the eluted fraction after the second wash was significantly higher for the wild-type strain. This suggests that the engineered strain is more retained on chitin beads. This was confirmed by a 40 times higher number of cells associated with the chitin beads for the engineered over the wild-type strain(Beads on Figure 17). <br />
<br/>Thus, we successfully engineered <i>B. subtilis</i> to promote its fixation on chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and engineered bacterium to chitin. The <span style="color:#0000FF">WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentrations have been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.<br />
</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We wanted to observe SubtiTree bound on the chitin coated beads. In order to perform a 3D reconstruction showing this interaction, we used confocal laser scanning microscope. Through the use of a green fluorochrome (Syto9), we highlighted the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We can notice the engineered bacterium is well attached to the surface of beads coated with chitin.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of engineered strain associated with beads surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and the engineered strain (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p><br />
<br />
<p class="texte">We then performed a wash step on the chitin beads. We measured the release of bacteria on the washing solution. When our bacterium has the binding module (Right on Figure 20), there is less release than without the module (Right on Figure 20). Therefore, the engineered bacterium is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of elution fraction of WT <i>B. subtilis</i> (Left) and engineered bacterium (Right). <br />
</p><br />
<br />
<p class="texte">To conclude, all the results are consistent with the successfull integration of a functional chitin binding system in <i>B. subtilis</i>. We thus validated the second module.</p><br />
<br />
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<br />
<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus. These tests were therefore performed with different non-pathogenic fungal strains from the same phylum as <i>Ceratocystis Platani</i>.<br />
</br><br />
After 1 to 6 days at 30°C depending on the fungal strain, the PDA (Potato Dextrose Agar) plates covered with fungus and containing a paper disk soaked with a commercial peptides solution were analyzed.</p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i> (Figure 21). Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. Copper Sulfate, a well-known chemical fungicide was used as a positive control.Inhibition of the fungal growth was complete with 20 mg/ml copper Sulfate, and at 10 mg/ml a darker halo appeared around the pad as can be seen on figure 21. This corresponds to a sporulating halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
<br />
<center><img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"></center><br />
<p class="legend"> Figure 21: Results of the preliminary tests of antifungal compounds</p><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth in the tested conditions. Following these tests, new conditions were adopted in order to avoid too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees, and a 'sap-like' medium was elaborated (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">protocols</a> for more informations). Incubations were performed at room temperature. These new conditions were used as standard for the the next experiment.<br><br />
Camille also concluded that turning blue the Canal du Midi using high copper sulfate concentrations is not such a good idea... Thereby strengthening our faith in SubtiTree :-) ! <br />
</p><br />
<br />
<p class="title2">2. Test with antifungal bacteria</p><br />
<br />
<p class="texte">In order to test our <i>Bacillus subtilis</i> engineered strains, it was essential to find the right balance between the fungal growth and the bacterial one which was achieved with the previous modifications. Furthermore, in our genetic constructions, the antifungal peptides were designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h before testings. Inhibitory effect of supernatant and cell pellets were tested separately. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i> growth was clearly observable for the strain expressing the D4E1 peptide. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (Figure 22).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, we thus suppose a putative synergistic effect between these two peptides.</br><br />
Regarding EcAMP-1, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP-1 and in addition, the sequencing results for these constructs showed several discrepancies with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of either low concentrations or of instability in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus, <i>Aspergillus brasiliensis</i> (figure 22). This effect is comparable to the one previously noted with a low concentration of copper sulfate (figure 21).</br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
After this set of experiments, the strains expressing D4E1 or GAFP-1 + D4E1 have been shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b>we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in association with Sylvain Raffaële and Marielle Barascud in the National Institute for the Agronomic Research.</p><br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of antifungal <i>B. subtilis</i> in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform preliminary<i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous sets of experiments. </br><br />
The strain expressing fungicides was first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) was placed on the leaves. </br><br />
</p><br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves could be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br><br />
Without proper treatment, the drop of the phytopathogenic fungus on <i>Nicotiana benthamiana</i> leaves caused a necrosis halo which could be measured after 40h. The number of necrotic sites and the lesion size appeared as reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike the WT bacterium. Two independant replicate of this experiments were performed successfully</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b><br />
</p><br />
<br />
<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">We could expect that bringing altogether the three modules (chemotaxis, binding and antifungal) should even improved the performance of SubtiTree. Thus, these results open the way towards the use of SubtiTree in plane tree</br><br />
More than ever, let's save our trees with SubtiTree!<br />
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<p class="texte">How did we validate the three modules and improve our new protocols? Click below to find out…</p><br />
<br />
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<br />
<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">We performed several assays to demonstrate the chemotaxis ability of our engineered <i>Bacillus subtilis</i> strain to move towards N-acetylglucosamine (NAG), the base unit of chitin. The ability of the <i>wild-type</i> strain to move towards glucose was the positive control. <br />
</p><br />
<br />
<p class="title2">1. Petri dishes Test</p><br />
<p class="texte"><br />
The first chemotaxis assay was done in Petri dishes filled with a growth medium containing 0,3% agar. This semi-solid medium was supposed to favor bacterial motility. A paper disk soaked with the attractive compound was placed in the middle of the dish, then cells were loaded into the medium (see Figure 1). This protocol was from the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Petri dishes chemotaxis assay. (A) pipetman was used to inject cells into the semi-solid medium. (B) Bacteria would move toward the attractive compound diffusing from a paper disk.<br />
</p><br />
<br />
<p class="texte">With this assay, we however failed to see any chemotaxis of the <i>wild-type</i> control toward glucose (Figure 2-A).As <i>B. subtilis</i> is attracted by many other glucides and amino-acids that can be found in rich medium such as in LB medium. Therefore, with the hope of improving the experimental conditions, we have challenged the cells with paper discs soaked in LB medium containing glucose (Figure 2-B).<br />
</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with paper discs soaked in either a Glucose solution (A) or in Glucose containing LB medium (B) as attractive compound. Paper discs soaked with water were used as negative controls.<br />
</p><br />
<br />
<p class="texte">No difference was observed between the Petri dishes with or without glucose. With glucose containing LB medium, a large halo around the paper disk was observed. This halo might be due to cells attracted by the solution, as it was not observed when cells were not inoculated in the Petri dish (data not shown). However this result was barely reproducible. Moreover with the addition of LB medium, it was hard to make the distinction between attractive effects and cell growth resulting from random diffusion. We have therefore given up this protocol and tested alternative protocols.<br />
</p><br />
<br />
<p class="title2">2. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol was from a PhD work (see references [1]). <i>B.subtilis</i> was grown overnight to a density of 8.10<sup>8</sup> cells/mL. 10mL of the culture was mixed with 15mL of LB medium containing 1.5% agar kept at 45°C. The final agar concentration was 0.9%. Tetracycline (25 µg/ml) was added to inhibit growth in order to only observe the chemotaxis phenomenon. Plates were cooled down and dried, before digging wells with either a punch or 1mL tips. The wells were then filled with the attractive compound (Figure 3). After one hour at room temperature, pictures of the plates were taken and the results analyzed.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Plug-in-pond test design.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
After one hour, no tangible results were obtained. After 12h we observed halos around the 1M glucose containing wells in the plates without tetracycline but not in the plates with tetracycline. Again, because cells could use glucose for growth, we could not distinguish between growth and chemotaxis. Making the hypothesis that the concentration of tetracycline could have been too high and inhibited any bacterial activity, we thereafter lowered the tetracycline concentration to 15µg/mL. We repeated this protocol with this new experimental condition. We made two wells per plate (Figure 5), one with either Glucose or n-acetyl-glucosamine and one with LB medium. As previously, no results were achieved after 1h, but after 12h we could notice halos.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>B. subtilis</i> WT. The upper well contains attractive compound and the lower well contains medium without any attractive compound.<br />
</p><br />
<br />
<p class="texte"><br />
Results were not as clear as in the previous assay (Figure 4), but halos around the wells with glucose at 250mM with and without tetracycline were observed. With N-acetyl-glucosamine (NAG) as the attractive compound, halos were observed for a concentration of 25mM with tetracycline and for a concentration of 250mM without tetracycline, thus suggesting that our <i>B. subtilis</i> 168 strain is attracted toward NAG and uses it to grow.<br />
</p><br />
<br />
<br />
<p class="texte"><br />
<b>References:</b></br><br />
[1]: Claudine Baraquet. <b>Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez <i>Escherichia coli</i> et <i>Shewanella oneidensis</i></b>. 2008, Université de la Méditerranée Aix-Marseille II.<br />
</p><br />
<br />
<br />
<br />
<br />
<p class="title2">4. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin dye towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse 2014 iGEM team asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte"><br />
This new system was tested with the fuchsin dye and the assay was made with WT <i>B. subtilis</i> and N-Acetylglucosamine as the attractive compound.<br />
<br><br><br />
<i>NB: We could not see any diffusion of the fushin dye from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to prevent the presence of any air bubbles which could have impeded diffusion.<br><br />
- Then, the tube 2 was plugged and maintained with the thumb while another iGEM mate was adding the bacterial suspention of WT <i>B. subtilis</i> into the tube 1.<br><br />
- The tube 1 was also plugged and only then the thumb could be removed from the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2.<br><br />
- The same process was made with xylose as a positive control.<br><br />
<br><br />
<i>NB: According to the paper "Chemotaxis towards sugars by </i>Bacillus subtilis", (Ordal et al., 1979),<i> glucose and xylose have the same attractant power. We have privileged a positive control instead of a negative one as we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2 hours. Every 40 minutes, samples from each were removed and streaked on solid medium (dilution 1/1,000) in order to estimate the bacterial concentration.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement. As we did not find any information in the litterature and did not have enough time to optimize this protocol, we dicided to test again the first protocol from the Imperial college 2011 iGEM team : the tips capillary test.<br />
</p><br />
<br />
<p class="title2"> 5. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol from Imperial College 2011 iGEM team was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br />
<br><br />
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br><br />
- 15µL of each chemo-attractant was pipetted.<br><br />
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tips.<br><br />
- The tip was then sealed with a piece of parafilm in order to keep the liquid sterile and inside the tip.<br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- When all the chemo-attractants were added, the were fixed on a green support. The whole process can be seen on Figure 10.<br><br />
- Each tip was then immersed into 300 µL of a bacterial suspension in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and kept it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria suspensions and the bacteria content of the tips was monitored with a Thoma cell under the microscope.<br><br />
<br><br />
We experienced several problems with this system:<br><br />
- The liquid level decreasing so much during the course of the experiment that we did not have enough liquid to fill the Thoma cell for counting.<br><br />
- The bacteria were moving, therefore preventing us from accurately counting them. Taking into account this probelem, we decided to estimate the bacterial concentration by streaking the tips content on agar plate instead of using Thoma cell and microscopy.<br />
<br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte">And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>B. subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than that of the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
At the beginning, the experiment was conducted with only one negative control, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis</i> 168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. In contrast, a cell layer was observed for the NAG plates with every concentration, indicating that a large number of cells have been inoculated in the plates.<br><br />
<br><br />
Thus, we assumed that WT <i>B. subtilis</i> was more attracted by NAG than by fuchsin. In addition we could neglect the bacterial growth because the test only lasted one hour. We also neglected diffusion and osmolality phenomena for the reasons explained above. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells and thus make the fuschin dye an appropriate negative control.<br><br />
<br><br />
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
Hopefully, we managed to find a negative control: galactose (25mM). The article "Chemotaxis towards sugars by <i>B. subtilis</i>" (<i>Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<p class="texte"> The miracle arrived! We managed to prove that our WT <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p><br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
<br />
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</div><br />
<br />
<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2">1. Preliminary experiments</p><br />
<p class="texte">For the first experiment we wanted to check if the buffer of the binding assay was compatible with <i>B. subtilis</i> survival. To do that, we tested four bacterial concentrations (from OD 0.1 to OD 0.01). These <i>B. subtilis</i> suspensions were incubated 1 hour at 4°C with 500µL of either Chitin Binding Buffer (CBB) or water. 100 µL cell suspension were plated on LB medium in order to count surviving cells.</br><br />
Cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025.<br />
</br>We observed similar survival rates between cells treated with CBB or water (data not shown).<br />
Thus, the experimental conditions of the chitin binding assay are compatible with the bacterial life.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 16</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein allowing the bacterium to bind chitin. It is composed of the Cell Wall Binding Domain of a <i>B. subtilis</i> protein LycT, and of the GbpA domain 4 of <i>Vibrio cholerae</i> able to bind chitin. The capacity to bind chitin was assessed by bringing together either the WT strain or the engineered bacterium with chitin beads. After several washes, bacteria still bound to the beads were counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We measured the quantity of bacteria before the binding assay (Direct), in the eluted fraction of the first (Wash A) and the second (Wash B) washing steps, and associated with the beads.</br> <br />
<br />
Both bacterial solutions of WT and engineered bacterium used for the binding assay had the initial same concentration before the assay (Direct on Figure 17).<br />
There is also no significant difference between both strains after the first wash (Wash A on Figure 17). However, the concentration of cells quantified in the eluted fraction after the second wash was significantly higher for the wild-type strain. This suggests that the engineered strain is more retained on chitin beads. This was confirmed by a 40 times higher number of cells associated with the chitin beads for the engineered over the wild-type strain(Beads on Figure 17). <br />
<br/>Thus, we successfully engineered <i>B. subtilis</i> to promote its fixation on chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and engineered bacterium to chitin. The <span style="color:#0000FF">WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentrations have been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.<br />
</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We wanted to observe SubtiTree bound on the chitin coated beads. In order to perform a 3D reconstruction showing this interaction, we used confocal laser scanning microscope. Through the use of a green fluorochrome (Syto9), we highlighted the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We can notice the engineered bacterium is well attached to the surface of beads coated with chitin.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of engineered strain associated with beads surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and the engineered strain (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p><br />
<br />
<p class="texte">We then performed a wash step on the chitin beads. We measured the release of bacteria on the washing solution. When our bacterium has the binding module (Right on Figure 20), there is less release than without the module (Right on Figure 20). Therefore, the engineered bacterium is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of elution fraction of WT <i>B. subtilis</i> (Left) and engineered bacterium (Right). <br />
</p><br />
<br />
<p class="texte">To conclude, all the results are consistent with the successfull integration of a functional chitin binding system in <i>B. subtilis</i>. We thus validated the second module.</p><br />
<br />
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</div><br />
<br />
<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus. These tests were therefore performed with different non-pathogenic fungal strains from the same phylum as <i>Ceratocystis Platani</i>.<br />
</br><br />
After 1 to 6 days at 30°C depending on the fungal strain, the PDA (Potato Dextrose Agar) plates covered with fungus and containing a paper disk soaked with a commercial peptides solution were analyzed.</p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i> (Figure 21). Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. Copper Sulfate, a well-known chemical fungicide was used as a positive control.Inhibition of the fungal growth was complete with 20 mg/ml copper Sulfate, and at 10 mg/ml a darker halo appeared around the pad as can be seen on figure 21. This corresponds to a sporulating halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
<br />
<center><img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"></center><br />
<p class="legend"> Figure 21: Results of the preliminary tests of antifungal compounds</p><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth in the tested conditions. Following these tests, new conditions were adopted in order to avoid too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees, and a 'sap-like' medium was elaborated (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">protocols</a> for more informations). Incubations were performed at room temperature. These new conditions were used as standard for the the next experiment.<br><br />
Camille also concluded that turning blue the Canal du Midi using high copper sulfate concentrations is not such a good idea... Thereby strengthening our faith in SubtiTree :-) ! <br />
</p><br />
<br />
<p class="title2">2. Test with antifungal bacteria</p><br />
<br />
<p class="texte">In order to test our <i>Bacillus subtilis</i> engineered strains, it was essential to find the right balance between the fungal growth and the bacterial one which was achieved with the previous modifications. Furthermore, in our genetic constructions, the antifungal peptides were designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h before testings. Inhibitory effect of supernatant and cell pellets were tested separately. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i> growth was clearly observable for the strain expressing the D4E1 peptide. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (Figure 22).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, we thus suppose a putative synergistic effect between these two peptides.</br><br />
Regarding EcAMP-1, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP-1 and in addition, the sequencing results for these constructs showed several discrepancies with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of either low concentrations or of instability in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus, <i>Aspergillus brasiliensis</i> (figure 22). This effect is comparable to the one previously noted with a low concentration of copper sulfate (figure 21).</br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
After this set of experiments, the strains expressing D4E1 or GAFP-1 + D4E1 have been shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b>we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in association with Sylvain Raffaële and Marielle Barascud in the National Institute for the Agronomic Research.</p><br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of antifungal <i>B. subtilis</i> in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform preliminary<i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous sets of experiments. </br><br />
The strain expressing fungicides was first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) was placed on the leaves. </br><br />
</p><br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves could be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br><br />
Without proper treatment, the drop of the phytopathogenic fungus on <i>Nicotiana benthamiana</i> leaves caused a necrosis halo which could be measured after 40h. The number of necrotic sites and the lesion size appeared as reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike the WT bacterium. Two independant replicate of this experiments were performed successfully</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b><br />
</p><br />
<br />
<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">We could expect that bringing altogether the three modules (chemotaxis, binding and antifungal) should even improved the performance of SubtiTree. Thus, these results open the way towards the use of SubtiTree in plane tree</br><br />
More than ever, let's save our trees with SubtiTree!<br />
</p><br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Team&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Our Team</p> <br />
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<p class="texte">Let us introduce you to our fabulous tree-friendly team , 100% biodegradable and certified pesticide-free.</p><br />
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<p class="title1">Students</p><br />
<p class="title3"> <i>(Put the mouse over the pictures to extend them!)</i> </p><br />
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<img src="https://static.igem.org/mediawiki/2014/d/da/Diane_gros.png" alt="Diane Barbay" border="0" style="margin-top:5px"/><br />
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<p class="title2"><b>Diane Barbay</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Diligent (Wait a minute, I have to write down these results in my notebook! ㅋㅋㅋ)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Hardworking, Diane is the most conscientious in her work: her notebook is so clean and organized! She is persevering and always tries to find the answer to the problem (except when plasmids shortens between two digestions…). She talks to bacteria to encourage them taking up plasmid during transformation. She is like a babysitter for our cells :-)<br />
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<p class="title2"><b>Emeline Flajollet</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in competent cells (Oh no, I have to make competent cells again…)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Master of Microbiology at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About her:</b> Emeline looks discreet at first, but when you meet her, you discover a frank person who knows how to express her opinions and how to be understood, which is a good thing for team work. She is diligent and involved in her work, always trying to do her best, and providing advice to others. She spends a lot of time on social networks, allowing us to keep in touch with other teams.<br />
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<img src="https://static.igem.org/mediawiki/2014/8/80/Mathieu_gros.png" alt="Mathieu Fournié" border="0" style="margin-top:5px"/><br />
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<p class="title2"><b>Mathieu Fournié</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in communication (I found someone else who wants to interview us!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Master of Microbiology at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About him:</b> Initial founder of the project, Mathieu is a fan of Midi Pyrenées's region and is eager to protect its natural environment and resources. He is devoted to the project and does his best to succeed. His leadership skills help us focus on the main goals and deadlines. Even if it makes him forget to do his own tasks! His good interpersonal skills and numerous contacts helped us to capture media attention. <br />
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<p class="title2"><b>Florie Gosseau</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our IT expert (Modelling? Ok let’s do it!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Bioinformatics and Biological Systems Master at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About her:</b> Florie is a smiling and spontaneous girl. She is this kind of person capable of giving a true smile when nothing works, and she can disentangle conflicts thanks to her patience and her kindness. She is really helpful and never says no when you ask for service. She is also frank and reports problems when there are some. Finally, she is the only one good at informatics, which makes her indispensable for us!<br />
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<img src="https://static.igem.org/mediawiki/2014/9/92/Camille_gros.png" alt="Mathieu Fournié" border="0" style="margin-top:5px"/><br />
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<p class="title2"><b>Camille Jourdan</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in cloning (Camille, which enzyme should I take?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About him:</b> Jovial and funny, Camille has sometimes eruptions of madness which makes him so special. Conversely, he is capable of incredible moments of intelligence and concentration (primers and plasmids hold no secret for him) and he is really diligent. He is sarcastic but definitely not nasty. He is the athlete of the team and never misses an opportunity to make gymnastics exercises or Plasmid Dance!<br />
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<p class="title2"><b>Aurélie Kanitzer</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Distracted (Oh I forgot my keys! I have to go back home, see you in 15 minutes!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Aurélie is a joyful and spontaneous person, which makes you laugh and smile. Often distracted, she can make blunders… She is natural and honest and does not try to play a role. She is open-minded and helpful: you can count on her. Strong and sensitive at the same time, she is able to face difficulties. Don’t go by appearances, this tiny girl has personality and she does not let others walk over her!<br />
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<p class="title2"><b>Laureen Mirassou</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our best negotiator (Don’t worry about prices for flight tickets, I deal with that)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Laureen is a mix of kindness, helpfulness and generosity. Always trying to help people, she is essential to ease tensions and to solve social issues: she says what is wrong gently and calmly. Her good interpersonal abilities helped us to make good business and to solve many administrative problems. Her high level of English proficiency is also something precious for the team.<br />
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<p class="title2"><b>Manon Molina</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Organized (Let’s make a to-do-list!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About her:</b> Manon is organized: she always makes to-do-lists to not forget anything and do tasks in time. Thank goodness, she is here to monitor the budget and do the accounting! She makes her work conscientiously and meticulously, not allowing mistakes. She is kind and lenient but can also be frank when she does not endorse your methods. In short: serious and devoted to the project.<br />
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<p class="title2"><b>Fanny Pineau</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Absent-minded (which cloning are we doing?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About her:</b> In the lab, Fanny is often lost in her works: too many cloning at the same time, you should not ask too much to her blond brain! Not enough concentrated, she can sometimes make blunders. Besides that, she is applied and perfectionist when she plunges into her work. Finally, she is a joyful and smiling person; also honest and frank, telling it like it is.<br />
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<p class="title2"><b>Pierre Reitzer</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Relaxed (Make a presentation barefoot, why not?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About him:</b> Pierre is the co-founder of the project. He is really friendly and is always willing to discuss and help people with their experiments. Moreover, he greets us with a smile each time we see him in the lab. He is spontaneous and forthright: we know what he thinks. Sometimes, he has his head in the clouds and fails the same experiment three times… But he is involved in his work and does not count hours. <br />
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<p class="title2"><b>Abdel Touré</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Luxury tastes (Why would we go elsewhere than the Hilton?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Master at ENSAT Toulouse agronomic school</p> <br />
<p class="textesimple"><b>About him:</b> Abdel is this kind of person that you remember: he is always jovial and running everywhere. He is a funny guy who likes making jokes. But, be careful! He is also sensitive, your jokes might not make him laugh at all. He takes care of his appearance, and likes smoking his e-cigarette. He always swears in English, but we like it because this is funny, and he is a good English speaker!<br />
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<p class="title1">Instructors</p><br />
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<p class="title2"><b>Gilles Truan</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our amazing instructor!</p><br />
<p class="textesimple"><b>Job:</b> Researcher at CNRS (Centre National de la Recherche Scientifique) and at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
<p class="textesimple"><b>About him:</b> He is with us from the beginning to the end as a father (and already a grandfather!!), our instructor is the master of cloning and the captain of PCR! He provided us valuable help and good tips he has known since the time he was supervising Marie Curie's doctoral thesis.</p><br />
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<p class="title2"><b>Brice Enjalbert</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> The right-hand man of our amazing instructor!</p><br />
<p class="textesimple"><b>Job:</b> Teacher-Researcher at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
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<p class="title2"><b>Florence Bordes</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> The only female advisor! </p><br />
<p class="textesimple"><b>Job:</b> Teacher-Researcher at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
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<p class="title2"><b>Kaymeuang Cam</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our bacteriologist advisor! </p><br />
<p class="textesimple"><b>Job:</b> Teacher-Researcher at IPBS (Institut de Pharmacologie et de Biologie Structurale)</p> <br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<br />
<p class="texte">The ethical questioning turned out to be one of the major starting points of our project. Acting on an established environment and modifying it is no mean feat and our thinking combines technical and philosophical point of views. The actual purpose of our project also leads us to undertake an ethical questioning about the role of the scientist regarding “useless” things such as the trees lining the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="texte" style="text-align:center"><B>Is it the scientists’ role to protect beauty?</B><br />
</p><br />
<br />
<p class="texte"> Beauty is a feeling of satisfaction and is selfless. It is more a feeling than the property of a thing, this is not a notion we can clearly understand. Indeed, we can find something beautiful even when we don’t know the purpose of the object. There is always a distinction between natural beauty and artistic beauty according to Hegel, the famous author. The artistic beauty is born from our mind and our spirit: it is an element of signification of the work of art whereas the natural beauty of the object is external. In a way, the Canal du Midi combines both types of beauty: a natural one regarding the nature, the centenary plane trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, scientists judge beauty as a superficial feature not deserving to undertake any kind of scientific efforts to maintain it. The traditional role of scientists is to solve global issues and to elaborate complex strategies in order to find useful solutions for everyone’s life. <br />
Once made this observation, one may wonder why synthetic biology would be used only to protect the useless beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial interrogation leads us to consider science and synthetic biology in another way. <B> What if the role of scientists was also to make people rediscovering the beauty of nature? What if the bases of new scientific challenges resulted from a more local scale? </B> Science does not have to be elitist, it has so much to gain opening itself to these challenges. First scientifically, as research is never useless and as we never know the impact and the scope of our results. Then, socially as we could measure the deep interest raised by our project within the population and the media. Adopting a new vision of synthetic biology, we probably make people change their mind about this innovative discipline. <br />
The traditional cold objectivity of science distances itself from the society. Scientists are also beings capable of feeling the beauty, sensitive to the charm of landscapes and <B>able to understand the usefulness of useless trees </B>…<br />
The design of a strategy to protect useless beauty may seem senseless but we believe that it is also the scientist’s duty. Thus, it becomes essential to protect the beauty of this site. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human intervention in the nature</p><br />
<p class="texte">Our main questioning aim to understand the complicated relationship between man and nature. Does the mankind have the proper right to operate in nature? Is modified nature considered as artificial?</p><br />
<p class="texte" style="text-align:center"><B> Mankind & Nature </B><br />
</p><br />
<br />
<p class="texte"> Nature is known as a creation of God. Human is linked to the nature and for that reason the nature deserves to be respected and loved. Mankind has always been linked to Nature as its survival depends on what comes out of the ground, the trees, the oceans… The nature is a source of wealth for the humankind. It ensures survival and development by giving men the wood, the rocks, the soil to build shelters. Being in contact with nature can allow men to feel strong emotion, as describe by poets like Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth of humanity, man himself understood well the importance of studying and mastering Nature to develop the civilization. Still today the most advanced technologies often try to mimic natural phenomena. With the development of the civilizations, men modified their environment, changing it for their own comfort depending on their own desire. By increasing their cities and acitvities, humans modify the natural environment. With the industrialization of the societies, the natural environment has suffered from human activities such as waste discharges, oil slicks, intensive fishing but also the introduction of devastating species such as the pathogen,<I> Ceratocystis platani</I>. However, despite these negative aspects, men are capable of favorable actions to help the environment and fix their mistakes. The current trend is to limit the impact of human interventions on the nature, and hopefully this trend is not transient and will not vanish. A new desire is born, a wish to protect the nature and the wildness. Man fits with his position: he takes advantage of the environment and the environment takes advantage of the reasoned human interventions. There is an adaptation of the mankind toward the nature. Moreover, humans have the capacity of empathy: people are able to understand the emotions and cognitive states of other organisms and to identify to them. To respond to these feelings, humans have technological tools allowing them to fight against enemies such as <I>Ceratocystis platani.</I><br />
</p><br />
<br />
<p class="texte">In conclusion, by destroying and hammering the nature, we jeopardize our lives. We need the nature, we come from the nature and we depend on nature for our survival, our food, our discoveries and our civilisation. Respecting, loving and preserving the environment is a question of survival.</p><br />
<br />
<p class="texte" style="text-align:center"><B> Nature and artifice </B><br />
</p><br />
<br />
<p class="texte">Talking about the nature refers to the whole world with an exception: all the transformations made by mankind. Thus, the nature consists in the real without all the artificial elements created by humans. The nature is existing regardless of men and his interventions whereas artificial is everything that exists thanks to humans.<br />
</p><br />
<br />
<p class="texte">However, pretending that natural and artificial are opposite does not seem to be true. Man cannot create without elements provided by the nature, he is just transforming the nature, changing the shape. Thus we may wonder if there is a true difference between natural and artificial. The border between these two notions is not as obvious as it seems. The landscapes are shaped by the hand of man, animals are domesticated, and now bacteria are considered as cell factories. A natural reserve is artificially preserved as the result of human actions. Is there still something natural since the birth of humankind? Actually, the artifice is a slight modification of Nature and couldn’t exist by itself. The distinction between natural and artificial seems sterile and we clearly understand that these notions are inextricably linked and need each other to exist. </p><br />
<br />
<p class="texte">In conclusion,isn't it our duty to use our unique position in the history of life and our human approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="texte" style="text-align:center"><B>Back to our project</B><br />
</p><br />
<br />
<br />
<p class="texte">These inextricable links are obviously the basis of our project. We aim to artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago. The modification of a naturally occurring form of life to strengthen it is maybe just the imitation of the natural evolution process. What is considered today as ‘non-natural’ may be one day regarded differently. To the extent that everything is done not to unbalance the ecosystem, our intervention can be judged rightful, even more than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="texte" style="text-align:center"><B> Potential strategies discussed</B><br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that SubtiTree will not survive and spread in the environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the environment thanks to a proline auxotrophy system<br />
<br>- Prevent the sporulation of <I> B. subtilis</I> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="texte" style="text-align:center"><B> Public perception </B><br />
</p><br />
<br />
<p class="texte"><I><CENTER>Political and public adhesion</I></CENTER></p><br />
<br />
<p class="texte">Because of the magnitude of our project,its application interested several civil services. Indeed some municipalities and regional councils supported our local engagement. Beyond that, our project interests the highest level of the “Canal du Midi” administration: the national navigation authority and the Ministry of agriculture funded this project. They are still looking for the best way to further this work after the iGEM competition. </p><br />
<br />
<p class="texte">This project also received the attention of the public through several articles in newspapers, television, radio and internet. First we had just a local coverage, but days after days there were more and more media intereseted in SubtiTree. This mediatic coverage allowed us to contact concerned citizens who participated to the development of this project. This interaction with the public allowed us to explain and promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="texte"><I><CENTER>Safety principle</I></CENTER></p><br />
<br />
<p class="texte">One single tree infected by Canker, and all the trees located in an area of a couple of hundred meters around are included in the prophylactic cut. We acted to preserve the surrounding trees. The modification of the endophytic microbial fauna generated by the introduction of the engineered bacterium has to be compared to the introduction of chemicals. They contain chlorine atom and aromatic hydrocarbon, so their remediation is complicated and they represent a source of pollution. By shortening the lifespan to one season and minimizing the risks of spreading, we plan a safe and environmental-friendly way to fight Canker. <br />
<br />
<br />
<p class="texte" style="text-align:center"><B> Feasability </B><br />
</p><br />
<br />
<p class="texte">We wonder about the feasibility of tree’s treatment. As we used endophytic bacteria, we can count on the natural growth of SubtiTree inside the sap. So we can inject few bacteria to be sure to have enough bacteria to protect the tree. Some researchers (Xianling Ji1 et al) already injected <i>Bacillus subtilis</i> in plants and observe an increase of bacteria concentration to a maximum of 10^5 bacteria/mL/p><br />
<br />
<p class="texte">As we aim to inject a poor quantity of bacteria, this treatment remains cheaper than the injection of several litter of fungicides. In addition, this injection prevents the preventive tree cutting, which is very expensive. Cutting one tree cost around €3000. The administration in charge of the protection of the “Canal du Midi” already plans to spend 220 million euros to cut and replant all trees along the Canal. Besides the important cost of cutting trees, it will destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the iGEM’s circumstances we could not have the time to go deeper. First, we can improve the fixation module. Using chitin as fixation anchor is simple but not enough specific to fix just one fungus type. That’s why we first think to fix SubtiTree to one protein included in the <I>Ceratocystis platani</I>’s membrane: CP. The bacterial prototype designed this summer can be optimized to trigger the fungicides production when the binding is completed, and to be more specific changing the peptides produced.</p><br />
<br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<br />
<p class="texte">The ethical questioning turned out to be one of the major starting points of our project. Acting on an established environment and modifying it is no mean feat and our thinking combines technical and philosophical point of views. The actual purpose of our project also leads us to undertake an ethical questioning about the role of the scientist regarding “useless” things such as the trees lining the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="texte" style="text-align:center"><B>Is it the scientists’ role to protect beauty?</B><br />
</p><br />
<br />
<p class="texte"> Beauty is a feeling of satisfaction and is selfless. It is more a feeling than the property of a thing, this is not a notion we can clearly understand. Indeed, we can find something beautiful even when we don’t know the purpose of the object. There is always a distinction between natural beauty and artistic beauty according to Hegel, the famous author. The artistic beauty is born from our mind and our spirit: it is an element of signification of the work of art whereas the natural beauty of the object is external. In a way, the Canal du Midi combines both types of beauty: a natural one regarding the nature, the centenary plane trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, scientists judge beauty as a superficial feature not deserving to undertake any kind of scientific efforts to maintain it. The traditional role of scientists is to solve global issues and to elaborate complex strategies in order to find useful solutions for everyone’s life. <br />
Once made this observation, one may wonder why synthetic biology would be used only to protect the useless beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial interrogation leads us to consider science and synthetic biology in another way. <B> What if the role of scientists was also to make people rediscovering the beauty of nature? What if the bases of new scientific challenges resulted from a more local scale? </B> Science does not have to be elitist, it has so much to gain opening itself to these challenges. First scientifically, as research is never useless and as we never know the impact and the scope of our results. Then, socially as we could measure the deep interest raised by our project within the population and the media. Adopting a new vision of synthetic biology, we probably make people change their mind about this innovative discipline. <br />
The traditional cold objectivity of science distances itself from the society. Scientists are also beings capable of feeling the beauty, sensitive to the charm of landscapes and <B>able to understand the usefulness of useless trees </B>…<br />
The design of a strategy to protect useless beauty may seem senseless but we believe that it is also the scientist’s duty. Thus, it becomes essential to protect the beauty of this site. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human intervention in the nature</p><br />
<p class="texte">Our main questioning aim to understand the complicated relationship between man and nature. Does the mankind have the proper right to operate in nature? Is modified nature considered as artificial?</p><br />
<p class="texte" style="text-align:center"><B> Mankind & Nature </B><br />
</p><br />
<br />
<p class="texte"> Nature is known as a creation of God. Human is linked to the nature and for that reason the nature deserves to be respected and loved. Mankind has always been linked to Nature as its survival depends on what comes out of the ground, the trees, the oceans… The nature is a source of wealth for the humankind. It ensures survival and development by giving men the wood, the rocks, the soil to build shelters. Being in contact with nature can allow men to feel strong emotion, as describe by poets like Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth of humanity, man himself understood well the importance of studying and mastering Nature to develop the civilization. Still today the most advanced technologies often try to mimic natural phenomena. With the development of the civilizations, men modified their environment, changing it for their own comfort depending on their own desire. By increasing their cities and acitvities, humans modify the natural environment. With the industrialization of the societies, the natural environment has suffered from human activities such as waste discharges, oil slicks, intensive fishing but also the introduction of devastating species such as the pathogen,<I> Ceratocystis platani</I>. However, despite these negative aspects, men are capable of favorable actions to help the environment and fix their mistakes. The current trend is to limit the impact of human interventions on the nature, and hopefully this trend is not transient and will not vanish. A new desire is born, a wish to protect the nature and the wildness. Man fits with his position: he takes advantage of the environment and the environment takes advantage of the reasoned human interventions. There is an adaptation of the mankind toward the nature. Moreover, humans have the capacity of empathy: people are able to understand the emotions and cognitive states of other organisms and to identify to them. To respond to these feelings, humans have technological tools allowing them to fight against enemies such as <I>Ceratocystis platani.</I><br />
</p><br />
<br />
<p class="texte">In conclusion, by destroying and hammering the nature, we jeopardize our lives. We need the nature, we come from the nature and we depend on nature for our survival, our food, our discoveries and our civilisation. Respecting, loving and preserving the environment is a question of survival.</p><br />
<br />
<p class="texte" style="text-align:center"><B> Nature and artifice </B><br />
</p><br />
<br />
<p class="texte">Talking about the nature refers to the whole world with an exception: all the transformations made by mankind. Thus, the nature consists in the real without all the artificial elements created by humans. The nature is existing regardless of men and his interventions whereas artificial is everything that exists thanks to humans.<br />
</p><br />
<br />
<p class="texte">However, pretending that natural and artificial are opposite does not seem to be true. Man cannot create without elements provided by the nature, he is just transforming the nature, changing the shape. Thus we may wonder if there is a true difference between natural and artificial. The border between these two notions is not as obvious as it seems. The landscapes are shaped by the hand of man, animals are domesticated, and now bacteria are considered as cell factories. A natural reserve is artificially preserved as the result of human actions. Is there still something natural since the birth of humankind? Actually, the artifice is a slight modification of Nature and couldn’t exist by itself. The distinction between natural and artificial seems sterile and we clearly understand that these notions are inextricably linked and need each other to exist. </p><br />
<br />
<p class="texte">In conclusion,isn't it our duty to use our unique position in the history of life and our human approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="texte" style="text-align:center"><B>Back to our project</B><br />
</p><br />
<br />
<br />
<p class="texte">These inextricable links are obviously the basis of our project. We aim to artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago. The modification of a naturally occurring form of life to strengthen it is maybe just the imitation of the natural evolution process. What is considered today as ‘non-natural’ may be one day regarded differently. To the extent that everything is done not to unbalance the ecosystem, our intervention can be judged rightful, even more than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="texte" style="text-align:center"><B> Potential strategies discussed</B><br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that SubtiTree will not survive and spread in the environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the environment thanks to a proline auxotrophy system<br />
<br>- Prevent the sporulation of <I> B. subtilis</I> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="texte" style="text-align:center"><B> Public perception </B><br />
</p><br />
<br />
<p class="texte"><I><CENTER>Political and public adhesion</I></CENTER></p><br />
<br />
<p class="texte">Because of the magnitude of our project,its application interested several civil services. Indeed some municipalities and regional councils supported our local engagement. Beyond that, our project interests the highest level of the “Canal du Midi” administration: the national navigation authority and the Ministry of agriculture funded this project. They are still looking for the best way to further this work after the iGEM competition. </p><br />
<br />
<p class="texte">This project also received the attention of the public through several articles in newspapers, television, radio and internet. First we had just a local coverage, but days after days there were more and more media intereseted in SubtiTree. This mediatic coverage allowed us to contact concerned citizens who participated to the development of this project. This interaction with the public allowed us to explain and promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="texte"><I><CENTER>Safety principle</I></CENTER></p><br />
<br />
<p class="texte">One single tree infected by Canker, and all the trees located in an area of a couple of hundred meters around are included in the prophylactic cut. We acted to preserve the surrounding trees. The modification of the endophytic microbial fauna generated by the introduction of the engineered bacterium has to be compared to the introduction of chemicals. They contain chlorine atom and aromatic hydrocarbon, so their remediation is complicated and they represent a source of pollution. By shortening the lifespan to one season and minimizing the risks of spreading, we plan a safe and environmental-friendly way to fight Canker. <br />
<br />
<br />
<p class="texte" style="text-align:center"><B> Feasability </B><br />
</p><br />
<br />
<p class="texte">We wonder about the feasibility of tree’s treatment. As we used endophytic bacteria, we can count on the natural growth of SubtiTree inside the sap. So we can inject few bacteria to be sure to have enough bacteria to protect the tree. Some researchers (Xianling Ji1 et al) already injected <i>Bacillus subtilis</i> in plants and observe an increase of bacteria concentration to a maximum of 10^5 bacteria/mL/p><br />
<br />
<p class="texte">As we aim to inject a poor quantity of bacteria, this treatment remains cheaper than the injection of several litter of fungicides. In addition, this injection prevents the preventive tree cutting, which is very expensive. Cutting one tree cost around €3000. The administration in charge of the protection of the “Canal du Midi” already plans to spend 220 million euros to cut and replant all trees along the Canal. Besides the important cost of cutting trees, it will destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the iGEM’s circumstances we could not have the time to go deeper. First, we can improve the fixation module. Using chitin as fixation anchor is simple but not enough specific to fix just one fungus type. That’s why we first think to fix SubtiTree to one protein included in the <I>Ceratocystis platani</I>’s membrane: CP. The bacterial prototype designed this summer can be optimized to trigger the fungicides production when the binding is completed, and to be more specific changing the peptides produced.</p><br />
<br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Safety</p> <br />
</div><br />
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<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">iGEM Safety</a></li><br />
<li><a href="#select2">Chassis Organism</a></li><br />
<li><a href="#select3">New and/or modified coding region</a></li><br />
<li><a href="#select4">Team safety</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Safety is not just a slogan, it is a way of life"<br />
</p><br />
<br />
<br><br />
<!--PARTIE iGEM SAFETY--><br />
<p class="title1" id="select1">iGEM Safety</p><br />
<p class ="texte">Our safety form was approved in September 2014 by the iGEM team. We filled it with the help of Nathalie Doubrovine, safety officer at the LISBP.<br />
</p><br />
<br />
<!--TABLE CHASSIS ORGANISM--><br />
<p class="title1" id="select2">Chassis organisms</p><br />
<table style="border-width:1px; <br />
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><br />
<!--1ere ligne--><br />
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<td style="border:1px solid black"><b><p class ="texte">Species name (including strain)</b></td><br />
<td style="border:1px solid black"><b><p class ="texte">Risk group</b></td><br />
<td style="border:1px solid black"><b><p class ="texte">Risk Group Source</b></td><br />
<td style="border:1px solid black"><b><p class ="texte">Disease risk for humans ?</b></td><br />
</tr><br />
<!--2eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><i><p class ="texte">Escherichia coli MC1061</i></p></td><br />
<td style="border:1px solid black"><p class ="texte">1</p></td><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-7140.html">DSMZ</p></a><br />
<td style="border:1px solid black"><p class ="texte">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--3eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><i>Bacillus subtilis 168</i></p></td><br />
<td style="border:1px solid black"><p class ="texte">1</p></td><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-23778.html">DSMZ</p></a><br />
<td style="border:1px solid black"><p class ="texte">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><i>Aspergillus brasiliensis 246.65 CBS</i></p></td><br />
<td style="border:1px solid black"><p class ="texte">1</p></td><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-63263.html">DSMZ</p></a><br />
<td style="border:1px solid black"><p class ="texte">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><i>Chaetomium globosum 148.51 CBS</i></p></td><br />
<td style="border:1px solid black"><p class ="texte">1</p></td><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-1962.html">DSMZ</p></a><br />
<td style="border:1px solid black"><p class ="texte">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><i>Trichoderma reesei CBS 383.78</i></p></td><br />
<td style="border:1px solid black"><p class ="texte">1</p></td><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-768.html">DSMZ</p></a><br />
<td style="border:1px solid black"><p class ="texte">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><i>Aspergillus nidulans CBS 124.59</i></p></td><br />
<td style="border:1px solid black"><p class ="texte">1</p></td><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-820.html">DSMZ</p></a><br />
<td style="border:1px solid black"><p class ="texte">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
</table><br />
<br />
<br><br><br />
<br />
<br />
<!--TABLE NEW AND/OR MODIFIED CODING REGION--><br />
<p class="title1" id="select3">New and/or modified coding region</p><br />
<table style="border-width:1px; <br />
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><br />
<!--1ere ligne--><br />
<tr><br />
<td style="border:1px solid black"><b><p class ="texte">Part number/name</p></b></td><br />
<td style="border:1px solid black"><b><p class ="texte">Natural function of part</p></b></td><br />
<td style="border:1px solid black"><b><p class ="texte">How did you acquire it?</p></b></td><br />
<td style="border:1px solid black"><b><p class ="texte">How will you use it?</p></b></td><br />
</tr><br />
<!--2eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</p></td><br />
<td style="border:1px solid black"><p class ="texte">N-acetylatedGlucosamine based chemotaxis for <i>Bacillus subtilis</i></p></td><br />
<td style="border:1px solid black"><p class ="texte">We ordered this gene from a synthesis company (Eurofins)</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part allows the bacterium to reach the fungus</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</p></td><br />
<td style="border:1px solid black"><p class ="texte">RBS - Antifungal GAFP-1</p></td><br />
<td style="border:1px solid black"><p class ="texte">We ordered this gene from a synthesis company (Eurofins)</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part produces a fungicide. By this way, the bacteria is able to kill the fungus.</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</p></td><br />
<td style="border:1px solid black"><p class ="texte">RBS - Antifungal D4E1 - Double terminator</p></td><br />
<td style="border:1px solid black"><p class ="texte">We ordered this gene from a synthesis company (Eurofins)</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part produces a fungicide. By this way, the bacteria is able to kill the fungus.</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</p></td><br />
<td style="border:1px solid black"><p class ="texte">P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>Bacillus subtilis</i></p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part allows the bacterium to reach the fungus</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</p></td><br />
<td style="border:1px solid black"><p class ="texte">P<sub>veg</sub> + Chitin Binding Protein for <i>Bacillus subtilis</i></p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part allow the bacterium to fix the fungus</p></td><br />
</tr><br />
<!--8eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364006">BBa_K1364006</p></td><br />
<td style="border:1px solid black"><p class ="texte">Double expression cassette</p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part allows the bacterium to reach and bind to the fungus</p></td><br />
</tr><br />
<!--9eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</p></td><br />
<td style="border:1px solid black"><p class ="texte">RBS + Antifungal GAFP-1 + Double terminator</p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--10eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</p></td><br />
<td style="border:1px solid black"><p class ="texte">P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--11eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</p></td><br />
<td style="border:1px solid black"><p class ="texte">P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--12eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</p></td><br />
<td style="border:1px solid black"><p class ="texte">P<sub>veg</sub>-SpoVG + EcAMP-1</p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--13eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</p></td><br />
<td style="border:1px solid black"><p class ="texte">P<sub>veg</sub>-spoVG + EcAMP-1 + Double terminator</p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</p></td><br />
<td style="border:1px solid black"><p class ="texte">P<sub>veg</sub> - RBS - Antifungal GAFP-1 - RBS - Antifungal D4E1 - Double terminator</p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part produces a fungicide. By this way, the bacterium is able to kill the fungus.</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</p></td><br />
<td style="border:1px solid black"><p class ="texte">P<sub>veg</sub> + RFP</p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part produces a fluorescent protein. By this way we can evaluate promotor P<sub>veg</sub> strength.</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</p></td><br />
<td style="border:1px solid black"><p class ="texte">P<sub>lepA</sub> + RFP</p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part produces a fluorescent protein. By this way we can evaluate promotor P<sub>lepA</sub> strength.</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</p></td><br />
<td style="border:1px solid black"><p class ="texte">Promotor P<sub>lepA</sub> + RBS spoVG</p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part provides us a high level of translation.</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</p></td><br />
<td style="border:1px solid black"><p class ="texte">P<sub>veg</sub> + RBS + Antifungal EcAMP-1 + Double terminator</p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part produces a fungicide. By this way, the bacterium is able to kill the fungus.</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td style="border:1px solid black"><p class ="texte"><a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</p></td><br />
<td style="border:1px solid black"><p class ="texte">Integrative plasmid for <i>Bacillus subtilis</i></p></td><br />
<td style="border:1px solid black"><p class ="texte">We made it</p></td><br />
<td style="border:1px solid black"><p class ="texte">This part is an integrative vector.</p></td><br />
</tr><br />
</table><br />
<br />
<br><br />
<br />
<p class="title1" id="select4">Team safety</p><br />
<p class="title2">Safety in INSA de Toulouse</p><br />
<p class="texte">INSA de Toulouse is a public school for engineers. The biosafety guidelines are not specific to our institution; the French regulations are applied.<br><br />
The regulation about the workers' prevention against risks resulting from their exposure to pathogenic biological agents (Decree No. 94-352 of 4 May 1994) includes microorganisms, cell cultures and human endoparasites which may cause infections, allergies or toxicity. <br><br />
This Decree is the French transposition of the Directive 90/679 / EEC and is also transcribed in the Labour Code (Articles L4421-1 R4421-1 to R4427-5.)<br><br />
The <a href="http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000465273&dateTexte=&categorieLien=id">Decree of the 16th July 2007</a> describes the technical preventive measuresto set up in research laboratories (including containment), education, analysis, anatomy and surgical pathology, autopsy rooms, and industrial and agricultural facilities where workers are likely to be exposed to biological pathogens.<br><br />
The rules of health, safety, and preventive medicine applied in public services in France (and thus in all public facilities working in scientific and technological domains) are set out in the <a href="http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=LEGITEXT000006063791">Decree No. 82-453</a>. This decree refers to the Labour Code, Public Health Code and Environmental Code.<br><br />
The <a href="http://legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000024584737&categorieLien=id">Decree No. 2011-1177</a> is related to the use of genetically modified organisms.<br><br />
<br><br />
There is no one in charge of the biological safety at the INSA de Toulouse. However there is an organization which includes one prevention advisor and several prevention assistants in every structure. As far as the LISBP (our structure) is concerned, Nathalie Doubrovine is the prevention assistant.<br><br />
We have already discussed our project with her. She has given us the safety training and advices about how to respond to an imminent danger.<br><br />
She raised the problem of GMO and the related policy. Indeed she told us there is a special procedure for the destruction of GMO: we cannot wash this kind of organism down the drain.<br><br />
We were also informed about the potential chemical and microbiological risks thanks to her.<br><br />
<br><br />
The laboratory safety training requirements of the LISBP are detailed into the Rules of Procedure of the LISBP.<br><br />
The legislation requires employers to inform "all new employees" of the risks that they may encounter as well as ways to protect themselves from those risks. Therefore any newcomer must pass a practical and appropriate training in hygiene and safety to ensure its own safety and the one of his colleagues.<br><br />
Every person that enters in the LISBP has to make this training whatever its status (researcher, PhD student, trainee, etc.).<br><br />
The training is divided into two parts. The first one is a training concerning general risk prevention into research laboratories. This one is made individually thanks to the software NEO and with the explanations of the prevention assistant. The second one is a training about the techniques used during the occupation.<br><br />
<br><br />
All the team members have received this safety training. It lasts 1h30. It concerns fire risks, biological and chemical risks and good laboratory practices. We have made this training individually with a software designed by the CNRS and INSERM named NEO. This training ends with a questionnaire to check our knowledge. This questionnaire reminds us the basic safety rules in a molecular biology lab and gives us new knowledge about safety.<br><br />
We have also made a training to know how to sort out biological waste and how they are treated. We also learned that we cannot use autoclave by ourselves because a specific training is needed. We did not do this specific training.<br><br />
</p><br />
<br />
<p class="title2">Safety in the lab</p><br />
<!--CITATION--><br />
<p class="citation"><br />
"If you don't think it's safe, it probably isn't"</p><br />
<p class="texte">We have organized our workspace. There is the relaxing room where no microbial material should enter. In this room, we can eat and drink (a fridge, a kettle and a coffeemaker are available) but it is also the room where we have our meetings and where we can work on our computers. There is the lab, where we have to wear protective equipment and respect basic rules.</p><br />
<br />
<p class="title3">Personal protective equipment</p><br />
<p class="texte">As soon as we manipulate in the lab, we have to wear the following personal protective equipment:</p><br />
<table><br />
<tr><td><p class="texte">- A conventional lab coat, closed with long sleeves</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/9/9c/Lab_coat.png"></td></tr><br />
<tr><td><p class="texte">- Closed shoes</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/8/82/Shoes.png"></td></tr><br />
<tr><td><p class="texte">- Gloves</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/3/37/Gloves.png"></td></tr><br />
<tr><td><p class="texte">- Glasses if needed (UV exposure, hot water or chemicals manipulation.) </p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/b/b4/Glasses.png"></td></tr><br />
</table><br />
<br />
<p class="title3">Basic rules in a lab</p><br />
<p class="texte">We have to apply the basic safety principles into a laboratory room:<br><br />
- It is forbidden to smoke in all the rooms.<br><br />
- It is forbidden to drink and eat in the laboratory rooms.<br><br />
- It is compulsory to wear a closed lab coat in cotton.<br><br />
- It is compulsory to wear closed shoes.<br><br />
- Long hair must be tied back.<br><br />
- Oral pipetting of any substance is prohibited in any laboratory.<br><br />
<br><br />
There is also others precaution when working with biological organisms:<br><br />
- We need to wash our hands regularly.<br><br />
- It is compulsory to wear gloves except with the use of an electric burner.<br><br />
- In some cases (UV light, projection risk), it is compulsory to wear protection glasses.<br><br />
<br><br />
Indeed, different machine are used to keep a sterile area.</p><br />
<br />
<p class="title3">Waste</p><br />
<p class="texte">Different trash containers are available in the lab:<br><br />
- One for biological waste (yellow). This waste will be autoclaved.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/f/f7/Yellow_trash.JPG"><br><br><br />
- One for common waste (green or orange).<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/c/c7/Black_garbage.png"><br><br><br />
- Special waste for chemicals.<br />
<br />
<p class="title3">Devices and Material</p><br />
<p class="texte">We use a lot of different devices, and each one involves a particular risk. Here we described how we use this material safely to reduce risks:</p><br />
<p class="title4">Chemical storage</p><br />
<p class="texte">We have three cupboards dedicated to the different kind of chemical products we use:<br><br />
- Flammable<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/3/35/Flammable.png"><br><br><br />
- Acids<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/3/36/Acids.png"><br><br><br />
- Bases<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/7/74/Bases.png"><br><br><br />
Those cupboards are key-closed.</p><br />
<br />
<p class="title4">Ethidium Bromide</p><br />
<p class="texte">We have a dark room dedicated to the use of EtBr and UV. This room is key-closed and everyone entering into the room must wear gloves, glasses and lab coat. Everything which is in direct contact with something in this room has to stay here.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/7/75/EtBr_room.JPG"><br><br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/4/45/EtBr_door.JPG"><br><br><br />
Two specific trash cans are dedicated to the gloves or paper and the agarose gels contaminated.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/f/fe/EtBr_garbage.JPG"><br><br></p><br />
<br />
<p class="title4">Biological safety cabinet</p><br />
<p class="texte">We use a Biological safety cabinet (FASTER – Ultrasafe) to manipulate into a sterile area and thus avoid external contamination by unwanted microorganisms. We clean the bench of the BSC with ethanol before and after each manipulation. We also clean the BSC completely every two weeks. Because we work with fungi, we have to be very careful with the cleanliness of the BSC.</p><br />
<br />
<p class="title4">Electric burner</p><br />
<p class="texte">We also use electric burners to manipulate into a sterile area. The burning and fire risks are high but weaker than with a Bunsen Burner. We are very cautious when we use electric burners, and we use in preference BSC if it is possible. We do not have to wear gloves et we have to check that the electric burner is turned off after the manipulation.</p><br />
<br />
<p class="title4">Chemical hood</p><br />
<p class="texte">We use chemical hood in case we have to manipulate dangerous volatile chemical compound.</p><br />
<br />
<p class="title4">Water-bathes</p><br />
<p class="texte">The water-bathes are used extensively (transformation, digestion, etc.). However, they can be dangerous because of the exposition of boiling or hot water. We use special gloves for protecting us from heat, steam and projections. We take care of turning off the water-bathes at the end of the day.</p><br />
<br />
<br />
<br />
<p class="title2">Safety of our project</p><br />
<!--CITATION--><br />
<p class="citation"><br />
"Precaution is better than cure"</p><br />
<p class="texte">Several risks exist when working with microorganisms and manipulating genes. We can identify different classes of risk of our project now: <br><br />
- risks to the safety and health of team members, or other people working in the lab,<br><br />
- risks to the safety and health of the general public,<br><br />
- risks to the environment,<br><br />
- risks to security through malicious mis-use by individuals, groups, or countries.<br><br />
<br><br />
There are also risks in the Future, linked to our project’s growth (new knowledge and methods development).<br><br />
<br />
<p class="title3">Risks to the safety and health of team members, or other people working in the lab</p><br />
<p class="texte">We work with the <I>B. subtilis </I> and <i>E. coli</i> chassis. These organisms are non-pathogenic. Moreover the used parts are also non-pathogenic (the bio-fungicides produced are harmless for humans).<br><br />
On one hand, there is a major biological risk because of the manipulation of DNA and RNA of bacterial organisms.<br><br />
On the other hand the main risk for humans is chemical: we use Ethidium Bromide, hydrochloric acid, soda, solutions from a Miniprep kit, ethanol and bleach.<br><br />
<br><br />
However, we have a black room dedicated to the manipulation of Ethidium Bromide. Everything that touches something in this room cannot get out of the room. The waste has a special treatment.<br><br />
For the other chemical solutions, we always use gloves and glasses, and if necessary a hood.<br><br />
Furthermore, there are other risks such as UVs when we analyze an agarose gel but also water-bath and electrical/fire risks.<br><br />
For protecting us from the UVs, we use adapted glasses.<br><br />
At the end of every day, the last persons to leave check that everything is ok in the lab (no water-bath stayed on, everything disconnected).</p><br />
<br />
<p class="title3">Risks to the safety and health of the general public</p><br />
<p class="texte">If biological materials escape from our lab, the risks regarding safety and health of the general public is low because the manipulated parts are harmless. The only risk is the contact with DNA or RNA from a bacterial organism.<br><br />
Moreover, we have to be cautious with the use of antibiotics and we must not wash them down the drain.</p><br />
<br />
<p class="title3">Risks to the environment</p><br />
<p class="texte">If biological materials escape from our lab, GMOs can spread in the environment and gene transfer can occur. Moreover, the aim of our bacterial system (called SubtiTree) is to kill a fungus (Ceratocyctis platani). The risk to the environment is high because our system could perturb ecological niche and unbalance the environment of some plants. All our waste is autoclaved to minimize the risk for the environment.<br><br />
We use three fungicides.<br><br />
The first one, D4E1, is a synthetic peptide.<br><br />
The second is EcAMP-1 (BBa_K1162001), an AntiMicrobial Peptide from banyard grass added to the Registry last year by the team Utah State.<br><br />
The last one is GAFP-1, the Gastrodia AntiFungal Protein.<br><br />
These fungicides are not authorized yet by the European Union because they are not commercialized.</p><br />
<br />
<p class="title3">Risks to security through malicious mis-use by individuals, groups, or countries</p><br />
<p class="texte">If someone steals the biological material it could be dangerous for the environment because our bacterium will produce three bio-fungicides. This could lead to the disturbing of some ecosystems. <br><br />
However, the fungicides are natural and one of them is used in agriculture. With these considerations, we can assume that the risk is not very high for the chosen parts.</p><br />
<br />
<br />
<p class="title3">How to reduce these risks?</p><br />
<p class="texte">We take care of our waste and our biological material.<br><br />
We have chosen only non-pathogenic species to work on: <i>E. coli</i> and <i>B. subtilis</i>.<br><br />
We use non-pathogenic fungus to test our SubtiTree system: <i>Aspergillus brasiliensis</i>, <i>Chaetomium globosum</i>, <i>Trichoderma reesei</i> and <i>Aspergillus nidulans</i>.<br><br />
Indeed the targeted fungus, Ceratocystis platani, is pathogenic. Therefore we avoid working with it.</p><br />
<br />
<p class="title3">Risks in the Future</p><br />
<p class="texte">The system designed could be applied to many fungi. The fungicides used in our project are biological and specifically kill the fungi, and no other eukaryotic cells. <br><br />
However, a malicious person could replace these fungicides by others which are more dangerous. Indeed our problem is that there is no regulation of the production of fungicides.</p><br />
<br />
<p class="title3">How to reduce these Future risks?</p><br />
<p class="texte">We have thought about several ways to minimize risks: <br><br />
- we choose an auxotrophic strain of <I>B. subtilis</I>. The auxotrophy is for the glutamic acid, which is very present in plant's sap.<br><br />
- we choose a non-spore-forming strain of<I>B. subtilis</I>. The sporulation is the endophyte bacteria mechanism used to survive through winter. Thus, our bacterial system will be time limited.<br><br />
- we want to use a toxin/antitoxin system to avoid gene transfer between our optimized bacterium and another wild-type bacteria.</p><br />
<br />
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<p class="texte">How did we validate the three modules and improve our new protocols? Click below to find out…</p><br />
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<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
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<p class="texte"> We used and developed several protocols to demonstrate the existence of chemotaxis in <i>B. subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine. <br />
</p><br />
<p class="title2">1. Petri Dishes Test </p><br />
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<p class="texte">We first wanted to visualize chemotaxis on Petri dishes. We hoped to obtain pictures with bacteria halos directed or around attractive components and thus tried different protocols. The first protocol was adapted from the one published by <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>. They put the attractive compound on a paper disk in the middle of a Petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Figure 1).<br />
</br><br />
</br><br />
<b>We first tried to test chemotaxis onto Petri Dishes filled with a 0.3% agar medium. This semi-solid medium allows the bacterial motility. A paper disk containing an attractive compound is placed in the middle of the dish and cells are then loaded in the medium (see Figure 1). This protocol was taken from the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a></b>.</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Scheme showing how cells are filled into the medium. (A) A pipet tip is used to deposit cells in the gelose. (B) Bacteria should move toward the attractive compound which diffuses.</p><br />
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<p class="texte">We did not have any result with WT <i>Bacillus subtilis</i> and glucose as attractive compound (Figure 2-A). <i>B. subtilis</i> is attracted by many other glucides and amino-acids, so we also tried to test diluted glucose in LB medium attractant (Figure 2-B).</p><br />
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<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with Glucose as attractive compound (A) and Glucose added to LB medium as attractant (B).</p><br />
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<p class="texte"> We could not notice any difference between the petri dish with or without glucose. With an addition of LB medium to sugar, a large halo around the paper disk was noticeable. This halo may correspond to cells attracted by the solution, as it is not noticeable when cells are not added (data not shown). Anyway we did not have enough reproducible and reliable results to be satisfied with this test.<br> <br />
Furthermore, with the addition of LB medium, it is hard to make the distinction between the attractive effects and the simple growth resulting from random diffusion.</br><br />
We have started new tries using different protocols.</p><br />
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<p class="title2">2. Plug in Pond system<br />
</p><br />
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<p class="texte"><br />
This protocol we worked on is taken from a thesis (see references [1]). . A solution of <i>B.subtilis</i> is grown overnight so as to obtain a cell density of 8x10⁸ cells/mL. 10mL of the solution is mixed with 15mL of LB medium with 1.5 % agar kept at 45°.The final concentration of the obtained medium is 0.9% agar. Tetracycline is aded at 25µg/mL, in order to inhibit growth and to only observe the chemotaxis phenomenon. Plates are cooled and dried, before digging wells with a punch or 1mL tips. The wells are filled with attractive compounds (Figure 3). After one hour at room temperature, photos of the plates are taken and the results are analyzed.<br />
</p><br />
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<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Schema showing how are made plug-in-pond tests.</p><br />
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<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.<br />
</p><br />
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<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
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<p class="texte"><br />
After an hour, no tangible results were obtained. It is only after 12hours that we were able to observe halos around the wells with glucose at 1M in the plates without tetracycline. Tetracycline concentration seems to be too high and inhibits any bacterial activity. Therfore, we have worked with tetracycline at 15µg/mL.<br />
We tried this protocol again with this new condition. We made two wells per plate (Figure 5), one with either Glucose or N-acetyl-glucosamine and one with LB medium. As previsously, no results were achieved after 1h, but after 12hours we could notice halos.<br />
</p><br />
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<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>Bacillus subtilis</i> WT. The upper wells contain attractive compound and the lower contain medium without attractive compound. </p><br />
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<p class="texte"><br />
Results are not as clear as the first time, but we observed halos around the well with glucose at 250mM with and without tetracycline. We have then tried the same experiment with N-acetyl-glucosamine and we did not see any halo in the tested conditions. Thus we assumed that our <i>B. subtilis</i> 168 strain was not attracted to N-acetyl-glucosamine.<br />
However, the results are not clear, reliable and reproducible enough with the plug-in-pond protocol. Another testing protocol was then adopted. <br />
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</p><br />
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<p class="texte"><br />
<b>References:</b></br><br />
[1]: Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez <i>Escherichia coli</i> et <i>Shewanella oneidensis</i>, 2008, Claudine Baraquet, Université de la Méditerranée Aix-Marseille II<br />
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<p class="title2">4. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
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<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, we asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
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<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacterial solution of WT <i>Bacillus subtilis</i> in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed from the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative one because we were not sure that this system was efficient.</i><br><br />
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- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
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<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the 2011 Imperial college iGEM team : the tips capillary test.</br><br />
</p><br />
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<p class="title2"> 5. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from 2011 Imperial College iGEM team and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br />
<br><br />
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br><br />
- 15µL of each chemo-attractant was pipetted. <br><br />
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tip.<br><br />
- The top of the tip was then sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
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<p class="texte">- After all the attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 10.<br><br />
- Each tip was immersed in 300 µL of a bacterial solution in the wells of an Elisa plate.<br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
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<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
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- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with a Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plates instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
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<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
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<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
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There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>Bacillus subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
At the beginning, the experiment was conducted with only one negative contraol, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
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However, our team did not give up on synthetic biology ! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
Hopefully, we managed to find a negative control: galactose (25mM). The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<p class="texte"> The miracle arrived! We managed to prove that our WT <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p><br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
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<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
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<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
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<p class="title2">1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the Chitin Beads Buffer (CBB) buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
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<p class="title3">Results</p><br />
<p class="texte">We could not count bacteria either because of the high number of bacteria with the 0.1 OD solution or the low number of bacteria with the 0.01 OD solution. Thus, the study is mostly focused on the intermediate values (Figure 16).<br />
<br/>First of all, cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio.<br />
</br>Thus the the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C are compatible with the bacterial life. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
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</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
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</br><br />
<p class="legend">Figure 16: CBB presence has no effect on bacterial survival. The bacterial concentration was measured in <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span> of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).</p><br />
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<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
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<p class="title3">Purpose</p><br />
<p class="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein composed of the Cell Wall Binding Domain of LycT to attach the chimeric protein to the cell wall, and the GbpA domain 4 of <i>Vibrio cholerae</i> to bind chitin. The synthetic bacterium is put in contact with chitin beads (chitin: polymer present on the fungal pathogen wall). After several washes, bacteria remaining on the beads are counted.</p><br />
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<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration: 10^5 bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it removes 40 times more wild-type bacteria than SubtiTree. This result correlates to the number of Subtitree bound to the beads. <br />
<br/>Thus, the binding system is validated: SubtiTree binds efficiently to chitin.</p><br />
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</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
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<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and Subtitree the to chitin. The <span style="color:#0000FF">WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentrations have been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.<br />
</p><br />
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<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We wanted to observe SubtiTree bound on the chitin coated beads. In order to perform a 3D reconstruction showing this interaction, we used confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we highlighted the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we can notice that SubtiTree is sitting (well, sort off!!) on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of beads surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center><br />
</br><br />
<p class="legend">A short movie of 3D bead surfaces coated with chitin and Subtitree (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p><br />
<br />
<p class="texte">We then performed a wash step on the chitin beads. We measured the release of bacteria on the washing solution. When our bacterium has the binding module, there is less release and therefore, SubtiTree is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of bacteria after washing <br />
</p><br />
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<p class="texte">Finally, all results are consistent with the presence of functional binding system. We thus validate the second module.</p><br />
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<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporulating halo in response to the stress generated by the fungicide.<br />
</p><br />
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<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<p class="legend"> Figure 21: Results of the preliminary tests</p><br />
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</br><br />
<br />
<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
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<p class="title2">2. Test with SubtiTree</p><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with a low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of SubtiTree in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p><br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem to be reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b><br />
</p><br />
<br />
<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.</p><br />
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<h2>Binding</h2><br />
<p>To be attached to the fungal wall</p><br />
</div><br />
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<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Binding</p> <br />
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<!--Short description : à changer!!!--><br />
<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/e/e1/Bindingresume.png"></center><br />
<br />
<p class="texte"> In order to be highly efficient in the fight against the pythopathogen, our optimized bacterium has to be anchored to the fungus. Thus, we designed a chimeric protein (<a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>) capable of building a <B>bridge between the bacterial peptidoglycan and the fungal chitin</B>, the main component of the pathogen’s cell wall. According to the work of the Imperial College 2010 iGEM team, we used the Cell Wall Binding (CWB) domain of the LytC protein (coding for a N-acetylmuramoyl-L-alanine amidase) to attach our chimeric protein to the <I>B. subtilis</I> cell wall. On the other side of our protein, we added the domain 4 of GbpA from <I>Vibrio cholerae</I>, which is known to recognize chitin.<br />
</p><br />
<br />
<br><br />
<p class="title1"> More information about this module </p><br />
<p class="texte"> <br />
The Binding Module ORF is composed of 3 sections:</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte"><B>Anchor section</B>: the CWB (Cell Wall Binding) domain is extracted from LytC gene and composes the 5' side of our binding module. As previously described by the Imperial College of London 2010 iGEM team, we kept the first 318 bp. We can note the presence of the signal peptide at the beginning of the sequence, from 1 to 24 bp.</p></li><br />
<li class="tree"><p class="texte"><B>Chitin Binding Domain (CBD) section</b>: the domain 4 of GbpA from <I>Vibrio cholerae </I> is able to bind to N-Acetyl Glucosamine oligosacchararides. Also, the 3' side of our gene is composed by a part of the GbpA sequence (from 423 to 484 bp).</p></li><br />
<li class="tree"><p class="texte"><B>Helical Linker</B>: According to the work of the 2010 Imperial College of London iGEM team, we used the same six amino acids sequence (SRGSRA) to make a bridge between the Anchor section and the Chitin Binding section.<br />
</p></li></ul><br />
<center style="margin:-30px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/a/a0/Binding.png"></center><br />
<br></br><br />
<p class="texte"> <br />
The sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i>. <br />
</p><br />
<br />
<B class="title1">Final construction</B> <br />
<br />
<B class="title2">(More details about the intermediate parts <a href="https://2014.igem.org/Team:Toulouse/Result/parts#select2"target="_blank">Here</a>)</B> <br />
<br />
<p class="texte"><br />
<br>The binding module has been placed under the control of Pveg (<a href="http://parts.igem.org/Part:BBa_K143012"target="_blank">BBa_K143012</a>), a strong constitutive promoter and we used a consensus RBS <a href="http://parts.igem.org/Part:BBa_K090505"target="_blank">BBa_K090505</a> and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015"target="_blank">B0015</a>). The construct has been inserted in an integrative plasmid, pSBBS4S (<a href="http://parts.igem.org/Part:BBa_K823022"target="_blank">BBa_K823022</a>) which comes from the LMU-Munich 2012 iGEM team.</p> <br />
<br />
<center style="margin:20px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"></center><br />
<br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">M. Desvaux, E. Dumas, I. Chafsey and M. Hébraud.<b> Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure </b>. FEMS Microbiol. Lett. 256, 1–15 (2006). </p></li><br />
<li class="tree"><p class="texte">E. Wong, G. Vaaje-Kolstad, A. Ghosh, R. Hurtado-Guerrero, PV. Konarev, AF. Ibrahim, DI. Svergun, VG. Eijsink, NS. Chatterjee and DM. van Aalten.<b>The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces</b>. PLoS Pathog. 8, e1002373 (2012).</p></li><br />
<br />
<li class="tree"><p class="texte">H. Yamamoto, S. Kurosawa and J. Sekiguchi. <b>Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases</b>. J. Bacteriol. 185, 6666–6677 (2003).</p></li><br />
</ul><br />
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<h2>Binding</h2><br />
<p>To be attached to the fungal wall</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Binding</p> <br />
</div><br />
<br />
<br />
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<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
<br />
<!--Short description : à changer!!!--><br />
<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/e/e1/Bindingresume.png"></center><br />
<br />
<p class="texte"> In order to be highly efficient in the fight against the pythopathogen, our optimized bacterium has to be anchored to the fungus. Thus, we designed a chimeric protein (<a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>) capable of building a <B>bridge between the bacterial peptidoglycan and the fungal chitin</B>, the main component of the pathogen’s cell wall. According to the work of the Imperial College 2010 iGEM team, we used the CWB (Cell Wall Binding) domain of the LytC protein (coding for a N-acetylmuramoyl-L-alanine amidase) to attach our chimeric protein to the <I>B. subtilis</I> cell wall. On the other side of our protein, we added the domain 4 of GbpA from <I>Vibrio cholerae</I>, which is known to recognize chitin.<br />
</p><br />
<br />
<br><br />
<p class="title1"> More information about this module </p><br />
<p class="texte"> <br />
The Binding Module ORF is composed of 3 sections:</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte"><B>Anchor section</B>: the CWB (Cell Wall Binding) domain is extracted from LytC gene and composes the 5' side of our binding module. As previously described by the Imperial College of London 2010 iGEM team, we kept the first 318 bp. We can note the presence of the signal peptide at the beginning of the sequence, from 1 to 24 bp.</p></li><br />
<li class="tree"><p class="texte"><B>Chitin Binding Domain (CBD) section</b>: the domain 4 of GbpA from <I>Vibrio cholerae </I> is able to bind to N-Acetyl Glucosamine oligosacchararides. Also, the 3' side of our gene is composed by a part of the GbpA sequence (from 423 to 484 bp).</p></li><br />
<li class="tree"><p class="texte"><B>Helical Linker</B>: According to the work of the 2010 Imperial College of London iGEM team, we used the same six amino acids sequence (SRGSRA) to make a bridge between the Anchor section and the Chitin Binding section.<br />
</p></li></ul><br />
<center style="margin:-30px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/a/a0/Binding.png"></center><br />
<br></br><br />
<p class="texte"> <br />
The sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i>. <br />
</p><br />
<br />
<B class="title1">Final construction</B> <br />
<br />
<B class="title2">(More details about the intermediate parts <a href="https://2014.igem.org/Team:Toulouse/Result/parts#select2"target="_blank">Here</a>)</B> <br />
<br />
<p class="texte"><br />
<br>The binding module has been placed under the control of Pveg (<a href="http://parts.igem.org/Part:BBa_K143012"target="_blank">BBa_K143012</a>), a strong constitutive promoter and we used a consensus RBS <a href="http://parts.igem.org/Part:BBa_K090505"target="_blank">BBa_K090505</a> and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015"target="_blank">B0015</a>). The construct has been inserted in an integrative plasmid, pSBBS4S (<a href="http://parts.igem.org/Part:BBa_K823022"target="_blank">BBa_K823022</a>) which comes from the LMU-Munich 2012 iGEM team.</p> <br />
<br />
<center style="margin:20px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"></center><br />
<br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">M. Desvaux, E. Dumas, I. Chafsey and M. Hébraud.<b> Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure </b>. FEMS Microbiol. Lett. 256, 1–15 (2006). </p></li><br />
<li class="tree"><p class="texte">E. Wong, G. Vaaje-Kolstad, A. Ghosh, R. Hurtado-Guerrero, PV. Konarev, AF. Ibrahim, DI. Svergun, VG. Eijsink, NS. Chatterjee and DM. van Aalten.<b>The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces</b>. PLoS Pathog. 8, e1002373 (2012).</p></li><br />
<br />
<li class="tree"><p class="texte">H. Yamamoto, S. Kurosawa and J. Sekiguchi. <b>Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases</b>. J. Bacteriol. 185, 6666–6677 (2003).</p></li><br />
</ul><br />
<br />
<br />
<!-- Navigation section --> <br />
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<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<br />
<p class="texte">How did we validate the three modules and improve our new protocols? Click below to find out…</p><br />
<br />
<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a> | <a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a></p><br />
<br />
<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte"> We used and developed several protocols to demonstrate the existence of chemotaxis in <i>B. subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine. <br />
</p><br />
<p class="title2">1. Petri Dishes Test </p><br />
<br />
<p class="texte">We first wanted to visualize chemotaxis on Petri dishes. We hoped to obtain pictures with bacteria halos directed or around attractive components and thus tried different protocols. The first protocol was adapted from the one published by <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>. They put the attractive compound on a paper disk in the middle of a Petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Figure 1).<br />
</br><br />
</br><br />
<b>We first tried to test chemotaxis onto Petri Dishes filled with a 0.3% agar medium. This semi-solid medium allows the bacterial motility. A paper disk containing an attractive compound is placed in the middle of the dish and cells are then loaded in the medium (see Figure 1). This protocol was taken from the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a></b>.</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Scheme showing how cells are filled into the medium. (A) A pipet tip is used to deposit cells in the gelose. (B) Bacteria should move toward the attractive compound which diffuses.</p><br />
<br />
<p class="texte">We did not have any result with WT <i>Bacillus subtilis</i> and glucose as attractive compound (Figure 2-A). <i>B. subtilis</i> is attracted by many other glucides and amino-acids, so we also tried to test diluted glucose in LB medium attractant (Figure 2-B).</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with Glucose as attractive compound (A) and Glucose added to LB medium as attractant (B).</p><br />
<br />
<p class="texte"> We could not notice any difference between the petri dish with or without glucose. With an addition of LB medium to sugar, a large halo around the paper disk was noticeable. This halo may correspond to cells attracted by the solution, as it is not noticeable when cells are not added (data not shown). Anyway we did not have enough reproducible and reliable results to be satisfied with this test.<br> <br />
Furthermore, with the addition of LB medium, it is hard to make the distinction between the attractive effects and the simple growth resulting from random diffusion.</br><br />
We have started new tries using different protocols.</p><br />
<br />
<p class="title2">2. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol we worked on is taken from a thesis (see references [1]). . A solution of <i>B.subtilis</i> is grown overnight so as to obtain a cell density of 8x10⁸ cells/mL. 10mL of the solution is mixed with 15mL of LB medium with 1.5 % agar kept at 45°.The final concentration of the obtained medium is 0.9% agar. Tetracycline is aded at 25µg/mL, in order to inhibit growth and to only observe the chemotaxis phenomenon. Plates are cooled and dried, before digging wells with a punch or 1mL tips. The wells are filled with attractive compounds (Figure 3). After one hour at room temperature, photos of the plates are taken and the results are analyzed.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Schema showing how are made plug-in-pond tests.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
After an hour, no tangible results were obtained. It is only after 12hours that we were able to observe halos around the wells with glucose at 1M in the plates without tetracycline. Tetracycline concentration seems to be too high and inhibits any bacterial activity. Therfore, we have worked with tetracycline at 15µg/mL.<br />
We tried this protocol again with this new condition. We made two wells per plate (Figure 5), one with either Glucose or N-acetyl-glucosamine and one with LB medium. As previsously, no results were achieved after 1h, but after 12hours we could notice halos.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>Bacillus subtilis</i> WT. The upper wells contain attractive compound and the lower contain medium without attractive compound. </p><br />
<br />
<p class="texte"><br />
Results are not as clear as the first time, but we observed halos around the well with glucose at 250mM with and without tetracycline. We have then tried the same experiment with N-acetyl-glucosamine and we did not see any halo in the tested conditions. Thus we assumed that our <i>B. subtilis</i> 168 strain was not attracted to N-acetyl-glucosamine.<br />
However, the results are not clear, reliable and reproducible enough with the plug-in-pond protocol. Another testing protocol was then adopted. <br />
<br />
</p><br />
<br />
<br />
<p class="texte"><br />
<b>References:</b></br><br />
[1] : Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez Escherichia coli et Shewanella oneidensis, 2008, Claudine Baraquet, université de la méditerranée Aix-Marseille II<br />
</p><br />
<br />
<br />
<br />
<br />
<p class="title2">4. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, we asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacterial solution of WT <i>Bacillus subtilis</i> in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed from the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative one because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the 2011 Imperial college iGEM team : the tips capillary test.</br><br />
</p><br />
<br />
<p class="title2"> 5. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from 2011 Imperial College iGEM team and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br />
<br><br />
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br><br />
- 15µL of each chemo-attractant was pipetted. <br><br />
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tip.<br><br />
- The top of the tip was then sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 10.<br><br />
- Each tip was immersed in 300 µL of a bacterial solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with a Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plates instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>Bacillus subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
At the beginning, the experiment was conducted with only one negative contraol, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology ! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
Hopefully, we managed to find a negative control: galactose (25mM). The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<p class="texte"> The miracle arrived! We managed to prove that our WT <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p><br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
<br />
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<br />
<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2">1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The bacterial solutions could not be counted because of two major problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 16).<br />
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which agrees with the dilution ratio. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="legend">Figure 16: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).<br />
</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. <br />
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of beads surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin</p><br />
<br />
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of bacteria after washing <br />
</p><br />
<br />
<p class="texte">Finally, overall results are consistent with the presence of functional binding system.</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 1); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporulating halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
<br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<p class="legend"> Figure 21: Results of the preliminary tests</p><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree</p><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with a low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of SubtiTree in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p><br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem to be reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b><br />
</p><br />
<br />
<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.</p><br />
<br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Team&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fun Facts</p> <br />
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<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
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<div style="float:left; width:525px;"><br />
<img src="https://static.igem.org/mediawiki/2014/3/30/Kilometers.jpg" style="margin-top:5px; width:470px" /><br />
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<br />
<p class="title2">Have you ever tried to estimate the kilometers traveled by your team during this summer?</p><br />
<p class="texte"><br />
According to our calculations, one member walks approximately 4 kilometers per day all around the <br />
laboratory. This represents 5,544 kilometers for the whole team during the 126 days of this epic <br />
adventure. <br />
What does that mean exactly? Simply that we could walk, cycle, swim or whatever you want until <br />
Kazakhstan, Russia, Kenya… We could even have reached the USA but we decided to stay in the lab <br />
and take the plane to go to Boston!</p><br />
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<br> </br><br />
<br />
<div style="float:right; width:500px;"><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Interview.jpg" style="margin-top:5px; margin-left: 62px; width:450px" /><br />
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<br />
<p class="title2">What do trees lining the “Canal du Midi” think about SubtiTree?</p><br />
<p class="texte"><br />
According to our homemade impartial survey, 94% of the questioned plane trees approve our project and are interested in <br />
serving as guinea pigs for our new bacterial medicine. This percentage represents 41,580 trees which <br />
are also gathered in the association called: “Happy tree friends“.</p><br />
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<p class="title2">Multidisciplinary… Yes we are!</p><br />
<p class="texte">Housework in our laboratory became necessary when most of people were in vacations except us. <br />
But do you know the novelty this year in our team? Times are changing because now men are <br />
cleaning! ;-)</p><br />
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<br> <br> <br> <br> <br> <br><br />
<p class="title2">The “can’t miss it” event of the summer: the 2014 Football (also called Soccer in some third zone countries!!) World Cup!</p><br />
<p class="texte">From 06/12/14 to 07/13/14, the Toulouse iGEM Team was cheering on the French team. During the <br />
games of the French team, our group was juggling with wet lab and large screen projections! Despite <br />
our support, the French team did not win the World Cup. However, we have not said our last world <br />
yet: let’s see what will happen in 2018… ;-)</p><br />
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<div style="margin-top:50px; text-align:center;"><br />
<p class="title2">Have you ever forgotten a culture tube or a petri dish?</p><br />
<p class="texte" style="text-align:center;">Never? Let us show you what happens in that case!</p><br />
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<tr><td><img src="https://static.igem.org/mediawiki/2014/d/d4/Old_petri1.png" width="370px"></td><br />
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<p class="title2" style="padding-bottom:15px;">To finish this part, let’s do the official Awards Ceremony of the 2014 Toulouse iGEM Team!</p><br />
<ul><br />
<li class="tree"><p class="texte">The Geek Award: The three nominees are Laureen, Manon, Florie. And the winner is… <b>Florie</b> <br />
who spent the longest time in front of her laptop for the modeling part!</p></li><br />
<li class="tree"><p class="texte">The latest survivor of weekly meetings: The four nominees are Fanny, Emeline, Diane, Pierre. <br />
And the winner is… <b>Diane</b> who stayed up until 3am because she was skyping from South Korea!<br />
</p></li><br />
<li class="tree"><p class="texte">The worst singer award: The two nominees are Abdel, Pierre. And the winner is… <b>Abdel</b> who <br />
spent the whole day singing badly in the lab!</p></li><br />
<li class="tree"><p class="texte">The dancer award: The three nominees are Florie, Camille, Diane. And the winner is… <b>Camille</b><br />
who did the famous Plasmid Dance!</p></li><br />
<li class="tree"><p class="texte">The “hello you” award: No nominees because the only winner is… <b>Pierre</b> who was saying <br />
“Hello you” each time he meets someone!</p></li><br />
<li class="tree"><p class="texte">The most tired award: The three nominees are Laureen, Manon, Aurélie. And the winner is... <br />
<b>Manon</b> but we still do not know why!</p></li><br />
<li class="tree"><p class="texte">The misplaced ideas award: The four nominees are Laureen, Camille, Mathieu, Pierre. And <br />
the winner is… <b>Mathieu</b> but you do not want to know why!</p></li><br />
<li class="tree"><p class="texte">The perseverance award: The three nominees are Emeline, Diane, Abdel. And the winner is... <br />
<b>Emeline</b> who succeeded a cloning after twelve trials!</p></li><br />
<li class="tree"><p class="texte">The drawing award: The three nominees are Florie, Fanny, Manon. And the winner is… <b>Fanny</b> <br />
who drew our first SubtiTree logo!</p></li><br />
<li class="tree"><p class="texte">The phone-call award: The two nominees are Laureen, Pierre. And the winner is… <b>Laureen</b><br />
who was our lab secretary!</p></li><br />
<li class="tree"><p class="texte">The biggest blunder in the lab award: The three nominees are Aurélie, Fanny, Abdel. And the <br />
winner is… <b>Aurélie</b> who poured an agarose gel without gel tray!</p></li><br />
<li class="tree"><p class="texte">And last but not least ... The Best Nervous breakdown Award goes to ... <b>Our deep freezer</b>! The whole team is grateful for its hard work during a hot summer! Three successive breakdowns during the hot days of summer: thank you freezer, so long chap, we’ll unplug you after iGEM is finished and you’ll retire, hopefully not in the Canal du Midi…</li></p><br />
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{{:Team:Toulouse/template/footer}}</div>Dbbyhttp://2014.igem.org/Team:Toulouse/Team/Fun_factsTeam:Toulouse/Team/Fun facts2014-10-16T19:48:24Z<p>Dbby: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Team&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fun Facts</p> <br />
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<img src="https://static.igem.org/mediawiki/2014/3/30/Kilometers.jpg" style="margin-top:5px; width:470px" /><br />
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<p class="title2">Have you ever tried to estimate the kilometers traveled by your team during this summer?</p><br />
<p class="texte"><br />
According to our calculations, one member walks approximately 4 kilometers per day all around the <br />
laboratory. This represents 5,544 kilometers for the whole team during the 126 days of this epic <br />
adventure. <br />
What does that mean exactly? Simply that we could walk, cycle, swim or whatever you want until <br />
Kazakhstan, Russia, Kenya… We could even have reached the USA but we decided to stay in the lab <br />
and take the plane to go to Boston!</p><br />
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<div style="float:right; width:500px;"><br />
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<p class="title2">What do trees lining the “Canal du Midi” think about SubtiTree?</p><br />
<p class="texte"><br />
According to our homemade impartial survey, 94% of the questioned plane trees approve our project and are interested in <br />
serving as guinea pigs for our new bacterial medicine. This percentage represents 41,580 trees which <br />
are also gathered in the association called: “Happy tree friends“.</p><br />
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<p class="title2">Multidisciplinary…Yes we are!</p><br />
<p class="texte">Housework in our laboratory became necessary when most of people were in vacations except us. <br />
But do you know the novelty this year in our team? Times are changing because now men are <br />
cleaning! ;-)</p><br />
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<p class="title2">The “can’t miss it” event of the summer: the 2014 Football (also called Soccer in some third zone countries!!) World Cup!</p><br />
<p class="texte">From 06/12/14 to 07/13/14, the Toulouse iGEM Team was cheering on the French team. During the <br />
games of the French team, our group was juggling with wet lab and large screen projections! Despite <br />
our support, the French team did not win the World Cup. However, we have not said our last world <br />
yet: let’s see what will happen in 2018… ;-)</p><br />
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<p class="title2">Have you ever forgotten a culture tube or a petri dish?</p><br />
<p class="texte" style="text-align:center;">Never? Let us show you what happens in that case!</p><br />
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<tr><td><img src="https://static.igem.org/mediawiki/2014/d/d4/Old_petri1.png" width="370px"></td><br />
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<p class="title2" style="padding-bottom:15px;">To finish this part, let’s do the official Awards Ceremony of the 2014 Toulouse iGEM Team!</p><br />
<ul><br />
<li class="tree"><p class="texte">The Geek Award: The three nominees are Laureen, Manon, Florie. And the winner is… <b>Florie</b> <br />
who spent the longest time in front of her laptop for the modeling part!</p></li><br />
<li class="tree"><p class="texte">The latest survivor of weekly meetings: The four nominees are Fanny, Emeline, Diane, Pierre. <br />
And the winner is… <b>Diane</b> who stayed up until 3am because she was skyping from South Korea!<br />
</p></li><br />
<li class="tree"><p class="texte">The worst singer award: The two nominees are Abdel, Pierre. And the winner is… <b>Abdel</b> who <br />
spent the whole day singing badly in the lab!</p></li><br />
<li class="tree"><p class="texte">The dancer award: The three nominees are Florie, Camille, Diane. And the winner is… <b>Camille</b><br />
who did the famous Plasmid Dance!</p></li><br />
<li class="tree"><p class="texte">The “hello you” award: No nominees because the only winner is… <b>Pierre</b> who was saying <br />
“Hello you” each time he meets someone!</p></li><br />
<li class="tree"><p class="texte">The most tired award: The three nominees are Laureen, Manon, Aurélie. And the winner is... <br />
<b>Manon</b> but we still do not know why!</p></li><br />
<li class="tree"><p class="texte">The misplaced ideas award: The four nominees are Laureen, Camille, Mathieu, Pierre. And <br />
the winner is… <b>Mathieu</b> but you do not want to know why!</p></li><br />
<li class="tree"><p class="texte">The perseverance award: The three nominees are Emeline, Diane, Abdel. And the winner is... <br />
<b>Emeline</b> who succeeded a cloning after twelve trials!</p></li><br />
<li class="tree"><p class="texte">The drawing award: The three nominees are Florie, Fanny, Manon. And the winner is… <b>Fanny</b> <br />
who drew our first SubtiTree logo!</p></li><br />
<li class="tree"><p class="texte">The phone-call award: The two nominees are Laureen, Pierre. And the winner is… <b>Laureen</b><br />
who was our lab secretary!</p></li><br />
<li class="tree"><p class="texte">The biggest blunder in the lab award: The three nominees are Aurélie, Fanny, Abdel. And the <br />
winner is… <b>Aurélie</b> who poured an agarose gel without gel tray!</p></li><br />
<li class="tree"><p class="texte">And last but not least ... The Best Nervous breakdown Award goes to ... <b>Our deep freezer</b>! The whole team is grateful for its hard work during a hot summer! Three successive breakdowns during the hot days of summer: thank you freezer, so long chap, we’ll unplug you after iGEM is finished and you’ll retire, hopefully not in the Canal du Midi…</li></p><br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Team&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Our Team</p> <br />
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<p class="texte">Let us introduce you to our fabulous tree-friendly team , 100% biodegradable and certified pesticide-free.</p><br />
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<p class="title1">Students</p><br />
<p class="title3"> <i>(Put the mouse over the pictures to extend them!)</i> </p><br />
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<p class="title2"><b>Diane Barbay</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Diligent (Wait a minute, I have to write down these results in my notebook! ㅋㅋㅋ)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Hardworking, Diane is the most conscientious in her work: her notebook is so clean and organized! She is persevering and always tries to find the answer to the problem (except when plasmid shortens between two digestions…). She talks to bacteria to encourage them taking up plasmid during transformation. She’s like a babysitter for our cells.<br />
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<p class="title2"><b>Emeline Flajollet</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in competent cells (Oh no, i have to make competent cells again… ㅋㅋㅋ)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Master of Microbiology at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About her:</b> Emeline looks discreet at first, but when you meet her, you discover a frank person who knows how to express her opinions and how to be understood, which is a good thing for team work. She is diligent and involved in her work, always trying to do her best, and providing advice to others. She spends a lot of time on social networks, allowing us to keep in touch with other teams.<br />
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<p class="title2"><b>Mathieu Fournié</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in communication (I found someone else who wants to interview us!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Master of Microbiology at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About him:</b> Initial founder of the project, Mathieu is a fan of Midi Pyrenées's region and is eager to protect its natural environment and resources. He is devoted to the project and does his best to succeed. His leadership skills help us focus on the main goals and deadlines. Even if it makes him forget to do his own tasks! His good interpersonal skills and numerous contacts helped us to capture media attention. <br />
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<p class="title2"><b>Florie Gosseau</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our IT expert (Modelling? Ok let’s do it!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Bioinformatics and Biological Systems Master at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About her:</b> Florie is a smiling and spontaneous girl. She is this kind of person capable of giving a true smile when nothing works, and she can disentangle conflicts thanks to her patience and her kindness. She is really helpful and never says no when you ask for service. She is also frank and reports problems when there are some. Finally, she is the only one good at informatics, which makes her indispensable for us!<br />
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<p class="title2"><b>Camille Jourdan</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in cloning (Camille, which enzyme should I take?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About him:</b> Jovial and funny, Camille has sometimes eruptions of madness which makes him so special. Conversely, he is capable of incredible moments of intelligence and concentration (primers and plasmids hold no secret for him) and he is really diligent. He is sarcastic but definitely not nasty. He is the athlete of the team and never misses an opportunity to make gymnastics exercises or Plasmid Dance!<br />
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<p class="title2"><b>Aurélie Kanitzer</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Distracted (Oh I forgot my keys! I have to go back home, see you in 15 minutes!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Aurélie is a joyful and spontaneous person, which makes you laugh and smile. Often distracted, she can make blunders… She is natural and honest and does not try to play a role. She is open-minded and helpful: you can count on her. Strong and sensitive at the same time, she is able to face difficulties. Don’t go by appearances, this tiny girl has personality and she does not let others walk over her!<br />
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<p class="title2"><b>Laureen Mirassou</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our best negotiator (Don’t worry about prices for flight tickets, I deal with that)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Laureen is a mix of kindness, helpfulness and generosity. Always trying to help people, she is essential to ease tensions and to solve social issues: she says what is wrong gently and calmly. Her good interpersonal abilities helped us to make good business and to solve many administrative problems. Her high level of English proficiency is also something precious for the team.<br />
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<p class="title2"><b>Manon Molina</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Organized (Let’s make a to-do-list!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About her:</b> Manon is organized: she always makes to-do-lists to not forget anything and do tasks in time. Thank goodness, she is here to monitor the budget and do the accounting! She makes her work conscientiously and meticulously, not allowing mistakes. She is kind and lenient but can also be frank when she does not endorse your methods. In short: serious and devoted to the project.<br />
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<p class="title2"><b>Fanny Pineau</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Absent-minded (which cloning are we doing?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About her:</b> In the lab, Fanny is often lost in her works: too many cloning at the same time, you should not ask too much to her blond brain! Not enough concentrated, she can sometimes make blunders. Besides that, she is applied and perfectionist when she plunges into her work. Finally, she is a joyful and smiling person; also honest and frank, telling it like it is.<br />
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<p class="title2"><b>Pierre Reitzer</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Relaxed (Make a presentation barefoot, why not?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About him:</b> Pierre is the co-founder of the project. He is really friendly and is always willing to discuss and help people with their experiments. Moreover, he greets us with a smile each time we see him in the lab. He is spontaneous and forthright: we know what he thinks. Sometimes, he has his head in the clouds and fails the same experiment three times… But he is involved in his work and does not count hours. <br />
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<p class="title2"><b>Abdel Touré</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Luxury tastes (Why would we go elsewhere than the Hilton?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Master at ENSAT Toulouse agronomic school</p> <br />
<p class="textesimple"><b>About him:</b> Abdel is this kind of person that you remember: he is always jovial and running everywhere. He is a funny guy who likes making jokes. But, be careful! He is also sensitive, your jokes might not make him laugh at all. He takes care of his appearance, and likes smoking his e-cigarette. He always swears in English, but we like it because this is funny, and he is a good English speaker!<br />
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<p class="title1">Instructors</p><br />
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<img src="https://static.igem.org/mediawiki/2014/b/b6/Advisor_Gilles.jpg" style="margin-top:5px"/><br />
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<p class="title2"><b>Gilles Truan</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our amazing instructor!</p><br />
<p class="textesimple"><b>Job:</b> Researcher at CNRS (Centre National de la Recherche Scientifique) and at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
<p class="textesimple"><b>About him:</b> With us from the beginning to the end as a father (and already a grandfather!!), our instructor is the master of cloning and the captain of PCR! </p><br />
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<div style="float:right; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Advisor_Brice.jpg" style="margin-top:5px" /><br />
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<p class="title2"><b>Brice Enjalbert</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> The right-hand man of our amazing instructor!</p><br />
<p class="textesimple"><b>Job:</b> Teacher-Researcher at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
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<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="title2"><b>Florence Bordes</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> The only female advisor! </p><br />
<p class="textesimple"><b>Job:</b> Teacher-Researcher at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
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<div style="float:right; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2d/Advisor_Cam.jpg" style="margin-top:5px" /><br />
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<p class="title2"><b>Kaymeuang Cam</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our bacteriologist advisor! </p><br />
<p class="textesimple"><b>Job:</b> Teacher-Researcher at IPBS (Institut de Pharmacologie et de Biologie Structurale)</p> <br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Team&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Our Team</p> <br />
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<p class="texte">Let us introduce you to our fabulous tree-friendly team , 100% biodegradable and certified pesticide-free.</p><br />
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<p class="title1">Students</p><br />
<p class="title3"> <i>(Put the mouse over the pictures to extend them!)</i> </p><br />
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<p class="title2"><b>Diane Barbay</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Diligent (Wait a minute, I have to write down these results in my notebook! ㅋㅋㅋ)</p><br />
<p class="textesimple"><b>Studies:</b> Second year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Hardworking, Diane is the most conscientious in her work: her notebook is so clean and organized! She is persevering and always tries to find the answer to the problem (except when plasmid shortens between two digestions…). She talks to bacteria to encourage them taking up plasmid during transformation. She’s like a babysitter for our cells.<br />
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<p class="title2"><b>Emeline Flajollet</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in competent cells (Oh no, i have to make competent cells again… ㅋㅋㅋ)</p><br />
<p class="textesimple"><b>Studies:</b> Second year of Professional Master of Microbiology at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About her:</b> Emeline looks discreet at first, but when you meet her, you discover a frank person who knows how to express her opinions and how to be understood, which is a good thing to work in team. She is diligent and involved in her work, always trying to do the best, and providing advice to others. She spends a lot of time on social networks, allowing us to keep in touch with other teams.<br />
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<p class="title2"><b>Mathieu Fournié</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in communication (I found someone else who wants to interview us!)</p><br />
<p class="textesimple"><b>Studies:</b> Second year of Professional Master of Microbiology at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About him:</b> Initial founder of the project, Mathieu is a fan of his region and is eager to protect its natural environment and resources. He is devoted to the project and do his best to succeed. His leadership skills help us keeping in mind the main goals and deadlines. Even if it makes him forget to do his own tasks! His good interpersonal skills and numerous contacts helped us to capture media attention. <br />
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<p class="title2"><b>Florie Gosseau</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our IT expert (Modelling? Ok let’s do it!)</p><br />
<p class="textesimple"><b>Studies:</b> Second year of Bioinformatics and Biological Systems Master at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About her:</b> Florie is a smiling and spontaneous girl. She is this kind of person capable of given back the smile when nothing works, and she can disentangle conflicts thanks to her patience and her kindness. She is really helpful and never says no when you ask for service. She is also frank and reports problems when there are some. Finally, she is the only one good at informatics, which makes her indispensable for us!<br />
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<p class="title2"><b>Camille Jourdan</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in cloning (Camille, which enzyme should I take?)</p><br />
<p class="textesimple"><b>Studies:</b> Second year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About him:</b> Jovial and funny, Camille has sometimes eruptions of madness which makes him so special. Conversely, he is capable of incredible moments of intelligence and concentration (primers and plasmids hold no secret for him) and he is really diligent. He is sarcastic but definitely not nasty. He is the athlete of the team and never misses an opportunity to make gymnastics exercises or Plasmid Dance!<br />
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<p class="title2"><b>Aurélie Kanitzer</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Distracted (Oh I forgot my keys! I have to go back home, see you in 15 minutes!)</p><br />
<p class="textesimple"><b>Studies:</b> Second year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Aurélie is a joyful and spontaneous person, which makes you laugh and smile. Often distracted, she can make blunders… She is natural and honest and does not try to play a role. She is open-minded and helpful: you can count on her. Strong and sensitive at the same time, she is able to face difficulties. Don’t go by appearances, this tiny girl has personality and she does not let others walk over her!<br />
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<p class="title2"><b>Laureen Mirassou</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our best negocatior (Don’t worry about prices for flight tickets, I deal with that)</p><br />
<p class="textesimple"><b>Studies:</b> Second year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Laureen is a mix of kindness, helpfulness and generosity. Always trying to help people, she is essential to ease tensions and to solve social issues: she says what is wrong gently and calmly. Her good interpersonal abilities helped us to make good business and to solve many administrative problems. Her high level of English proficiency is also something precious for the team.<br />
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<p class="title2"><b>Manon Molina</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Organized (Let’s make a to-do-list!)</p><br />
<p class="textesimple"><b>Studies:</b> Second year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About her:</b> Manon is organized: she always makes to-do-lists to not forget anything and do tasks in time. Thank goodness, she is here to monitor the budget and do the counts! She makes her work conscientiously and meticulously, not allowing mistakes. She is kind and lenient but can also be frank when she does not endorse your methods. In short: serious and devoted to the project.<br />
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<p class="title2"><b>Fanny Pineau</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Absent-minded (which cloning are we doing?)</p><br />
<p class="textesimple"><b>Studies:</b> Second year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About her:</b> In the lab, Fanny is often lost in her works: too many clonings at the same time, you should not ask too much to her blond brain! Not enough concentrated, she can sometimes make blunders. Besides that, she is applied and perfectionist when she plunges into her work. Finally, she is a joyful and smiling person; also honest and frank, telling it like it is.<br />
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<p class="title2"><b>Pierre Reitzer</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Relaxed (Make a presentation barefoot, why not?)</p><br />
<p class="textesimple"><b>Studies:</b> Second year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About him:</b> Pierre is the co-founder of the project. He is really friendly and is always willing to discuss and help people with their experiments. Moreover, he greets us with a smile each time we see him in the lab. He is spontaneous and forthright: we know what he thinks. Sometimes, he has his head in the clouds and fails the same experiment three times… But he is involved in his work and does not count hours. <br />
</p><br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div style="float:left; width:215px;"><br />
<input type="image" name="page" value="main" <br />
onMouseOut="src='https://static.igem.org/mediawiki/2014/f/fa/Abdel.png'" <br />
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</div> <br />
<div style="float: right; width:720px;"> <br />
<p class="title2"><b>Abdel Touré</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Luxury tastes (Why would not we go to the Hilton?)</p><br />
<p class="textesimple"><b>Studies:</b> Second year of Master at ENSAT Toulouse agronomic school</p> <br />
<p class="textesimple"><b>About him:</b> Abdel is this kind of person that you remember: he is always jovial and running everywhere. He is a funny guy who likes making jokes. But, be careful! He is also sensitive, your jokes might not make him laught at all. He takes care of his appearence, and likes smoking his e-cigarette. He always swears in English, but we like it because this is funny, and he is a good English speaker!<br />
</p><br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<p class="title1">Instructors</p><br />
<br />
<div style="float:left; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/b/b6/Advisor_Gilles.jpg" style="margin-top:5px"/><br />
</div> <br />
<br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="title2"><b>Gilles Truan</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our amazing instructor!</p><br />
<p class="textesimple"><b>Job:</b> Researcher at CNRS (Centre National de la Recherche Scientifique) and at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
<p class="textesimple"><b>About him:</b> With us from the beginning to the end as a father, our instructor is the master of cloning and the captain of PCR! </p><br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div style="float:right; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Advisor_Brice.jpg" style="margin-top:5px" /><br />
</div> <br />
<br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"><br />
<p class="title2"><b>Brice Enjalbert</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> The right-hand man of our amazing instructor!</p><br />
<p class="textesimple"><b>Job:</b> Teacher-Researcher at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div style="float:left; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Advisor_Florence.jpg" style="margin-top:5px" /><br />
</div> <br />
<br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="title2"><b>Florence Bordes</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> The only advisor female! </p><br />
<p class="textesimple"><b>Job:</b> Teacher-Researcher at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div style="float:right; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2d/Advisor_Cam.jpg" style="margin-top:5px" /><br />
</div> <br />
<br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"><br />
<p class="title2"><b>Kaymeuang Cam</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our bacteriologist advisor! </p><br />
<p class="textesimple"><b>Job:</b> Teacher-Researcher at IPBS (Institut de Pharmacologie et de Biologie Structurale)</p> <br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>Dbbyhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-16T16:05:57Z<p>Dbby: </p>
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<h2>Spreading</h2><br />
<p>How to keep control on SubtiTree?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte">Our engineered bacterium is designed to be inoculated in a tree and to cure fungal diseases. To target the possible environmental issues resulting of using a modified organism directly on trees bordering the Canal du Midi, our team worked on different aspects to ensure a safe use of SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these issues, we imagined three modules.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center style="margin-bottom:80px;"><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/2/27/Spreading_sch%C3%A9ma.jpg"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<p class="title1">Survival in the environment: proline auxotroph <i>B. subtilis</i></p><br />
<p class="texte">SubtiTree will live in sap tree, thus we will use an endophyte <I>B. subtilis</I> strain. In order to contain our bacteria in this area during a short period of time, we thought of modifying some of its survival characteristics. To make the bacterium growth dependant on the presence of the tree (and therefore avoid spreading in the environment), we planned to use a <i>B.subtilis</i> strain auxotroph for proline. The bacterium should then be unable to synthesize this essential amino acid. Proline is the most abundant amino acid in the phloem sap. If the bacterium is in the sap, it should grow normally without any deficiency, but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a long time (proline is present only in very low quantities in the ground). <br />
<br/> <br />
Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore they should be easy to find.</p> DIRE QUE CE N'EST PAS CE QU'ON A UTILISE??<br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
It is known that endophyte bacteria must sporulate to survive to winter. In order to limit the spreading of our bacterium, we planned to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should die in the fall.<br\> <br />
<i>B. subtilis</i> is a sporing bacterium: sporulation enables the microorganism to resist very harsh conditions and to spread from tree to tree.<br/> <br />
To control any unwanted long-term development of SubtiTree, our strain should therefore be deficient for sporulating. We USED or COULD USE a <I>B.subtilis</I> strain deficient in the late genes for sporulation. Thus, during fall, when the sap become less nutritious and the temperature is low, the engineered bacterium will die and not pass through the following winter.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation, thus allowing a better faith on the genetic constructions.<br />
</p><br />
<br />
<p class="texte">These two first characteristics of SubtiTree demonstrate that it should be an annual bacterium, growing only in sap tree. By combining them, they should prevent any long term colonization of any other ecological niche than the plane trees</p><br />
<br />
<br />
<p class="title1">Gene transfer: toxin-antitoxin system</p><br />
<br />
<p class="texte">We also wondered about horizontal genes transfer. The goal of this module is to prevent horizontal transfers between bacteria and any exchange of synthetic genetic material that could be dangerous between wild type organisms and optimized organisms.<br />
<br>We thought about a system limiting such transfers: a toxin-antitoxin module. It involves the addition of two genes to the bacterium: a gene encoding for a toxin (for example <i>tse2</i>) and a gene encoding for the antitoxin (<i>tsi1</i>), placing them in an opposite way on the genome. The large space between them prevents simultaneous transfers: if the optimized bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/><br />
Therefore, if anther host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not possess the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
In summary, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin. <br />
</p><br />
<br />
<p class="texte">Our synthetic genes are not the only problem in the design of SubtiTree. One of the side effects of our cloning method is the persistence of antibiotic resistance genes. This is incompatible with the introduction of SubtiTree in the environment. It is possible to delete this resistance in chromosome.<br />
<br><br />
While we have not constructed yet these modules, we definitely think that the measures that we designed should render the use of SubtiTree acceptable in the environment. <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
All our constructions are carried by integrative plasmids. Consequently, our different genetic modules should be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependent on a selective pressure, allowing a high level of transcription <i>in planta</i>. <br />
</p><br />
<br />
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color:#666; font-size:18px;">Modeling</br><br />
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{{:Team:Toulouse/template/footer}}</div>Dbbyhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-16T15:59:57Z<p>Dbby: </p>
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<h2>Spreading</h2><br />
<p>How to keep control on SubtiTree?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte">Our engineered bacterium is designed to be inoculated in a tree and to cure fungal diseases. To target the possible environmental issues resulting of using a modified organism directly on trees bordering the Canal du Midi, our team worked on different aspects to ensure a safe use of SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these issues, we imagined three modules.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center style="margin-bottom:80px;"><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/2/27/Spreading_sch%C3%A9ma.jpg"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<p class="title1">Survival in the environment: proline auxotroph <i>B. subtilis</i></p><br />
<p class="texte">SubtiTree will live in sap tree, thus we will use an endophyte <I>B. subtilis</I> strain. In order to contain our bacteria in this area during a short period of time, we thought of modifying some of its survival characteristics. To make the bacterium growth dependant on the presence of the tree (and therefore avoid spreading in the environment), we planned to use a <i>B.subtilis</i> strain auxotroph for proline. The bacterium should then be unable to synthesize this essential amino acid. Proline is the most abundant amino acid in the phloem sap. If the bacterium is in the sap, it should grow normally without any deficiency, but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a long time (proline is present only in very low quantities in the ground). <br />
<br/> <br />
Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore they should be easy to find.</p> DIRE QUE CE N'EST PAS CE QU'ON A UTILISE??<br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
It is known that endophyte bacteria must sporulate to survive to winter. In order to limit the spreading of our bacterium, we planned to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should die in the fall.<br\> <br />
<i>B. subtilis</i> is a sporing bacterium: sporulation enables the microorganism to resist very harsh conditions and to spread from tree to tree.<br/> <br />
To control any unwanted long-term development of SubtiTree, our strain should therefore be deficient for sporulating. We USED or COULD USE a <I>B.subtilis</I> strain deficient in the late genes for sporulation. Thus, during fall, when the sap become less nutritious and the temperature is low, the engineered bacterium will die and not pass through the following winter.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation, thus allowing a better faith on the genetic constructions.<br />
</p><br />
<br />
<p class="texte">These two first characteristics of SubtiTree demonstrate that it should be an annual bacterium, growing only in sap tree. By combining them, they should prevent any long term colonization of any other ecological niche than the plane trees</p><br />
<br />
<br />
<p class="title1">Gene transfer: toxin-antitoxin system</p><br />
<br />
<p class="texte">We also wondered about horizontal genes transfer. The goal of this module is to prevent horizontal transfers between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms: it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.<br />
<br>Considering this issue, we thought about a system to avoid such transfers: a toxin-antitoxin module. It involves the addition of two genes to the bacterium: a gene encoding for a toxin (for example <i>tse2</i>) and a gene encoding for the antitoxin (<i>tsi1</i>), placing them in an opposite way on the genome. The large space between them prevents simultaneous transfers: if the optimized bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/><br />
Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin. <br />
</p><br />
<br />
<p class="texte">Our synthetic genes are not the only problem in the design of SubtiTree. One of the side effects of our cloning method is the persistence of antibiotic resistance genes. This is incompatible with the introduction of SubtiTree in the environment. It is possible to delete this resistance in chromosome. To conclude, the spreading limitation shown previously makes the use of SubtiTree acceptable in the environment. <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
All our constructions are carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependent on a selective pressure, allowing a high level of transcription <i>in planta</i>. <br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Fungicides<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
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<h2>Fungicides</h2><br />
<p>To eradicate fungal diseases</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fungicides</p> <br />
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<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/0/0c/Recap_fungicides.jpg"><br />
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<p class="legend">Figure 1: Scheme of the fungicide module</p></center><br />
<br />
<p class="textesimple">The main objective of SubtiTree is to ensure the <b> destruction of the pathogenic fungi </b> inside the tree. In order to achieve this goal, we built a genetic module to produce three different peptides with antifungal activities. This triple therapy minimizes the risk of the introduction of a resistance mechanism.</p> <br><br />
<br />
<p class="textesimple">Originally from plants, these peptides have different targets to maximize the lethality on <i>C. platani</i>.</p><br />
<br></br> <br />
<ul><br />
<li class="tree"><p class="texte"><b>D4E1</b> is a synthetic peptide analog to Cecropin B AMPs (AntiMicrobial Peptides) made of 17 amino acids which has been shown to have an antifungal activity by complexing with a sterol present in the conidia’s wall of numerous fungi.</p></li><br />
<br />
<li class="tree"><p class="texte"><b>GAFP-1 </b>(<i>Gastrodia</i> Anti Fungal Protein 1), also known as gastrodianin, is a mannose and chitin binding lectin originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years. GAFP-1 accumulates in nutritive corms where the fungal infection takes place, and <i>in vitro</i> assays demonstrated it can inhibit the growth of ascomycete and basidiomycete fungal plant pathogens.</p></li><br />
<br />
<li class="tree"><p class="texte"><b>EcAMP-1 </b>(<i>Echinochloa crus-galli</i> AntiMicrobial Peptide) consists in 37 amino acids inhibiting hyphae elongation. EcAMP-1 is the first example of AMP with a novel disulfide-stabilized-α helical hairpin fold. It is isolated from kernels of barnyard grass. EcAMP-1 exhibits high activity against fungi of the genus <i>Fusarium</i>.</p></li><br />
</ul><br />
</p><br />
<br><br />
<p class="title1" style="margin-top:30px;">More information about this module </p><br />
<p class="texte"><br />
We built different genetic constructions to test each fungicide separately and to test them all together on the same operon where the three genes coding for the antifungal peptides are placed under the control of the constitutive promoter P<sub>veg</sub> in <i>Bacillus subtilis</i>.</p><br />
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<center><img style="width:930px; float:left; margin: 30px 0 45px;" src="https://static.igem.org/mediawiki/parts/d/d0/Fungicideprod.jpg"><br />
<p class="legend">Figure 2: Fungicide operon</p></center><br />
<br />
<p class="title2">Added parts</p><br />
<p class="title3">EcAMP-1</p><br />
<p class="texte">EcAMP-1 was already present in the Registry, added by the Utah State 2013 iGEM team (<a href="http://parts.igem.org/Part:BBa_K1162001"_blank">BBa_K1162001</a>). This part has been modified and improved by our team (<a href="http://parts.igem.org/Part:BBa_K1364019"_blank">BBa_K1364019</a>). </p><br />
<p class="title3">D4E1 and GAFP-1</p><br />
<p class="texte">We added D4E1 and GAFP-1 to the Registry of Standard Biological Parts (See <a href="https://2014.igem.org/Team:Toulouse/Result/parts/Submitted_parts"_blank">Submitted parts</a>). <br>We ordered the genes to a synthesis company and did cloning. These new BioBricks were designed in order to be expressed and secreted with <i>Bacillus subtilis</i>. </p><br />
<br><br />
<p class="title2">Secretion</p><br />
<p class="texte">In order to export the peptides outside the bacteria, the coding sequence of D4E1 and GAFP-1 was flanked on the N-terminal end with a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process.</p><br><br />
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<center><img style="width:400px; " src="https://static.igem.org/mediawiki/2014/2/2e/Secretion.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/2014/d/d7/Fongpep.jpg"><br />
<br><p class="legend">Figure 3: Design of GAFP-1 and D4E1</p></center><br />
<br><br />
<br />
<br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">A. J De Lucca, J.M Bland, C. Grimm, T.J Jacks.<b> Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1 </b>. Canadian Journal of Microbiology. 1998, Vol. 44:514-520. </p></li><br />
<li class="tree"><p class="texte">Kanniah Rajasekaran, Kurt D. Stromberg, Jeffrey W. Cary, and Thomas E. Cleveland.<b> Broad-Spectrum Antimicrobial Activity in vitro of the Synthetic Peptide D4E1</b>. J. Agric. Food Chem. 2001, Vol. 49, 2799-2803.</p></li><br />
<li class="tree"><p class="texte">M. Visser, D. Stephan, J.M. Jaynes and J.T. Burger.<b> A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens</b>. Letters in Applied Microbiology. 2012, Vol. 54, 543–551.</p></li><br />
<li class="tree"><p class="texte">K. D. Cox, D. R. Layne, R. Scorza, G Schnabel. <b>Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco</b>. Planta. 2006, Vol. 224:1373–1383</p></li><br />
<li class="tree"><p class="texte">Xiaochen Wang, Guy Bauw, Els J.M. Van Damme, Willy J. Peumans, Zhang-Liang Chen, Marc Van Montagu and Willy Dillen. <b>Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties</b>. The Plant Journal. 2001, Vol. 25(6), 651±661</p></li><br />
<li class="tree"><p class="texte">Svetlana B. Nolde, Alexander A. Vassilevski, Eugene A. Rogozhin, Nikolay A. Barinov, Tamara A. Balashova, Olga V. Samsonova, Yuri V. Baranov, Alexey S. Arseniev and Eugene V. Grishin. <b>Disulfide-stabilized Helical Hairpin Structure and Activity of a Novel Antifungal Peptide EcAMP1 from Seeds of Barnyard Grass (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p></li><br />
</ul><br />
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<h2>Binding</h2><br />
<p>To be attached to the fungal wall</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Binding</p> <br />
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<p class="texte"> In order to be highly efficient in the fight against the pythopathogen, our optimized bacterium has to be anchored to the fungus. Thus, we designed a chimeric protein (<a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>) capable of building a <B>bridge between the bacterial peptidoglycan and the fungal chitin</B>, the main component of the pathogen’s cell wall. According to the work of the Imperial College 2010 iGEM team, we used the CWB domain of the LytC protein (coding for a N-acetylmuramoyl-L-alanine amidase) to attach our chimeric protein to the <I>B. subtilis</I> cell wall. On the other side of our protein, we added the domain 4 of GbpA from <I>Vibrio cholerae</I>, which is known to recognize chitin.<br />
</p><br />
<br />
<br><br />
<p class="title1"> More information about this module </p><br />
<p class="texte"> <br />
The Binding Module ORF is composed of 3 sections:</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte"><B>Anchor section</B>: the CWB (Cell Wall Binding) domain is extracted from LytC gene and composes the 5' side of our binding module. As previously described by the Imperial College of London 2010 iGEM team, we kept the first 318 bp. We can note the presence of the signal peptide at the beginning of the sequence, from 1 to 24 bp.</p></li><br />
<li class="tree"><p class="texte"><B>Chitin Binding Domain (CBD) section</b>: the domain 4 of GbpA from <I>Vibrio cholerae </I> is able to bind to N-Acetyl Glucosamine oligosacchararides. Also, the 3' side of our gene is composed by a part of the GbpA sequence (from 423 to 484 bp).</p></li><br />
<li class="tree"><p class="texte"><B>Helical Linker</B>: According to the work of the 2010 Imperial College of London iGEM team, we used the same six amino acids sequence (SRGSRA) to make a bridge between the Anchor section and the Chitin Binding section.<br />
</p></li></ul><br />
<center style="margin:-30px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/a/a0/Binding.png"></center><br />
<br></br><br />
<p class="texte"> <br />
The sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i>. <br />
</p><br />
<br />
<B class="title1">Final construction</B> <br />
<br />
<B class="title2">(More details about the intermediate parts <a href="https://2014.igem.org/Team:Toulouse/Result/parts#select2"target="_blank">Here</a>)</B> <br />
<br />
<p class="texte"><br />
<br>The binding module has been placed under the control of Pveg (<a href="http://parts.igem.org/Part:BBa_K143012"target="_blank">BBa_K143012</a>), a strong constitutive promoter and we used a consensus RBS <a href="http://parts.igem.org/Part:BBa_K090505"target="_blank">BBa_K090505</a> and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015"target="_blank">B0015</a>). The construct has been inserted in an integrative plasmid, pSBBS4S (<a href="http://parts.igem.org/Part:BBa_K823022"target="_blank">BBa_K823022</a>) which comes from the LMU-Munich 2012 iGEM team.</p> <br />
<br />
<center style="margin:20px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"></center><br />
<br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">M. Desvaux, E. Dumas, I. Chafsey and M. Hébraud.<b> Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure </b>. FEMS Microbiol. Lett. 256, 1–15 (2006). </p></li><br />
<li class="tree"><p class="texte">E. Wong, G. Vaaje-Kolstad, A. Ghosh, R. Hurtado-Guerrero, PV. Konarev, AF. Ibrahim, DI. Svergun, VG. Eijsink, NS. Chatterjee and DM. van Aalten.<b>The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces</b>. PLoS Pathog. 8, e1002373 (2012).</p></li><br />
<br />
<li class="tree"><p class="texte">H. Yamamoto, S. Kurosawa and J. Sekiguchi. <b>Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases</b>. J. Bacteriol. 185, 6666–6677 (2003).</p></li><br />
</ul><br />
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<div class="banniere-content"><br />
<h2>Binding</h2><br />
<p>To be attached to the fungal wall</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Binding</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
<br />
<!--Short description : à changer!!!--><br />
<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/e/e1/Bindingresume.png"></center><br />
<br />
<p class="texte"> In order to be highly efficient in the fight against the pythopathogen, our optimized bacterium has to be anchored to the fungus. Thus, we designed a chimeric protein (<a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>) capable of building <B>a bridge between the bacterial peptidoglycan and the fungal chitin</B>, the main component of the pathogen’s cell wall. According to the work of the Imperial College 2010 iGEM team, we used the CWB domain of the LytC protein (coding for a N-acetylmuramoyl-L-alanine amidase) to attach our chimeric protein to the <I>B. subtilis</I> cell wall. On the other side of our protein, we added the domain 4 of GbpA from <I>Vibrio cholerae</I>, which is known to recognize chitin.<br />
</p><br />
<br />
<br><br />
<p class="title1"> More information about this module </p><br />
<p class="texte"> <br />
The Binding Module ORF is composed of 3 sections:</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte"><B>Anchor section</B>: the CWB (Cell Wall Binding) domain is extracted from LytC gene and composes the 5' side of our binding module. As previously described by the Imperial College of London 2010 iGEM team, we kept the first 318 bp. We can note the presence of the signal peptide at the beginning of the sequence, from 1 to 24 bp.</p></li><br />
<li class="tree"><p class="texte"><B>Chitin Binding Domain (CBD) section</b>: the domain 4 of GbpA from <I>Vibrio cholerae </I> is able to bind to N-Acetyl Glucosamine oligosacchararides. Also, the 3' side of our gene is composed by a part of the GbpA sequence (from 423 to 484 bp).</p></li><br />
<li class="tree"><p class="texte"><B>Helical Linker</B>: According to the work of the 2010 Imperial College of London iGEM team, we used the same six amino acids sequence (SRGSRA) to make a bridge between the Anchor section and the Chitin Binding section.<br />
</p></li></ul><br />
<center style="margin:-30px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/a/a0/Binding.png"></center><br />
<br></br><br />
<p class="texte"> <br />
The sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i>. <br />
</p><br />
<br />
<B class="title1">Final construction</B> <br />
<br />
<B class="title2">(More details about the intermediate parts <a href="https://2014.igem.org/Team:Toulouse/Result/parts#select2"target="_blank">Here</a>)</B> <br />
<br />
<p class="texte"><br />
<br>The binding module has been placed under the control of Pveg (<a href="http://parts.igem.org/Part:BBa_K143012"target="_blank">BBa_K143012</a>), a strong constitutive promoter and we used a consensus RBS <a href="http://parts.igem.org/Part:BBa_K090505"target="_blank">BBa_K090505</a> and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015"target="_blank">B0015</a>). The construct has been inserted in an integrative plasmid, pSBBS4S (<a href="http://parts.igem.org/Part:BBa_K823022"target="_blank">BBa_K823022</a>) which comes from the LMU-Munich 2012 iGEM team.</p> <br />
<br />
<center style="margin:20px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"></center><br />
<br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">M. Desvaux, E. Dumas, I. Chafsey and M. Hébraud.<b> Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure </b>. FEMS Microbiol. Lett. 256, 1–15 (2006). </p></li><br />
<li class="tree"><p class="texte">E. Wong, G. Vaaje-Kolstad, A. Ghosh, R. Hurtado-Guerrero, PV. Konarev, AF. Ibrahim, DI. Svergun, VG. Eijsink, NS. Chatterjee and DM. van Aalten.<b>The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces</b>. PLoS Pathog. 8, e1002373 (2012).</p></li><br />
<br />
<li class="tree"><p class="texte">H. Yamamoto, S. Kurosawa and J. Sekiguchi. <b>Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases</b>. J. Bacteriol. 185, 6666–6677 (2003).</p></li><br />
</ul><br />
<br />
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<!-- Navigation section --> <br />
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<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
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color:#666; font-size:18px;">Fungicides</br><br />
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<div class="banniere-content"><br />
<h2>Binding</h2><br />
<p>To be attached to the fungal wall</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Binding</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
<br />
<!--Short description : à changer!!!--><br />
<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/e/e1/Bindingresume.png"></center><br />
<br />
<p class="texte"> In order to be highly efficient in the fight against the pythopathogen, our optimized bacterium has to be anchored to the fungus. Thus, we designed a chimeric protein (<a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>) capable of building <B>a bridge between the bacterial peptidoglycan and the fungal chitin</B>, the main component of the pathogen’s cell wall. According to the work of the Imperial College 2010 iGEM team, we used the CWB domain of the LytC protein (coding for a N-acetylmuramoyl-L-alanine amidase) to attach our chimeric protein to the <I>B. subtilis</I> cell wall. On the other side of our protein, we added the domain 4 of GbpA from <I>Vibrio cholerae</I>, which is known to recognize chitin.<br />
</p><br />
<br />
<br><br />
<p class="title1"> More information about this module </p><br />
<p class="texte"> <br />
The Binding Module ORF is composed of 3 sections:</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte"><B>Anchor section</B>: the CWB (Cell Wall Binding) domain is extracted from LytC gene and composes the 5' side of our binding module. As previously described by the Imperial College of London 2010 iGEM team, we kept the first 318 bp. We can note the presence of the signal peptide at the beginning of the sequence, from 1 to 24 bp.</p></li><br />
<li class="tree"><p class="texte"><B>Chitin Binding Domain (CBD) section</b>: the 4th domain of GbpA from <I>Vibrio cholerae </I> is able to bind to N-Acetyl Glucosamine oligosacchararides. Also, the 3' side of our gene is composed by a part of the GbpA sequence (from 423 to 484 bp).</p></li><br />
<li class="tree"><p class="texte"><B>Helical Linker</B>: According to the work of the 2010 Imperial College of London iGEM team, we used the same six amino acids sequence (SRGSRA) to make a bridge between the Anchor section and the Chitin Binding section.<br />
</p></li></ul><br />
<center style="margin:-30px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/a/a0/Binding.png"></center><br />
<br></br><br />
<p class="texte"> <br />
The sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i>. <br />
</p><br />
<br />
<B class="title1">Final construction</B> <br />
<br />
<B class="title2">(More details about the intermediate parts <a href="https://2014.igem.org/Team:Toulouse/Result/parts#select2"target="_blank">Here</a>)</B> <br />
<br />
<p class="texte"><br />
<br>The binding module has been placed under the control of Pveg (<a href="http://parts.igem.org/Part:BBa_K143012"target="_blank">BBa_K143012</a>), a strong constitutive promoter and we used a consensus RBS <a href="http://parts.igem.org/Part:BBa_K090505"target="_blank">BBa_K090505</a> and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015"target="_blank">B0015</a>). The construct has been inserted in an integrative plasmid, pSBBS4S (<a href="http://parts.igem.org/Part:BBa_K823022"target="_blank">BBa_K823022</a>) which comes from the LMU-Munich 2012 iGEM team.</p> <br />
<br />
<center style="margin:20px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"></center><br />
<br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">M. Desvaux, E. Dumas, I. Chafsey and M. Hébraud.<b> Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure </b>. FEMS Microbiol. Lett. 256, 1–15 (2006). </p></li><br />
<li class="tree"><p class="texte">E. Wong, G. Vaaje-Kolstad, A. Ghosh, R. Hurtado-Guerrero, PV. Konarev, AF. Ibrahim, DI. Svergun, VG. Eijsink, NS. Chatterjee and DM. van Aalten.<b>The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces</b>. PLoS Pathog. 8, e1002373 (2012).</p></li><br />
<br />
<li class="tree"><p class="texte">H. Yamamoto, S. Kurosawa and J. Sekiguchi. <b>Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases</b>. J. Bacteriol. 185, 6666–6677 (2003).</p></li><br />
</ul><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
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<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
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<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>Dbbyhttp://2014.igem.org/Team:Toulouse/Project/ChemotaxisTeam:Toulouse/Project/Chemotaxis2014-10-16T15:18:25Z<p>Dbby: </p>
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<h2>Chemotaxis</h2><br />
<p>To target the pathogenic fungus</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Chemotaxis</p> <br />
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<br />
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<br />
<br />
<center><img style="width:420px; " src="https://static.igem.org/mediawiki/parts/e/e9/Recap_chemotax.jpg"></center><br />
<br />
<p class="title1">What is chemotaxis?</p><br />
<br />
<p class="texte"><br />
Chemotaxis is a bacterial function which allows moving toward a gradient of concentration of a molecule that is present in the medium. With this system bacteria can swim to a location containing higher concentrations of molecules such as sugar, amino acid, vitamins... Chemotactic-signal transducers respond to changes in the concentration of attractants and repellents in the environment, transduce the signal from the outside to the inside of the cell, and facilitate sensory adaptation through the variation of the level of methylation. <br />
<br />
<p class="title1">More information on this module</p><br />
<p class="texte"><br />
Chemotaxis is used as a way to detect and come close to the location of fungi infection. During its growth, fungi release N-acetylglucosamine (NAG), the basic unit of chitin which composed its cell wall. Thus, there should exist a gradient of the concentration of NAG around the fungi.</p><br />
<p class="texte"><br />
It is known that <i>B. subtilis</i> is able to detect and to swim towards glucose using the Methyl-accepting chemotaxis protein, called <b>McpA</b>.<br><br />
Some bacteria are attracted by NAG, like <i>Vibrio cholerae</i> which has a NAG regulated methyl-accepting chemotaxis protein: <b>VCD</b>.</p><br />
<br />
<center><img width="500px" SRC="https://static.igem.org/mediawiki/2014/4/47/Chimio1.png" alt="schema" style="margin-bottom:60px;"></center><br />
<p class="texte"><br />
Therefore, our idea is to switch the natural glucose specificity of <i>B. subtilis'</i> McpA to a NAG specificity. To achieve this, we need to change the extracellular domain of McpA, the domain responsible for the specificity, by the extracellular domain of VCD.<br />
The whole sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i> before its synthesis.</p><br />
<br />
<center><img width="600px" SRC="https://static.igem.org/mediawiki/2014/e/e4/Chimio2.png" alt="gene construct" style="margin-bottom:40px;"></center><br />
<br />
<p class="title1">References</p><br />
<ul><br />
<br />
<li class="tree"><p class="texte">K. Meibom,L. Xibing, A. Nielsen, CY. Wu, S. Roseman and G. Schoolnik.<b> The Vibrio cholerae chitin utilization program </b>. The National Academy of Sciences of the USA (2004).</p></li><br />
<li class="tree"><p class="texte">C. Kristich and GW. Ordal. <b><i>Bacillus subtilis</i> CheD is a chemoreceptor modification enzyme required for chemotaxis</b>. J Biol Chem. 2002 Jul 12;277(28):25356-62. Epub 2002 May 13.<br></p></li><br />
</ul><br />
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<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
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color:#666; font-size:18px;">Binding</br><br />
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{{:Team:Toulouse/template/footer}}</div>Dbbyhttp://2014.igem.org/Team:Toulouse/Project/OverviewsTeam:Toulouse/Project/Overviews2014-10-16T15:09:01Z<p>Dbby: </p>
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<h2>Overview</h2><br />
<p>SubtiTree, a bacterium to save our trees</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Overview</p> <br />
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Among many other things, Southern France is well-known for the gorgeousness of its landscapes. Plane trees (<i>Platanus sp.</i>) are widely present and participate to the charm of this area, especially along the famous “Canal du Midi”. It is impossible to imagine this UNESCO World Heritage masterpiece without its trees. Unfortunately, these trees are threatened by a severe fungal infection called Canker Stain, and today the only treatment consists in a costly preventive tree-cutting and implies significant ecological troubles.<br />
</p><br />
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<p class="texte"><br />
Facing this emergency, the students from the iGEM Toulouse Team decided to be committed to the protection of their local heritage. Using a bacterium vector naturally present in the trees, our team offers an alternative solution originated from synthetic biology. Using different genetic modules, we engineered a bacterium (SubtiTree) capable of heading towards the pathogen, binding to its cell wall and finally delivering different fungicides to save the tree from its invaders. Our team also began to design genetic modules preventing any accidental spreading of the optimized microorganism, thus limiting the ecological and ethical footprints of SubtiTree. Although our project originated from a very local and specific tree disease, it could be transposable to other plant diseases.<br />
</p><br />
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<p class="title1">Choice of the chassis </p><br />
<p class="texte"><i>Bacillus subtilis</i> has been reported to be an endophyte bacterium of a large variety of plants and trees. Therefore, this model organism is a perfect chassis for our project. Already used to treat plant diseases and fungal pathogens, we aim to engineer <i>Bacillus subtilis</i> to fight Canker Stain inside of the tree. Injected directly in the tree sap, our smart bacterium will act as a curative and preventive drug.</p><br />
<br />
<center><img style="width:700px; margin: -10px 0 55px 130px;" ; src="https://static.igem.org/mediawiki/parts/2/2b/Overview_.jpg"></center><br />
<br />
<p class="title1">Chemotaxis<a href="https://2014.igem.org/Team:Toulouse/Project/Chemotaxis"; style="font-size: 13px; cursor: pointer; color: #888; margin-left: 10px;"> Show more</a></p><br />
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<p class="texte">First, the bacterium targets the pathogen with a chemotaxis module which recognizes the soluble molecules released by the fungus' cell wall (N-acetylglucosamine).</p><br />
<br />
<br />
<p class="title1">Binding<a href="https://2014.igem.org/Team:Toulouse/Project/binding"; style="font-size: 13px; cursor: pointer; color: #888; margin-left: 10px;"> Show more</a></p><br />
<br />
<p class="texte">Then, SubtiTree binds onto the pathogen using a chimeric protein anchored to the bacterium peptidoglycan and which can make a bridge between bacterial cell wall and fungal chitin, the main component of the pathogen's cell wall.</p><br />
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<p class="title1">Fungicides<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides"; style="font-size: 13px; cursor: pointer; color: #888; margin-left: 10px;"> Show more</a></p><br />
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<p class="texte">In the third step, our designed bacterium fights the pathogen by production of a powerful treatment based on the release of three different fungicides.</p><br />
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<p class="title1">Spreading <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading"; style="font-size: 13px; cursor: pointer; color: #888; margin-left: 10px;">Show more</a></p><br />
<p class="texte">Our team worked on different aspects to control SubtiTree release in the environment. The aim is to prevent horizontal transfers between different bacteria and limit the growth and survival of the engineered bacterium inside the tree during one season.</p><br />
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<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
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color:#666; font-size:18px;">Chemotaxis</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
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<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>Dbbyhttp://2014.igem.org/Team:ToulouseTeam:Toulouse2014-10-16T14:49:19Z<p>Dbby: </p>
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<div class="text-presentation"><br />
<h1 class="title1" style="font-size:28px;"><br />
SubtiTree project<br />
</h1><br />
<p class="texte"><br />
Fungal diseases lead to major economic losses all over the world. Some pathogens trigger trachemycosis by disturbing the vascular system of the plant. Canker is one of these infections especially affecting plane trees (<i>Platanus sp.</i>). Plane trees are widely present in Southern France and in particular along the famous Canal du Midi, participating to the site gorgeousness. Today, the only treatment consists in a costly preventive tree-cutting and implies significant ecological troubles. Facing this emergency, our team proposes an innovative solution originated from synthetic biology. <br />
</p><br />
<a class="button-home" href="https://2014.igem.org/Team:Toulouse/Project/project-context" style="border: 1px solid #282828;-webkit-border-radius: 5px;-moz-border-radius: 5px;border-radius: 5px; padding: 13px 25px 12px; color: #282828; text-decoration: none; font-size: 17px; background: none; display: block; width: 89px;">Learn more</a><br />
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<div class="title-part-projet">Overview</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">Let's save our trees with SubtiTree!</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class="part-large-projet" style="background: url('https://static.igem.org/mediawiki/2014/a/aa/Chimio.jpg') no-repeat center;-webkit-background-size: cover;-moz-background-size: cover;-o-background-size: cover;background-size: cover;"><br />
<div class="title-part-projet" style="color:white; ">Chemotaxis</div><br />
<div style="position:absolute; top:90px; left:50px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">To target the pathogenic fungus</div><br />
<div style="border-bottom: 1px solid #white; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; left:50px; font-size:16px;"><u>Read more</u></div><br />
</a><br />
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<a href="https://2014.igem.org/Team:Toulouse/Project/binding" class="part-large-projet" style="background: url('https://static.igem.org/mediawiki/2014/0/0b/Billes3D.png') no-repeat center;-webkit-background-size: cover;-moz-background-size: cover;-o-background-size: cover;background-size: cover;"><br />
<div class="title-part-projet style="color:white; ">Binding</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">To be attached to the fungal pathogen wall</div><br />
<div style="border-bottom: 1px solid #515553; color:white; font-family:'Open Sans'; position:absolute; bottom:28px; right:28px; font-size:16px;">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="part-little-projet"><br />
<div class="title-part-projet">Modeling</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">To develop a predictive model</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:28px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
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<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="part-little-projet"><br />
<div class="title-part-projet">Spreading</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">How do we keep control on Subtitree?</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;"">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="part-large-projet" style="background:url('https://static.igem.org/mediawiki/2014/3/38/Fungi.jpg') no-repeat center;-webkit-background-size: cover;-moz-background-size: cover;-o-background-size: cover;background-size: cover;"><br />
<div class="title-part-projet" style="color:white;">Fungicides</div><br />
<div style="position:absolute; top:90px; left:50px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">To eradicate fungal diseases</div><br />
<div style="border-bottom: 1px solid white; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
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<h2 style="color:green; text-align:center; border:none; font-weight:bold; margin:30px 0px 50px 0px;">HUMAN PRACTICE</h2><br />
<br />
<div class="sticker-aside" style="margin-right:20px;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/Template-igem2014-HP-1.jpg" alt="image safety" /><br />
<h2>Safety</h2><br />
<p>Safety is not just a slogan, it is a way of life.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/Safety"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
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<img src="https://static.igem.org/mediawiki/2014/e/ea/Ethics.jpg" alt="" /><br />
<h2>Ethics</h2><br />
<p>Ethics are more important than laws.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/ethics"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
</div><br />
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<img src="https://static.igem.org/mediawiki/2014/2/20/Bacteria_talk.png" alt="" style="margin-top:26px;" /><br />
<h2>Communication</h2><br />
<p>Good words are worth much, and cost little.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/Communication"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
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<!-------------------------------- SPONSOR ---------------------------------><br />
<div class="Sponsor" style="margin:0 auto; width:960px; padding:60px 0 70px;"><p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d5/Toulouse_sponsors.png" alt="Bandeau Sponsor"></p><br />
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{{:Team:Toulouse/template/footer}}</div>Dbbyhttp://2014.igem.org/Team:Toulouse/Team/Fun_factsTeam:Toulouse/Team/Fun facts2014-10-16T14:38:51Z<p>Dbby: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Team&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fun Facts</p> <br />
<br />
</div> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div style="float:left; width:525px;"><br />
<img src="https://static.igem.org/mediawiki/2014/3/30/Kilometers.jpg" style="margin-top:5px; width:470px" /><br />
</div><br />
<br />
<p class="title2">Have you ever tried to estimate the kilometers travelled by your team during this summer?</p><br />
<p class="texte"><br />
According to our calculations, one member walks approximately 4 kilometers per day all around the <br />
laboratory. This represents 5544 kilometers for the whole team during the 126 days of this epic <br />
adventure. <br />
What does that mean exactly? Simply that we could walk, cycle, swim or whatever you want until <br />
Kazakhstan, Russia, Kenya… We could even have reached the USA but we decided to stay in the lab <br />
and take the plane to go to Boston!</p><br />
<br />
<br> </br><br />
<br />
<div style="float:right; width:500px;"><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Interview.jpg" style="margin-top:5px; margin-left: 62px; width:450px" /><br />
</div> <br />
<br />
<p class="title2">What do trees lining the “Canal du Midi” think about SubtiTree?</p><br />
<p class="texte"><br />
According to our homemade impartial survey, 94% of the questioned plane trees approve our project and are interested in <br />
serving as guinea pigs for our new bacterial medicine. This percentage represents 41 580 trees which <br />
are also gathered in the association called: “Happy tree friends“.</p><br />
<br />
<br />
<div style="float:left; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Multidisciplinary_yes_we_are.jpg" style="margin-top:5px; width:450px" /><br />
</div> <br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br> <br />
<p class="title2">Multidisciplinary…Yes we are!</p><br />
<p class="texte">Housework in our laboratory became necessary when most of people were in vacations except us. <br />
But do you know the novelty this year in our team? Times are changing because now men are <br />
cleaning! ;-)</p><br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br> <br> <br><br />
<br />
<div style="float:right; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/World_cup.jpg" style="margin-top:5px; width:450px; margin-left:62px;" /><br />
</div> <br />
<br> <br> <br> <br> <br> <br><br />
<p class="title2">The unmissable event this summer: The 2014 football World Cup !</p><br />
<p class="texte">From 06/12/14 to 07/13/14, the Toulouse iGEM Team was cheering on the French team. During the <br />
games of the French team, our group was juggling with wet lab and large screen projections! Despite <br />
our support, the French team did not win the World Cup. However, we have not said our last world <br />
yet: let’s see what will happen in 2018… ;-)</p><br />
<br />
<div class="clear"></div><br />
<br />
<div style="margin-top:50px; text-align:center;"><br />
<p class="title2">Have you ever forgetten a culture tube or a petri dish?</p><br />
<p class="texte" style="text-align:center;">Never? Let us show you what happens in that case!</p><br />
</div><br />
<center><br />
<table><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/f/fa/Old_tube1.png" width="160px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/1/1b/Old_tube2.JPG" width="400px"></td></tr><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/d/d4/Old_petri1.png" width="370px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/d/d7/Old_petri2.JPG" width="400px"></td></tr><br />
</table><br />
</center><br />
<br />
<br><br />
<br><br />
<br> <br />
<br />
<div style="text-align:center; width:760px; margin:0 auto; margin-top:15px; border-top:1px solid #555; padding-top:60px;"><br />
<p class="title2" style="padding-bottom:15px;">To finish this part, let’s do the official Awards Ceremony of the Toulouse iGEM Team 2014!</p><br />
<ul><br />
<li class="tree"><p class="texte">The Geek Award: The three nominees are Laureen, Manon, Florie. And the winner is… <b>Florie</b> <br />
who spent the longest time in front of her laptop for the modeling part!</p></li><br />
<li class="tree"><p class="texte">The latest survivor of weekly meetings: The four nominees are Fanny, Emeline, Diane, Pierre. <br />
And the winner is… <b>Diane</b> who stayed up until 3am because she was skyping from South <br />
Korea!</p></li><br />
<li class="tree"><p class="texte">The worst singer award: The two nominees are Abdel, Pierre. And the winner is… <b>Abdel</b> who <br />
spent the whole day singing badly in the lab!</p></li><br />
<li class="tree"><p class="texte">The dancer award: The three nominees are Florie, Camille, Diane. And the winner is… <b>Camille</b><br />
who did the famous Plasmid Dance!</p></li><br />
<li class="tree"><p class="texte">The “hello you” award: No nominees because the only winner is… <b>Pierre</b> who was saying <br />
“Hello you” each time he met someone!</p></li><br />
<li class="tree"><p class="texte">The most tired award: The three nominees are Laureen, Manon, Aurélie. And the winner is... <br />
<b>Manon</b> but we still do not know why!</p></li><br />
<li class="tree"><p class="texte">The misplaced ideas award: The four nominees are Laureen, Camille, Mathieu, Pierre. And <br />
the winner is… <b>Mathieu</b> but you do not want to know why!</p></li><br />
<li class="tree"><p class="texte">The perseverance award: The three nominees are Emeline, Diane, Abdel. And the winner is... <br />
<b>Emeline</b> who succeeded a cloning after twelve trials!</p></li><br />
<li class="tree"><p class="texte">The drawing award: The three nominees are Florie, Fanny, Manon. And the winner is… <b>Fanny</b> <br />
who drew our first SubtiTree logo!</p></li><br />
<li class="tree"><p class="texte">The phone-call award: The two nominees are Laureen, Pierre. And the winner is… <b>Laureen</b><br />
who was our lab secretary!</p></li><br />
<li class="tree"><p class="texte">The biggest blunder in the lab award: The three nominees are Aurélie, Fanny, Abdel. And the <br />
winner is… <b>Aurélie</b> who poured an agarose gel without gel tray!</p></li><br />
<li class="tree"><p class="texte">And last but not least ... The Best Nervous breakdown Award goes to ... <b>Our deep freezer</b>! The whole team is grateful for its hard work during a hot summer!</li></p><br />
</ul><br />
</div><br />
<br />
</div><br />
</div><br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
<br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Checking of the genetic constructions after plasmid integration in <i>Bacillus subtilis</i> </a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
</div><br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an Escherichia coli cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1 M CaCl2 and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl2 <br />
<br/><br />
- Centrifuge 10 minutes at 4 500 RPM<br />
<br/><br />
- Resuspend the pellet in 500µL of 0.1 M CaCl2<br />
<br/><br />
- Add glycerol for a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3µL of kit plate DNA <br />
<br/><br />
NB: for kit plate, resuspend the well in 10µL of sterile water<br />
- Put the tubes 20minutes in the ice<br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13 000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50µL on the second plate.<br />
<br/><br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B></p><br />
<p class="texte"><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C…)<br />
<br/><br />
- Resuspend one colony/culture tube in 5mL of LB medium with antibiotic<br />
<br/><br />
- Leave the culture shakes overnight at 37°C <br />
<p class="texte"><B> Day 1 </B></p><br />
<p class="texte"><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with a hot elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200µL of buffer 1 is added to resuspend the pellet, 400µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
60µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. <br />
<br> Buffer 1: Tris 10 mM pH 8 + EDTA 1mM<br />
<br> Buffer 2: NaOH 2mM + SDS 1%<br />
<br> Bufer 3: A COOK 3M + A COOH 15% <br />
</I><br />
<br />
<p class="title1" id="select4"> Cloning </p><br />
<p class="texte"><br />
<br>After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.<br />
<br><br />
<p class="texte"><B> First Step </B><br />
<br> <B> BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE </B><br />
<br> 1) Digestion mix<br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte">2) Gel extraction<br />
<br><br />
- Prepare a 1% of 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100V or 1hour at 50V.<br />
<br><br />
- The revelation is made in BET (10minutes) and then 5minutes in water.<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit.<br />
<br />
<p class="texte">3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> THE TWO PARTS HAVE A DIFFERENT ANTIBIOTIC RESISTANCE </B><br />
<br>1) Digestion mix <br />
<br>For each part, add : <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
2) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> Second step </B><br />
<br><B> Ligation </B><br />
<br/><br />
- Mix 10 µL of insert + 4µL of vector + 2µL of 10x T4 buffer + 0.5µL of T4 ligase + 3.5µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation<br />
<br/><br />
</p><br />
<br />
<p class="texte"><br />
2) Transformation<br />
<br/><br />
- Take 5µL of the ligation mix for 50µL of competent cells and use the Toulouse iGEM Team 2014 transformation protocol.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.<br />
<br/><br />
</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="texte"><br />
1) Colony PCR<br />
<br/><br />
- Add 0.5µL of plasmid + 25µL of DreamTAQ MasterMix + 2µL of each 10µM primer (VR and VF2) + H20 qsp 25µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C 5min<br />
<br/>- Then 4°C<br />
<br/><br />
<br />
<p class="texte"><br />
2) Analytic digestion<br />
<br/><br />
- Put a colony in 5mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2µL of plasmid + 2µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100V.<br />
<br/><br />
<br />
<p class="texte"><br />
<br/>3) Sequencing<br />
<br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.<br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<p class="texte"><br />
<br/>Strain: <I>Bacillus subtilis</I> 168. <br />
<br/>Plasmid : pSBBS4S given by the Munich University iGEM Team. l’équipe iGEM de l’université de Munich. This plasmid is replicative in <I>E. coli</I> and integrative in <I>Bacillus subtilis</I>.<br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the Bacillus strain and plate this on an LB agar plate overnight at 37°C<br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2 ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400 µl of culture in a fresh tube ( tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight <br />
<br/><br />
<br />
<p class="texte"><br />
<b> Preparation of solutions </b><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> 10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="texte"><br />
<I> <br> 1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="title1" id="select7">Checking of the genetic constructions after plasmid integration in <I>Bacillus subtilis</I></p><br />
<p class="texte"><br />
<br>Test of the pSBBS4S plasmid integration in Bacillus subtilis genome on the threonine site:<br />
<br>- Plate the transformed Bacillus strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + spectinomycine. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium without threonine but can not grow on the other media.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="title2">Chemotaxis test</p><br />
<p class="texte"><br />
Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the « Capillary essay » from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium so they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b1/Installation_1.gif"></center><br />
<p class="texte"><br />
<br>- Put 200µl of the different chemoattractants in the wells of the ELISA plate and pipette 15µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). <br />
NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on Ceratocystis platani wall. The volume in the tips must be marked.<br />
<br>- Put the tips with chemoattractants in 300µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
<br />
<p class="title2">Binding test</p><br />
<p class="texte"><I>CBB (Chitin Binding Buffer):</I><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
</p><br />
<br />
<p class="texte"><br />
<I>Column activation:</I> <br />
<br>- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
</p><br />
<br />
<br />
<p class="texte"><I>Bacterial fixation on the chitin beads:</I><br />
<br>- Add 200 µL of bacteria solution (105 bactéria/mL)to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500 µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB to recover the beads directly<br />
</p><br />
<br />
<p class="texte"><I>Bacteria count:</I><br />
<br>- Make different dilutions : 10-1, 10-3, 10-5 of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10-2, 10-4 of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
</p><br />
<br />
<p class="title2">Fungicide test: anti-fungal activities</p><br />
<p class="texte"><br />
CAUTION : all the lab equipment must be desinfected before the manipulations with the fungi. <br />
<br>Three different funguss strains were used : <I>Aspergillus brasiliensis, Aspergillus nidulans, Trichoderma reesei</I><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water.<br />
<br>- a microscopy count can be performed thanks to Toma cell to determine the conidia concentration.<br />
<br>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.<br />
<br>- After 72hours of liquid culture of different clones of B. subtilis with the fungicides module, the culture can be centrifuged.<br />
<br>- 130µL of supernatant is used to soak a plug on a fungus plate. The bacterial pellet is resuspended in 130µL of LB medium and put on a tampon. <br />
<br>- The plates containing 10,000 conidia and the supernatant or bacteria soaked plugs are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wilt type strains or copper sulfate at 10 and 20mg/mL.<br />
</p><br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay</p><br />
<p class="texte"><br />
The first step involves doing the inoculation of SubtiTree in plants through stomata (opened in wet condition). We dilute our bacterial samples for two concentrations: 5.10^6 and 10^8 bacteria per mL. The bacterial solutions of WT, having the expression cassette of the D4E1 or D4E1-operon GAFP1 are introduced into plants (a control test without bacteria was performed). Thanks to a 1 ml syringe (without needle),we inject plant with bacteria by pressure. We use five leaves of each plant, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Compared for Agrobacterium species, bacterial growth in the plant is left for 24 h.<br />
</br><br />
<br />
The next step begins with preparation of the fungal samples. PDA cores containing the fungal culture is crushed and left in culture in the PDB . Then it pass through a filter of 100 µm (to remove large aggregates) and through a 40 µm filter to allow small molecules. The caught hyphae are placed in the PDB for 24 to 48 hours. This liquid culture is filtered to retain the living fragment and place in solution. Then, 2.5 of OD is adjusted. Obtained leaves above are detached from the plants using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf is deposited 5μL of fungal solution (using beveled tips because it is too viscous), as control we keep inoculated leaf without fungus. Pictures are achieved at different times. All the plants are destroyed by autoclaving.<br />
</p><br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
<br />
</ul><br />
</div><br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Checking of the genetic constructions after plasmid integration in <i>Bacillus subtilis</i> </a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
</div><br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an Escherichia coli cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1 M CaCl2 and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl2 <br />
<br/><br />
- Centrifuge 10 minutes at 4 500 RPM<br />
<br/><br />
- Resuspend the pellet in 500µL of 0.1 M CaCl2<br />
<br/><br />
- Add glycerol for a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3µL of kit plate DNA <br />
<br/><br />
NB: for kit plate, resuspend the well in 10µL of sterile water<br />
- Put the tubes 20minutes in the ice<br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13 000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50µL on the second plate.<br />
<br/><br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B></p><br />
<p class="texte"><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C…)<br />
<br/><br />
- Resuspend one colony/culture tube in 5mL of LB medium with antibiotic<br />
<br/><br />
- Leave the culture shakes overnight at 37°C <br />
<p class="texte"><B> Day 1 </B></p><br />
<p class="texte"><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with a hot elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200µL of buffer 1 is added to resuspend the pellet, 400µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
60µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. <br />
<br> Buffer 1: Tris 10 mM pH 8 + EDTA 1mM<br />
<br> Buffer 2: NaOH 2mM + SDS 1%<br />
<br> Bufer 3: A COOK 3M + A COOH 15% <br />
</I><br />
<br />
<p class="title1" id="select4"> Cloning </p><br />
<p class="texte"><br />
<br>After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.<br />
<br><br />
<p class="texte"><B> First Step </B><br />
<br> <B> BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE </B><br />
<br> 1) Digestion mix<br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte">2) Gel extraction<br />
<br><br />
- Prepare a 1% of 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100V or 1hour at 50V.<br />
<br><br />
- The revelation is made in BET (10minutes) and then 5minutes in water.<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit.<br />
<br />
<p class="texte">3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> THE TWO PARTS HAVE A DIFFERENT ANTIBIOTIC RESISTANCE </B><br />
<br>1) Digestion mix <br />
<br>For each part, add : <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
2) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> Second step </B><br />
<br><B> Ligation </B><br />
<br/><br />
- Mix 10 µL of insert + 4µL of vector + 2µL of 10x T4 buffer + 0.5µL of T4 ligase + 3.5µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation<br />
<br/><br />
</p><br />
<br />
<p class="texte"><br />
2) Transformation<br />
<br/><br />
- Take 5µL of the ligation mix for 50µL of competent cells and use the Toulouse iGEM Team 2014 transformation protocol.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.<br />
<br/><br />
</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="texte"><br />
1) Colony PCR<br />
<br/><br />
- Add 0.5µL of plasmid + 25µL of DreamTAQ MasterMix + 2µL of each 10µM primer (VR and VF2) + H20 qsp 25µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C 5min<br />
<br/>- Then 4°C<br />
<br/><br />
<br />
<p class="texte"><br />
2) Analytic digestion<br />
<br/><br />
- Put a colony in 5mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2µL of plasmid + 2µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100V.<br />
<br/><br />
<br />
<p class="texte"><br />
<br/>3) Sequencing<br />
<br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.<br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<p class="texte"><br />
<br/>Strain: <I>Bacillus subtilis</I> 168. <br />
<br/>Plasmid : pSBBS4S given by the Munich University iGEM Team. l’équipe iGEM de l’université de Munich. This plasmid is replicative in <I>E. coli</I> and integrative in <I>Bacillus subtilis</I>.<br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the Bacillus strain and plate this on an LB agar plate overnight at 37°C<br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2 ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400 µl of culture in a fresh tube ( tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight <br />
<br/><br />
<br />
<p class="texte"><br />
<b> Preparation of solutions </b><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> 10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="texte"><br />
<I> <br> 1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="title1" id="select7"> Checking of the genetic constructions after plasmid integration in <I>Bacillus subtilis</I> </p><br />
<p class="texte"><br />
<br>Test of the pSBBS4S plasmid integration in Bacillus subtilis genome on the threonine site:<br />
<br>- Plate the transformed Bacillus strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + spectinomycine. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium without threonine but can not grow on the other media.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="title2">Chemotaxis test</p><br />
<p class="texte"><br />
<br>Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the « Capillary essay » from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium so they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b1/Installation_1.gif"></center><br />
<p class="texte"><br />
<br>- Put 200µl of the different chemoattractants in the wells of the ELISA plate and pipette 15µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). <br />
NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on Ceratocystis platani wall. The volume in the tips must be marked.<br />
<br>- Put the tips with chemoattractants in 300µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
<br />
<p class="title2">Binding test</p><br />
<p class="texte"><I>CBB (Chitin Binding Buffer):</I><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
</p><br />
<br />
<p class="texte"><br />
<I>Column activation:</I> <br />
<br>- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
</p><br />
<br />
<br />
<p class="texte"><I>Bacterial fixation on the chitin beads:</I><br />
<br>- Add 200 µL of bacteria solution (105 bactéria/mL)to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500 µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB to recover the beads directly<br />
</p><br />
<br />
<p class="texte"><I>Bacteria count:</I><br />
<br>- Make different dilutions : 10-1, 10-3, 10-5 of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10-2, 10-4 of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
</p><br />
<br />
<p class="title2">Fungicide test: anti-fungal activities</p><br />
<p class="texte"><br />
CAUTION : all the lab equipment must be desinfected before the manipulations with the fungi. <br />
<br>Three different funguss strains were used : <I>Aspergillus brasiliensis, Aspergillus nidulans, Trichoderma reesei</I><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water.<br />
<br>- a microscopy count can be performed thanks to Toma cell to determine the conidia concentration.<br />
<br>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.<br />
<br>- After 72hours of liquid culture of different clones of B. subtilis with the fungicides module, the culture can be centrifuged.<br />
<br>- 130µL of supernatant is used to soak a plug on a fungus plate. The bacterial pellet is resuspended in 130µL of LB medium and put on a tampon. <br />
<br>- The plates containing 10,000 conidia and the supernatant or bacteria soaked plugs are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wilt type strains or copper sulfate at 10 and 20mg/mL.<br />
</p><br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay</p><br />
<p class="texte"><br />
The first step involves doing the inoculation of SubtiTree in plants through stomata (opened in wet condition). We dilute our bacterial samples for two concentrations: 5.10^6 and 10^8 bacteria per mL. The bacterial solutions of WT, having the expression cassette of the D4E1 or D4E1-operon GAFP1 are introduced into plants (a control test without bacteria was performed). Thanks to a 1 ml syringe (without needle),we inject plant with bacteria by pressure. We use five leaves of each plant, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Compared for Agrobacterium species, bacterial growth in the plant is left for 24 h.<br />
</br><br />
<br />
The next step begins with preparation of the fungal samples. PDA cores containing the fungal culture is crushed and left in culture in the PDB . Then it pass through a filter of 100 µm (to remove large aggregates) and through a 40 µm filter to allow small molecules. The caught hyphae are placed in the PDB for 24 to 48 hours. This liquid culture is filtered to retain the living fragment and place in solution. Then, 2.5 of OD is adjusted. Obtained leaves above are detached from the plants using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf is deposited 5μL of fungal solution (using beveled tips because it is too viscous), as control we keep inoculated leaf without fungus. Pictures are achieved at different times. All the plants are destroyed by autoclaving.<br />
</p><br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
<br />
</ul><br />
</div><br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Checking of the genetic constructions after plasmid integration in <i>Bacillus subtilis</i> </a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
</div><br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an Escherichia coli cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1 M CaCl2 and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl2 <br />
<br/><br />
- Centrifuge 10 minutes at 4 500 RPM<br />
<br/><br />
- Resuspend the pellet in 500µL of 0.1 M CaCl2<br />
<br/><br />
- Add glycerol for a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3µL of kit plate DNA <br />
<br/><br />
NB: for kit plate, resuspend the well in 10µL of sterile water<br />
- Put the tubes 20minutes in the ice<br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13 000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50µL on the second plate.<br />
<br/><br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B></p><br />
<p class="texte"><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C…)<br />
<br/><br />
- Resuspend one colony/culture tube in 5mL of LB medium with antibiotic<br />
<br/><br />
- Leave the culture shakes overnight at 37°C <br />
<p class="texte"><B> Day 1 </B></p><br />
<p class="texte"><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with a hot elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200µL of buffer 1 is added to resuspend the pellet, 400µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
60µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. <br />
<br> Buffer 1: Tris 10 mM pH 8 + EDTA 1mM<br />
<br> Buffer 2: NaOH 2mM + SDS 1%<br />
<br> Bufer 3: A COOK 3M + A COOH 15% <br />
</I><br />
<br />
<p class="title1" id="select4"> Cloning </p><br />
<p class="texte"><br />
<br>After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.<br />
<br><br />
<p class="texte"><B> First Step </B><br />
<br> <B> BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE </B><br />
<br> 1) Digestion mix<br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte">2) Gel extraction<br />
<br><br />
- Prepare a 1% of 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100V or 1hour at 50V.<br />
<br><br />
- The revelation is made in BET (10minutes) and then 5minutes in water.<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit.<br />
<br />
<p class="texte">3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> THE TWO PARTS HAVE A DIFFERENT ANTIBIOTIC RESISTANCE </B><br />
<br>1) Digestion mix <br />
<br>For each part, add : <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
2) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> Second step </B><br />
<br><B> Ligation </B><br />
<br/><br />
- Mix 10 µL of insert + 4µL of vector + 2µL of 10x T4 buffer + 0.5µL of T4 ligase + 3.5µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation<br />
<br/><br />
</p><br />
<br />
<p class="texte"><br />
2) Transformation<br />
<br/><br />
- Take 5µL of the ligation mix for 50µL of competent cells and use the Toulouse iGEM Team 2014 transformation protocol.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.<br />
<br/><br />
</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="texte"><br />
1) Colony PCR<br />
<br/><br />
- Add 0.5µL of plasmid + 25µL of DreamTAQ MasterMix + 2µL of each 10µM primer (VR and VF2) + H20 qsp 25µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C 5min<br />
<br/>- Then 4°C<br />
<br/><br />
<br />
<p class="texte"><br />
2) Analytic digestion<br />
<br/><br />
- Put a colony in 5mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2µL of plasmid + 2µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100V.<br />
<br/><br />
<br />
<p class="texte"><br />
<br/>3) Sequencing<br />
<br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.<br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<p class="texte"><br />
<br/>Strain: <I>Bacillus subtilis</I> 168. <br />
<br/>Plasmid : pSBBS4S given by the Munich University iGEM Team. l’équipe iGEM de l’université de Munich. This plasmid is replicative in <I>E. coli</I> and integrative in <I>Bacillus subtilis</I>.<br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the Bacillus strain and plate this on an LB agar plate overnight at 37°C<br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2 ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400 µl of culture in a fresh tube ( tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight <br />
<br/><br />
<br />
<p class="texte"><br />
<b> Preparation of solutions </b><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> 10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="texte"><br />
<I> <br> 1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="title1" id="select7"> Checking of the genetic constructions after plasmid integration in <I>Bacillus subtilis</I> </p><br />
<p class="texte"><br />
<br>Test of the pSBBS4S plasmid integration in Bacillus subtilis genome on the threonine site:<br />
<br>- Plate the transformed Bacillus strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + spectinomycine. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium without threonine but can not grow on the other media.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="title2">Chemotaxis test</p><br />
<p class="texte"><br />
<br>Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the « Capillary essay » from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium so they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b1/Installation_1.gif"></center><br />
<p class="texte"><br />
<br>- Put 200µl of the different chemoattractants in the wells of the ELISA plate and pipette 15µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). <br />
NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on Ceratocystis platani wall. The volume in the tips must be marked.<br />
<br>- Put the tips with chemoattractants in 300µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
<br />
<p class="texte"><br />
<B> Binding test </B><br />
<p class="texte"><I><br>CBB (Chitin Binding Buffer):</I><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
<br />
<p class="texte"><br />
<I><br>Column activation:</I> <br />
<br>- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
<br />
<br />
<p class="texte"><I><br>Bacterial fixation on the chitin beads:</I><br />
<br>- Add 200 µL of bacteria solution (105 bactéria/mL)to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500 µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB to recover the beads directly<br />
<br />
<p class="texte"><I><br> Bacteria count:</I><br />
<br>- Make different dilutions : 10-1, 10-3, 10-5 of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10-2, 10-4 of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
<br />
<p class="title2">Fungicide test: anti-fungal activities</p><br />
<p class="texte"><br />
CAUTION : all the lab equipment must be desinfected before the manipulations with the fungi. <br />
<br>Three different funguss strains were used : <I>Aspergillus brasiliensis, Aspergillus nidulans, Trichoderma reesei</I><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water.<br />
<br>- a microscopy count can be performed thanks to Toma cell to determine the conidia concentration.<br />
<br>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.<br />
<br>- After 72hours of liquid culture of different clones of B. subtilis with the fungicides module, the culture can be centrifuged.<br />
<br>- 130µL of supernatant is used to soak a plug on a fungus plate. The bacterial pellet is resuspended in 130µL of LB medium and put on a tampon. <br />
<br>- The plates containing 10,000 conidia and the supernatant or bacteria soaked plugs are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wilt type strains or copper sulfate at 10 and 20mg/mL.<br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay</p><br />
<p class="texte"><br />
The first step involves doing the inoculation of SubtiTree in plants through stomata (opened in wet condition). We dilute our bacterial samples for two concentrations: 5.10^6 and 10^8 bacteria per mL. The bacterial solutions of WT, having the expression cassette of the D4E1 or D4E1-operon GAFP1 are introduced into plants (a control test without bacteria was performed). Thanks to a 1 ml syringe (without needle),we inject plant with bacteria by pressure. We use five leaves of each plant, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Compared for Agrobacterium species, bacterial growth in the plant is left for 24 h.</br><br />
<br />
<br>The next step begins with preparation of the fungal samples.PDA cores containing the fungal culture is crushed and left in culture in the PDB . Then it pass through a filter of 100 µm (to remove large aggregates) and through a 40 µm filter to allow small molecules. The caught hyphae are placed in the PDB for 24 to 48 hours. This liquid culture is filtered to retain the living fragment and place in solution. Then, 2.5 of OD is adjusted. Obtained leaves above are detached from the plants using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf is deposited 5μL of fungal solution (using beveled tips because it is too viscous), as control we keep inoculated leaf without fungus. Pictures are achieved at different times. All the plants are destroyed by autoclaving.<br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
<br />
</ul><br />
</div><br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Checking of the genetic constructions after plasmid integration in <i>Bacillus subtilis</i> </a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
</div><br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an Escherichia coli cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1 M CaCl2 and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl2 <br />
<br/><br />
- Centrifuge 10 minutes at 4 500 RPM<br />
<br/><br />
- Resuspend the pellet in 500µL of 0.1 M CaCl2<br />
<br/><br />
- Add glycerol for a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3µL of kit plate DNA <br />
<br/><br />
NB: for kit plate, resuspend the well in 10µL of sterile water<br />
- Put the tubes 20minutes in the ice<br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13 000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50µL on the second plate.<br />
<br/><br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B></p><br />
<p class="texte"><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C…)<br />
<br/><br />
- Resuspend one colony/culture tube in 5mL of LB medium with antibiotic<br />
<br/><br />
- Leave the culture shakes overnight at 37°C <br />
<p class="texte"><B> Day 1 </B></p><br />
<p class="texte"><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with a hot elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200µL of buffer 1 is added to resuspend the pellet, 400µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
60µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. <br />
<br> Buffer 1: Tris 10 mM pH 8 + EDTA 1mM<br />
<br> Buffer 2: NaOH 2mM + SDS 1%<br />
<br> Bufer 3: A COOK 3M + A COOH 15% <br />
</I><br />
<br />
<p class="title1" id="select4"> Cloning </p><br />
<p class="texte"><br />
<br>After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.<br />
<br><br />
<p class="texte"><B> First Step </B><br />
<br> <B> BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE </B><br />
<br> 1) Digestion mix<br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte">2) Gel extraction<br />
<br><br />
- Prepare a 1% of 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100V or 1hour at 50V.<br />
<br><br />
- The revelation is made in BET (10minutes) and then 5minutes in water.<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit.<br />
<br />
<p class="texte">3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> THE TWO PARTS HAVE A DIFFERENT ANTIBIOTIC RESISTANCE </B><br />
<br>1) Digestion mix <br />
<br>For each part, add : <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
2) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> Second step </B><br />
<br><B> Ligation </B><br />
<br/><br />
- Mix 10 µL of insert + 4µL of vector + 2µL of 10x T4 buffer + 0.5µL of T4 ligase + 3.5µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation<br />
<br/><br />
</p><br />
<br />
<p class="texte"><br />
2) Transformation<br />
<br/><br />
- Take 5µL of the ligation mix for 50µL of competent cells and use the Toulouse iGEM Team 2014 transformation protocol.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.<br />
<br/><br />
</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="texte"><br />
1) Colony PCR<br />
<br/><br />
- Add 0.5µL of plasmid + 25µL of DreamTAQ MasterMix + 2µL of each 10µM primer (VR and VF2) + H20 qsp 25µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C 5min<br />
<br/>- Then 4°C<br />
<br/><br />
<br />
<p class="texte"><br />
2) Analytic digestion<br />
<br/><br />
- Put a colony in 5mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2µL of plasmid + 2µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100V.<br />
<br/><br />
<br />
<p class="texte"><br />
<br/>3) Sequencing<br />
<br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.<br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<p class="texte"><br />
<br/>Strain: <I>Bacillus subtilis</I> 168. <br />
<br/>Plasmid : pSBBS4S given by the Munich University iGEM Team. l’équipe iGEM de l’université de Munich. This plasmid is replicative in <I>E. coli</I> and integrative in <I>Bacillus subtilis</I>.<br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the Bacillus strain and plate this on an LB agar plate overnight at 37°C<br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2 ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400 µl of culture in a fresh tube ( tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight <br />
<br/><br />
<br />
<p class="texte"><br />
<b> Preparation of solutions </b><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> 10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="texte"><br />
<I> <br> 1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="title1" id="select7"> Checking of the genetic constructions after plasmid integration in <I>Bacillus subtilis</I> </p><br />
<p class="texte"><br />
<br>Test of the pSBBS4S plasmid integration in Bacillus subtilis genome on the threonine site:<br />
<br>- Plate the transformed Bacillus strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + spectinomycine. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium without threonine but can not grow on the other media.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="texte"><br />
<B> Chemotaxis test </B><br />
<br>Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the « Capillary essay » from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium so they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b1/Installation_1.gif"></center><br />
<p class="texte"><br />
<br>- Put 200µl of the different chemoattractants in the wells of the ELISA plate and pipette 15µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). <br />
NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on Ceratocystis platani wall. The volume in the tips must be marked.<br />
<br>- Put the tips with chemoattractants in 300µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
<br />
<p class="texte"><br />
<B> Binding test </B><br />
<p class="texte"><I><br>CBB (Chitin Binding Buffer):</I><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
<br />
<p class="texte"><br />
<I><br>Column activation:</I> <br />
<br>- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
<br />
<br />
<p class="texte"><I><br>Bacterial fixation on the chitin beads:</I><br />
<br>- Add 200 µL of bacteria solution (105 bactéria/mL)to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500 µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB to recover the beads directly<br />
<br />
<p class="texte"><I><br> Bacteria count:</I><br />
<br>- Make different dilutions : 10-1, 10-3, 10-5 of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10-2, 10-4 of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
<br />
<p class="texte"><br />
<B> Fungicide test: anti-fungal activities </B><br />
<br>CAUTION : all the lab equipment must be desinfected before the manipulations with the fungi. <br />
<br>Three different funguss strains were used : <I>Aspergillus brasiliensis, Aspergillus nidulans, Trichoderma reesei</I><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water.<br />
<br>- a microscopy count can be performed thanks to Toma cell to determine the conidia concentration.<br />
<br>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.<br />
<br>- After 72hours of liquid culture of different clones of B. subtilis with the fungicides module, the culture can be centrifuged.<br />
<br>- 130µL of supernatant is used to soak a plug on a fungus plate. The bacterial pellet is resuspended in 130µL of LB medium and put on a tampon. <br />
<br>- The plates containing 10,000 conidia and the supernatant or bacteria soaked plugs are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wilt type strains or copper sulfate at 10 and 20mg/mL.<br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay </p><br />
<p class="texte"><br />
The first step involves doing the inoculation of SubtiTree in plants through stomata (opened in wet condition). We dilute our bacterial samples for two concentrations: 5.10^6 and 10^8 bacteria per mL. The bacterial solutions of WT, having the expression cassette of the D4E1 or D4E1-operon GAFP1 are introduced into plants (a control test without bacteria was performed). Thanks to a 1 ml syringe (without needle),we inject plant with bacteria by pressure. We use five leaves of each plant, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Compared for Agrobacterium species, bacterial growth in the plant is left for 24 h.</br><br />
<br />
<br>The next step begins with preparation of the fungal samples.PDA cores containing the fungal culture is crushed and left in culture in the PDB . Then it pass through a filter of 100 µm (to remove large aggregates) and through a 40 µm filter to allow small molecules. The caught hyphae are placed in the PDB for 24 to 48 hours. This liquid culture is filtered to retain the living fragment and place in solution. Then, 2.5 of OD is adjusted. Obtained leaves above are detached from the plants using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf is deposited 5μL of fungal solution (using beveled tips because it is too viscous), as control we keep inoculated leaf without fungus. Pictures are achieved at different times. All the plants are destroyed by autoclaving.<br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Project monitoring</p> <br />
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<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a> | <a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a></p><br />
<br />
<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">1. Amplification of Binding Module (pEX-K4) into E.coli</p><br />
<p class="title3">Transformation of Binding module (pEX-K4) into E. coli</p><br />
<p class="texte">Gene "Binding module" synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br><b>Result:</b> We obtained distinct colonies on plates LB + Kanamycin (50 µg/mL)</p><br />
<br />
<p class="title3">Liquid culture of 2 clones : 1 et 2 (pEX-K4) transformed into E. coli</p><br />
<p class="texte"><b>Date:</b> 01/08/2014</p><br />
<br />
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit: 2 clones of Binding Module (pEX-K4) into E. coli</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br><b>Result:</b> Binding Module (pEX-K4) obtained</p><br />
<br />
<p class="title3">Digestion of Binding Module (pEX-K4) with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br><b>Result:</b> <br />
<br>- 3 bands : 1500bp (vector with Kanamycin resistance), 1300bp (Module Binding) and 1000p bp (vector with pUC Ori)<br />
<br>- The two Binding Module clones are ok</p><br />
<br />
<br />
<p class="title2">2. Cloning Binding Module on pEX-K4 with pSB1C3 (BBa_K606013 without RFP) into E. coli</p><br />
<p class="title3">Digestion Binding Module on pEX-K4 and BBa_K606013</p><br />
<p class="texte"><b>Date:</b> 23/08/2014<br />
<br><b>Expected bands after digestion for:</b><br />
<br>- BBa_K606013 : 860 bp for RFP and 2100 bp for vector pSB1C3<br />
<br>- Binding Module : 1400 bp for Binding Module and 1500bp+1000bp for vector pEX_K4<br />
<br>We decide to do a ligation between pSB1C3 and Binding Module without gel cutting and purification but after a PCR clean-up (Thermo scientific, GeneJET PCR purifiction kit #K0701) to remove the enzymes.</p><br />
<br />
<p class="title3">Ligation Binding Module on pSB1C3</p><br />
<p class="texte"><b>Date:</b> 04/08/2014</p><br />
<br />
<p class="title3">Transformation of Binding Module on pSB1C3 into E. coli</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>Transformation with 10 µL of the ligation mix and plate on Chloremphenicol LA plate<br />
<br>Result : many wrong clones</p><br />
<br />
<p class="title2">3. Cloning Binding Module on pEX-K4 with pVeg on pSB1C3 (BBa_K823003) into E. coli</p><br />
<p class="title3">Digestion Binding Module on pEX-K4 and BBa_K823003</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>BBa_K823003 digested by XbaI and PstI and Binding Module digested by SpeIand PstI<br />
<br>Gel Electrophoresis<br />
<br><b>Result:</b><br />
<br>- Binding Module : 1400 bp for Binding Module and 1500bp+1000bp for vector pEX_K47<br />
<br>We decide to do a ligation between pVeg and Binding Module without gel cutting and purification but after a PCR clean-up (Thermo scientific, GeneJET PCR purifiction kit #K0701) to remove the enzymes.</p><br />
<br />
<p class="title3">Ligation Binding Module on pVeg with pSB1C3</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>BBa_K823003 digested by XbaI and PstI and Binding Module digested by SpeIand PstI and ligation</p><br />
<br />
<p class="title3">Transformation of Binding Module on pVeg into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br><b>Result:</b> Many good clones (check on 06/08/2014)</p><br />
<br />
<p class="title2">4. Cloning Binding Module with Pveg (BBa-K1364005) on pSBBS4S (BBa-K823022) into <i>E. coli</i></p><br />
<br />
<p class="title3">Digestion Binding Module with Pveg (BBa-K1364005) and pSBBS4S (BBa-K823022)</p><br />
<p class="texte"><b>Date:</b> 07/08/2014<br />
<br>BBa_K823022 and Binding Module with Pveg (BBa_K1364005) digested by EcoRIand PstIGel Electrophoresis<br />
<br><b>Result:</b><br />
<br>- Binding Module with Pveg 1600 bp for Binding Module and 2100bp for vector pSB1C3</p><br />
<br />
<p class="title3">Ligation Binding Module with Pveg on pSBbs4S</p><br />
<p class="texte"><b>Date:</b> 07/08/2014<br />
<br>BBa_K823022 and Binding Module with Pveg (BBa_K1364005) digested by EcoRIand PstI<br />
<br>Ligation<br />
<br><b>Result:</b> Ligation between Binding Module with Pveg on pSBBS4S</p><br />
<br />
<p class="title3">Transformation of Binding Module with Pveg on pSBBS4S into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>Binding Module with Pveg on pSBbs4S<br />
<br>Plate on Ampicillin LA plate<br />
<br><b>Result:</b> Many good clones (check on 13/08/2014)</p><br />
<br />
<p class="title3">Transformation of Binding Module with Pveg on pSBBS4S into <i>B. subtilis</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>After 5 hours of incubation and the linearization of 6µl of DNA with 1µl of ScaI, we make the transformation. Plate on Spectinomycin LA plate.<br />
<br>Result : One good clone : PCR (check on 09/09/2014) and threonine test (check on 09/09/2014)</p><br />
<br />
<p class="title2">5. Binding Test</p><br />
<p class="texte">See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results#select2">here</a></p><br />
<br />
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<br />
<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">1. Transformation of chemotaxis (Puc-57) into <i>E. coli</i></p><br />
<p class="texte">Gene chemotaxis synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br><b>Date:</b> 01/08/2014<br />
<br><b>Result:</b> We obtained distinct colonies on LA + Ampicillin (100 µg/mL) plates</p><br />
<br />
<p class="title3">Culture of 4 clones: A, B, C, D of chemotaxis (Puc57) transformed into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014</p><br />
<br />
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit Using a Microcentrifuge: 4 clones of chemotaxis (Puc57) into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 05/08/2014<br />
<br><b>Result:</b> 4*50µL of chemotaxis (Puc57) obtained</p><br />
<br />
<p class="title2">2. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested pSB1C3 with EcoRI and PstI into <i>E. coli</i></p><br />
<br />
<p class="title3">Digestion BBa_K1364000 (chemotaxis_Puc57) with EcoRI and PstI - PCR kit Clean up</p><br />
<p class="texte"><b>Date:</b> 07/08/2014<br />
<br><b>Result:</b> Expected band after digestion for BBa_K1364000: 2300 bp<br />
<br><b>Problem:</b> We can't distinguish the vector band (2500 bp)</p><br />
<br />
<p class="title3">Gel extraction of BBa_K1364000</p><br />
<p class="texte"><b>Date:</b> 07/08/2014</p><br />
<br />
<p class="title3">Ligation BBa_K1364000 and digested PsB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 08/08/2014</p><br />
<br />
<p class="title3">Transformation BBa_K1364000 in E.coli</p><br />
<p class="texte"><b>Date:</b> 08/08/2014<br />
<br><b>Result:</b> We obtained distinct colonies on LA + Cm (15 µg/mL) plates and resuspended 15 colonies in LB+Cm (15 µg/mL)</p><br />
<br />
<p class="title3">Test of sensibility on Ampicillin</p><br />
<p class="texte"><b>Date:</b> 10/08/2014<br />
<br><b>Result:</b> we can determine which colonies are sensible for ampicillin and know which bacterium is carrying the chemotaxis gene.</p><br />
<br />
<p class="title3">PCR</p><br />
<p class="texte"><b>Date:</b> 11/08/2014</p><br />
<br />
<p class="title3">Digestion BBa_K1364000 on pSB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 11/08/2014<br />
<br><b>Result:</b> There is one colony which presents the right construction.</p><br />
<br />
<br />
<br />
<p class="title2">3. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested BBa_823003 (Pveg) on pSB1C3 with SpeI and PstI into <i>E. coli</i></p><br />
<p class="title3">Ligation BBa_K1364000 and digested BBa_823003 on PsB1C3 with SpeI and PstI</p><br />
<p class="texte"><b>Date</b>: 08/08/2014</p><br />
<p class="title3">Transformation BBa_1364004 in E.coli</p><br />
<p class="texte"><b>Date:</b> 8/08/2014</p><br />
<p class="title3">Test of sensibility on Ampicillin</p><br />
<p class="texte"><b>Date:</b> 10/08/2014<br />
<br><b>Result:</b> We can determine which colony is sensible for ampicillin and know which bacterium is carrying the chemotaxis gene. There is one colony which resists on ampicillin.</p><br />
<br />
<p class="title3">Digestion BBa_K1364004 on pSBC3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 12/08/2014<br />
<br><b>Result:</b> We did not see any colony with chemotaxis insert.</p><br />
<br />
<p class="title2">4. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested BBa_823003 (Pveg) on pSB1C3 with SpeI and PstI into <i>E. coli</i></p><br />
<p class="title3">Ligation BBa_K1364000 and digested BBa_823003 on PsB1C3 with SpeI and PstI</p><br />
<p class="texte"><b>Date:</b> 11/08/2014</p><br />
<p class="title3">Transformation BBa_1364004 in E.coli</p><br />
<p class="texte"><b>Date:</b> 11/08/2014</p><br />
<p class="title3">Test of sensibility on Ampicillin</p><br />
<p class="texte"><b>Date:</b> 14/08/2014<br />
<br><b>Result:</b> we obtained 4 colonies sensible at Ampicilline</p><br />
<br />
<p class="title3">Digestion BBa_K1364004 on PsB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 18/08/2014</p><br />
<p class="title3">Gel extraction of BBa_K1364000</p><br />
<p class="texte"><b>Date:</b> 19/08/2014</p><br />
<br />
<p class="title2">5. Cloning chemotaxis BBa_K1364004 with digested pSBBS4S with EcorI and PstI into <i>E. coli</i></p><br />
<p class="title3">Ligation BBa_K1364004 digested EcorI and PstI and digested PsB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 19/08/2014</p><br />
<p class="title3">Transformation BBa_1364004 in pSBBS4S in E.coli</p><br />
<p class="texte"><b>Date:</b> 19/08/2014 <br />
<br><b>Result:</b> We obtained one colony and resuspended it in LB+ Amp</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 1); return false">Collapse</a></p><br />
</div><br />
<br />
<br />
<div class="technology">Fungicides</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">D4E1</p><br />
<br />
<p class="title2">1. Amplification of synthetic gene (D4E1 on pEX-A2)</p><br />
<p class="title3">Transformation of D4E1 (pEX-A2) into <i>E. coli</i></p><br />
<p class="texte">Gene D4E1 synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br>Concentration of D4E1: 115ng/µL<br />
<br><b>Date:</b> 07/21//2014<br />
<br><b>Result:</b> We obtained distinct colonies on LA + Ampicillin (100 µg/mL)</p><br />
<br />
<p class="title3">Culture of 4 clones: A, B, C, D of D4E1 (pEX-A2) transformed into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/22/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of D4E1 (pEX-A2) into E. coli</p><br />
<p class="texte">Buffer EB at 50-55°C<br />
<br><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up</p><br />
<p class="texte"><b>Date:</b> 07/23/2014<br />
<br><b>Result:</b> 4*20µL D4E1 digested with EcoRI and PstI all the clones seem to have the right D4E1 gene.</p><br />
<br />
<p class="title2">2. Cloning D4E1 in pSB1C3</p><br />
<p class="title3">Digestion of D4E1 on pEX-A2 and pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<p class="title3">Ligation of D4E1 in pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<p class="title3">Transformation in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of D4E1 (pSB1C3) transformed into <i>E. coli</i></p><br />
<p class="texte">Processing details: 5mL of LB + chloramphenicol (15µg/mL) , using sterile plastic loop to culture colonies<br />
<br><b>Date:</b> 07/24/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<p class="title3">Digestion of D4E1 (pSB1C3) A, B, C, D, E, F with EcoRI and PstI - PCR kit Clean up + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014<br />
<br><b>Result:</b> clones C, D have the expected construction</p><br />
<p class="title3">PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014<br />
<br><b>Result:</b> clones C, D have the expected construction, and placed in cryopreservation.</p><br />
<br />
<p class="title2">3. Cloning Pveg+D4E1 on Pveg plasmid (pSB1C3)</p><br />
<br />
<p class="title3">Digestion of D4E1 on pEX-A2 and K823003 (Pveg on pSB1C3)</p><br />
<p class="texte"><b>Dated:</b></b> 07/24/2014<br />
<br><b>Result:</b> 20µL digestion of D4E1 on pEX-A2 and of K823003</p><br />
<br />
<p class="title3">Ligation of D4E1 in K823003 (Pveg on pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/24/2014<br />
<br><b>Result:</b> 20µL ligation of D4E1 in K823003</p><br />
<br />
<p class="title3">Transformation of ligation products in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 07/24/2014<br />
<br><b>Result:</b> E.coli transformed by D4E1+K823003</p><br />
<br />
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of transformed <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/26/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Dated:</b> 07/28/2014<br />
<br><b>Result:</b> clones E, F seem to have the expected construction</p><br />
<p class="title3">Digestion of clones E, F of D4E1+K823003 (Pveg on pSB1C3) with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 28/07/2014<br />
<br><b>Result:</b> clones C, D have the expected construction and are placed in cryopreservation.</p><br />
<br />
<p class="title2">4. Cloning Pveg+D4E1 on pSBBS4S (K823022) </p><br />
<p class="texte"><b>Date:</b> 08/13/2014</p><br />
<br />
<p class="title2">5. Cloning Pveg + D4E1 on pSBBS1C lacZ (23)</p><br />
<br />
<p class="title2">GAFP1</p><br />
<p class="title2">1. Amplification of synthetic gene (GAFP1 on pEX-A2)</p><br />
<br />
<p class="title3">Transformation of GAFP1 (pEX-A2) into E.coli</p><br />
<p class="texte">Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br>Concentration of GAFP1 : 145ng/µL<br />
<br><b>Date:</b> 07/21/2014<br />
<br><b>Result:</b> We obtained distinct colonies on plates LB + Ampicillin (100 µg/mL)</p><br />
<br />
<p class="title3">Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/22/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel Electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/23/2014 </p><br />
<br />
<p class="title2">2. Cloning GAFP1 gene on PSB1C3 = K1364002 in E. coli</p><br />
<br />
<p class="title3">Digestion GAFP1_pEX-A2 and BBa_K606013 (RFP_pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/23/2014<br />
<br><b>Result:</b> BBa_K606013 : 860 bp<br />
<br>We decide to conserve the miniprep B for BBa_K606013</p><br />
<br />
<p class="title3">Ligation GAFP1 and BBa_K606013</p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Tranformation of ligation products into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Culture of x clones of GAFP1+K606013 in <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/24/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge: 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<br />
<p class="title3">PCR on 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014<br />
<br><b>Result:</b> clones A, C, D, F seem to have the right construction</p><br />
<br />
<p class="title3">Digestion of 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) by EcoR1 and Pst1 + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<br />
<br />
<br />
<p class="title2">3. Cloning GAFP1+terminator(B0015) = K1364007 (pSB1C3)</p><br />
<br />
<p class="title3">Digestion of GAFP1 on pEX-A2 and B0015 (terminator on pSB1C3)<br />
<p class="texte"><b>Date:</b> 07/24/2014<br />
<br />
<p class="title3">Ligation of GAFP1 in B0015 (terminator on pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/24/2014</p><br />
<br />
<p class="title3">Transformation of ligation products in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 07/24/2014</p><br />
<br />
<p class="title3">Culture of 6 clones: A, B, C, D, E, F transformed in <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/26/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">PCR of GAFP1+B0015 = K1364007 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/28/2014<br />
<br><b>Result:</b> clones B, C, D, E, F seem to have the expected construction.</p><br />
<br />
<p class="title3">Digestion of clones E, F of GAFP1+Ter B0015 = K1364007 with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/28/2014<br />
<br><b>Result:</b> clones B, C, D, E, F have the expected construction.</p><br />
<br />
<br />
<br />
<p class="title2">4. Cloning GAFP1+terminator with Pveg on pSB1C3 (K1364008) in <i>E. coli</i></p><br />
<br />
<p class="title3">Digestion of GAFP1+ter = K1364007 on pSB1C3 and K823003 (terminator on pSB1C3) + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/29/2014</p><br />
<br />
<p class="title3">Gel extraction of K1364007 (extraction of GAFP1+ter gene)</p><br />
<p class="texte"><b>Date:</b> 07/29/2014</p><br />
<br />
<p class="title3">Ligation of GAFP1+ter in K823003 (Pveg on pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/29/2014<br />
<br><b>Result:</b> 20µL ligation of GAFP1+ter in K823003</p><br />
<br />
<p class="title3">Transformation of ligation products in E.coli</p><br />
<p class="texte"><b>Date:</b> 07/29/2014</p><br />
<br />
<p class="title3">Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli</p><br />
<p class="texte"><b>Date:</b> 07/30/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/31/2014</p><br />
<br />
<p class="title3">PCR of GAFP1+B0015 + K823003 = K1364008 (pSB1C3) A, B, C, D, E, F, G, H + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 31/07/2014<br />
<br><b>Result:</b> clones A, B, C, D, E, G, H seem to have the expected construction</p><br />
<br />
<p class="title3">Digestion of clones A, B, C, D, E, G, H of Pveg+K1364007 = K1364008 with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 31/07/2014<br />
<br><b>Result:</b> clones A, B, C, D, E, G, H have the expected construction</p><br />
<br />
<br />
<br />
<p class="title2">5. Cloning Pveg+GAFP1+Ter B0015 (BBa_K1364008) on pSBBS4S (BBa_K823022)</p><br />
<br />
<p class="title3">Digestion of Pveg+GAFP1+ ter B0015 (K1364008) on pSB1C3 and PsBBs4S (K823022)</p><br />
<p class="texte"><b>Date:</b> 08/01/2014</p><br />
<br />
<p class="title3">Ligation of K134008 (Pveg+GAFP1+ter fragment) and K823022 (PsBBs4S)</p><br />
<p class="texte"><b>Date:</b> 08/01/2014</p><br />
<br />
<p class="title3">Transformation of ligation products in E.coli</p><br />
<p class="texte"><b>Date:</b> 08/01/2014</p><br />
<br />
<p class="title3">Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli</p><br />
<p class="texte"><b>Date:</b> 08/02/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 08/04/2014</p><br />
<br />
<p class="title3">Digestion of clones A, B, C, D, E, G, H of K1364008+K823022 with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 08/04/2014<br />
<br><b>Result:</b> clones A, E, F have the right construction</p><br />
<br />
<br />
<br />
<br />
<p class="title2">EcAMP</p><br />
<br />
<p class="texte">The EcAMP part was sent by the Utah iGEM team. It was on a pUC plasmid (ampicilin resistance) with biobrick suffix and prefix.</p><br />
<br />
<p class="title2">1. Transformation of EcAMP in Escherichia coli MC 1061</p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<br />
<p class="title2">2. Spreading of coli cells transformed with pUC + Utah </p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title2">3. Liquid culture + Miniprep + Test of the miniprep</p><br />
<p class="texte"><b>Date:</b> 07/30/2014</p><br />
<br />
<br />
<br />
<p class="title2">4. Cloning 1: EcAMP + Pveg + RBS</p><br />
<br />
<p class="title3">Digestion of EcAMP (INSERT) by XbaI and PstI</p><br />
<p class="texte"><b>Date:</b> 07/31/2014</p><br />
<br />
<p class="title3">Digestion of Pveg + RBS (VECTOR)</p><br />
<p class="texte"><b>Date:</b> 07/31/2014</p><br />
<br />
<p class="title3">Ligation and transformation</p><br />
<p class="texte"><b>Date:</b> 08/04/2014</p><br />
<br />
<p class="title3">PCR test</p><br />
<p class="texte"><b>Date:</b> 08/05/2014</p><br />
<br />
<p class="title3">Analytical digestion</p><br />
<p class="texte"><b>Date:</b> 08/05/2014<br />
<br />
<br>The first cloning show a lack of DNA in the Miniprep EcAMP. The bands are hardly visible on the gel for the linearization of EcAMP + Pveg + RBS. The problem came from the PCR cleanup step: the fragment size of EcAMP is lower than 200bp.</p><br />
<br />
<br />
<br />
<p class="title2">5. Cloning 2: EcAMP + Pveg + RBS </p><br />
<p class="texte"><b>Date:</b> 06/08/2014</p><br />
<p class="title3">Digestion of EcAMP (INSERT) by XbaI and PstI</p><br />
<p class="title3">Digestion of Pveg + RBS (VECTOR)</p><br />
<p class="title3">Heat inactivation of the enzymes</p><br />
<p class="title3">Ligation and transformation</p><br />
<p class="title3">PCR test</p><br />
<p class="texte"><b>Date:</b> 07/08/2014</p><br />
<br />
<br />
<p class="title3">Striation on a petri dish to purify the clone</p><br />
<p class="texte">Purpose: to isolate a clone with vector+insert<br />
<br><b>Date:</b> 08/072014</p><br />
<br />
<p class="title3">Miniprep of Pveg+SpoVG+EcAMP and analytic digestion</p><br />
<p class="texte"><b>Date:</b> 08/08/2014</p><br />
<br />
<p class="title3">Ligation of Pveg SpoVG EcAMP with double terminateur B0015 +Transformation and liquid culture</p><br />
<p class="texte"><b>Date:</b> 08/11/2014</p><br />
<br />
<p class="title3">Miniprep of Pveg SpoVG EcAMP + analytic digestion</p><br />
<p class="texte"><b>Date:</b> 08/13/2014</p><br />
<br />
<p class="title3">Cloning K1364011 (EcAMP+Pveg +SpoVG + B0015) + K823022 (psBbs4S) : Digestion, ligation, transformation</p><br />
<p class="texte"><b>Date:</b> 08/19/2014</p><br />
<br />
<p class="title3">Cloning K1364011 + K823023 (pSBBS1C) : Digestion, ligation, transformation</p><br />
<p class="texte"><b>Date:</b> 08/18/2014</p><br />
<br />
<p class="title3">Verification of the insertion of K1364011 + K823022 (pSBBS4S) into the subtilis genome by threonine test </p><br />
<p class="texte"><b>Date:</b> 08/21/2014<br />
<br />
<br><br />
<br>Assembling the fungicides module : </p><br />
<br />
<br />
<br />
<p class="title2">D4E1 + GAFP1 </p><br />
<p class="title2">1. Cloning GAFP1+D4E1 on pSB1C3: BBa_K1364012 </p><br />
<br />
<p class="title3">Digestion of D4E1 on pEX-A2 and GAFP1 on pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/28/2014<br />
<br><b>Result:</b> We obtained 40µL digestion of D4E1 on pEX-A2 and of GAFP1 on pSB1C3.</p><br />
<br />
<p class="title3">Ligation of digestions of D4E1 on pEX-A2 and of GAFP1 on pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">Transformation of ligation of GAFP1 and D4E1 on pSB1C3 into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">PCR of GAFP1+D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/29/2014<br />
<br><b>Result:</b> clones A, C, F, G seem to have the expected construction.</p><br />
<br />
<p class="title3">Digestion of GAFP1+D4E1 (pSB1C3) A, C, F, G + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/30/2014<br />
<br><b>Result:</b> clone F (BBa_K1364012) has the expected construction and is placed on cryopreservation.</p><br />
<br />
<br />
<br />
<p class="title2">2. Cloning GAFP1+D4E1 (BBa_K1364012) on Pveg plasmid (BBa_K823003): BBa_K1364013</p><br />
<p class="title2">3. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS4S (K823022)</p><br />
<p class="title2">4. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS1C lacZ (K823023)</p><br />
<p class="title2">5. Fungicides tests</p><br />
<br />
<br />
<p class="title2">Cloning D4E1-GAFP1-EcAMP: BBa_K1364014</p><br />
<br />
<p class="title2">1. Construction of BBa_K1364014 in PsB1C3, in E. coli</p><br />
<br />
<p class="title3">Digestion of K1364010 and K1364012</p><br />
<p class="texte">Digestion of K13664010 by Spe1 and Pst1, and digestion of K1364012 by XbaI and Pst1.<br />
<br><b>Date:</b> 08/11/2014<br />
<br><b>Result:</b> We obtained digested fragments of K1364012 and ~500 bp fragment of K1364010</p><br />
<br />
<p class="title3">Ligation of ~500 bp fragment from K1364010 and K1364012</p><br />
<p class="texte"><b>Date:</b> 08/11/2014<br />
<br><b>Result:</b> We obtained ligation of K1364012 and ~500 bp fragment of K1364010</p><br />
<br />
<p class="title3">Transformation of ligation of ~500 bp fragment from K1364010 and K1364012 = K1364014 in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 08/12/2014</p><br />
<br />
<br />
<p class="title3">Testing 8 E.coli+K1364014 clones</p><br />
<p class="texte"><b>Date:</b> 08/14/2014 </p><br />
<br />
<p class="title3">Testing 15 E.coli+K1364014 clones</p><br />
<p class="texte"><b>Date:</b> 08/18/2014<br />
<br><b>Result:</b> clones H, J, O, Q, U, V might have the right K1364014 construction</p><br />
<br />
<br />
<p class="title2">2. BBa_K1364014 in PsBs4s (K823022) and PsBs1C (K823023)</p><br />
<p class="title3">Digestion of K1364014, K823022 and K823023</p><br />
<p class="texte"><b>Date:</b> 08/22/2014<br />
<br><b>Result</b>: digestion of K13664014, K823022 and K823023 by EcoR1 and Pst1 were well performed.</p><br />
<br />
<p class="title3">Ligation of K1364014 with K823022 and K1364014 with K823023</p><br />
<p class="texte"><b>Date:</b> 08/22/2014<br />
<br><b>Result:</b> We obtained 20µL ligation of K1364014 with K823022 and of K1364014 with K823023.<br />
</p><br />
<p class="title3">Transformation of ligations in E.coli</p><br />
<p class="texte">E. coli transformed by K1364014+K823022 spread on LB+Amp 100µg/mL agar plate</p><br />
<p class="texte">E. coli transformed by K1364014+K823023 spread on LB+Amp 100µg/mL agar plate<br />
<br><b>Date:</b> 08/25/2014</p><br />
<br />
<p class="title3">Testing E.coli+K1364014+K823022 and E.coli+K1364014+K823023 clones</p><br />
<p class="texte"><b>Date:</b> 08/27/2014</p><br />
<br />
<br />
<br />
<p class="title2">3. Transformation of K1364014+K823022 and K1364014+K823023 in <i>B. subtilis</i></p><br />
<p class="texte"><i>B. subtilis</i> transformed by 10µL K1364014+K823022 spread on LB+Spec 75µg/mL agar plate<br />
<br><i>B. subtilis</i> transformed by 10µL K1364014+K823023 spread on LB+Cm 15µg/mL agar plate<br />
<br><b>Date:</b> 08/28/2014</p><br />
<br />
<p class="title2">Fungicides tests</p><br />
<p class="title2">1. D4E1-GAFP1</p><br />
<p class="title3">Transformation in bacillus Pveg-D4E1-GAFP1 on pSBBS4S </p><br />
<p class="texte"><b>Date:</b> 08/12/2014</p> <br />
<p class="title3">Integration threonine test + fungicide test </p><br />
<p class="texte"><b>Date:</b> 08/13/2014 </p><br />
<br />
<p class="title2"2. D4E1</p><br />
<p class="title3"><br />
Cloning D4E1 into pSBBS1C + fungicide test</p><br />
<p class="texte"><b>Date:</b>08/15/2014</p><br />
<p class="title3"><br />
D4E1 on pSB1C3 + fungicide test</p><br />
<p class="texte"><b>Date:</b>08/19/2014</p><br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Project monitoring</p> <br />
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<br />
<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">1. Amplification of Binding Module (pEX-K4) into E.coli</p><br />
<p class="title3">Transformation of Binding module (pEX-K4) into E. coli</p><br />
<p class="texte">Gene "Binding module" synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br><b>Result:</b> We obtained distinct colonies on plates LB + Kanamycin (50 µg/mL)</p><br />
<br />
<p class="title3">Liquid culture of 2 clones : 1 et 2 (pEX-K4) transformed into E. coli</p><br />
<p class="texte"><b>Date:</b> 01/08/2014</p><br />
<br />
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit: 2 clones of Binding Module (pEX-K4) into E. coli</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br><b>Result:</b> Binding Module (pEX-K4) obtained</p><br />
<br />
<p class="title3">Digestion of Binding Module (pEX-K4) with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br><b>Result:</b> <br />
<br>- 3 bands : 1500bp (vector with Kanamycin resistance), 1300bp (Module Binding) and 1000p bp (vector with pUC Ori)<br />
<br>- The two Binding Module clones are ok</p><br />
<br />
<br />
<p class="title2">2. Cloning Binding Module on pEX-K4 with pSB1C3 (BBa_K606013 without RFP) into E. coli</p><br />
<p class="title3">Digestion Binding Module on pEX-K4 and BBa_K606013</p><br />
<p class="texte"><b>Date:</b> 23/08/2014<br />
<br><b>Expected bands after digestion for:</b><br />
<br>- BBa_K606013 : 860 bp for RFP and 2100 bp for vector pSB1C3<br />
<br>- Binding Module : 1400 bp for Binding Module and 1500bp+1000bp for vector pEX_K4<br />
<br>We decide to do a ligation between pSB1C3 and Binding Module without gel cutting and purification but after a PCR clean-up (Thermo scientific, GeneJET PCR purifiction kit #K0701) to remove the enzymes.</p><br />
<br />
<p class="title3">Ligation Binding Module on pSB1C3</p><br />
<p class="texte"><b>Date:</b> 04/08/2014</p><br />
<br />
<p class="title3">Transformation of Binding Module on pSB1C3 into E. coli</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>Transformation with 10 µL of the ligation mix and plate on Chloremphenicol LA plate<br />
<br>Result : many wrong clones</p><br />
<br />
<p class="title2">3. Cloning Binding Module on pEX-K4 with pVeg on pSB1C3 (BBa_K823003) into E. coli</p><br />
<p class="title3">Digestion Binding Module on pEX-K4 and BBa_K823003</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>BBa_K823003 digested by XbaI and PstI and Binding Module digested by SpeIand PstI<br />
<br>Gel Electrophoresis<br />
<br><b>Result:</b><br />
<br>- Binding Module : 1400 bp for Binding Module and 1500bp+1000bp for vector pEX_K47<br />
<br>We decide to do a ligation between pVeg and Binding Module without gel cutting and purification but after a PCR clean-up (Thermo scientific, GeneJET PCR purifiction kit #K0701) to remove the enzymes.</p><br />
<br />
<p class="title3">Ligation Binding Module on pVeg with pSB1C3</p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>BBa_K823003 digested by XbaI and PstI and Binding Module digested by SpeIand PstI and ligation</p><br />
<br />
<p class="title3">Transformation of Binding Module on pVeg into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br><b>Result:</b> Many good clones (check on 06/08/2014)</p><br />
<br />
<p class="title2">4. Cloning Binding Module with Pveg (BBa-K1364005) on pSBBS4S (BBa-K823022) into <i>E. coli</i></p><br />
<br />
<p class="title3">Digestion Binding Module with Pveg (BBa-K1364005) and pSBBS4S (BBa-K823022)</p><br />
<p class="texte"><b>Date:</b> 07/08/2014<br />
<br>BBa_K823022 and Binding Module with Pveg (BBa_K1364005) digested by EcoRIand PstIGel Electrophoresis<br />
<br><b>Result:</b><br />
<br>- Binding Module with Pveg 1600 bp for Binding Module and 2100bp for vector pSB1C3</p><br />
<br />
<p class="title3">Ligation Binding Module with Pveg on pSBbs4S</p><br />
<p class="texte"><b>Date:</b> 07/08/2014<br />
<br>BBa_K823022 and Binding Module with Pveg (BBa_K1364005) digested by EcoRIand PstI<br />
<br>Ligation<br />
<br><b>Result:</b> Ligation between Binding Module with Pveg on pSBBS4S</p><br />
<br />
<p class="title3">Transformation of Binding Module with Pveg on pSBBS4S into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>Binding Module with Pveg on pSBbs4S<br />
<br>Plate on Ampicillin LA plate<br />
<br><b>Result:</b> Many good clones (check on 13/08/2014)</p><br />
<br />
<p class="title3">Transformation of Binding Module with Pveg on pSBBS4S into <i>B. subtilis</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014<br />
<br>After 5 hours of incubation and the linearization of 6µl of DNA with 1µl of ScaI, we make the transformation. Plate on Spectinomycin LA plate.<br />
<br>Result : One good clone : PCR (check on 09/09/2014) and threonine test (check on 09/09/2014)</p><br />
<br />
<p class="title2">5. Binding Test</p><br />
<p class="texte">See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results#select2">here</a></p><br />
<br />
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<br />
<br />
<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">1. Transformation of chemotaxis (Puc-57) into <i>E. coli</i></p><br />
<p class="texte">Gene chemotaxis synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br><b>Date:</b> 01/08/2014<br />
<br><b>Result:</b> We obtained distinct colonies on LA + Ampicillin (100 µg/mL) plates</p><br />
<br />
<p class="title3">Culture of 4 clones: A, B, C, D of chemotaxis (Puc57) transformed into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 04/08/2014</p><br />
<br />
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit Using a Microcentrifuge: 4 clones of chemotaxis (Puc57) into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 05/08/2014<br />
<br><b>Result:</b> 4*50µL of chemotaxis (Puc57) obtained</p><br />
<br />
<p class="title2">2. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested pSB1C3 with EcoRI and PstI into <i>E. coli</i></p><br />
<br />
<p class="title3">Digestion BBa_K1364000 (chemotaxis_Puc57) with EcoRI and PstI - PCR kit Clean up</p><br />
<p class="texte"><b>Date:</b> 07/08/2014<br />
<br><b>Result:</b> Expected band after digestion for BBa_K1364000: 2300 bp<br />
<br><b>Problem:</b> We can't distinguish the vector band (2500 bp)</p><br />
<br />
<p class="title3">Gel extraction of BBa_K1364000</p><br />
<p class="texte"><b>Date:</b> 07/08/2014</p><br />
<br />
<p class="title3">Ligation BBa_K1364000 and digested PsB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 08/08/2014</p><br />
<br />
<p class="title3">Transformation BBa_K1364000 in E.coli</p><br />
<p class="texte"><b>Date:</b> 08/08/2014<br />
<br><b>Result:</b> We obtained distinct colonies on LA + Cm (15 µg/mL) plates and resuspended 15 colonies in LB+Cm (15 µg/mL)</p><br />
<br />
<p class="title3">Test of sensibility on Ampicillin</p><br />
<p class="texte"><b>Date:</b> 10/08/2014<br />
<br><b>Result:</b> we can determine which colonies are sensible for ampicillin and know which bacterium is carrying the chemotaxis gene.</p><br />
<br />
<p class="title3">PCR</p><br />
<p class="texte"><b>Date:</b> 11/08/2014</p><br />
<br />
<p class="title3">Digestion BBa_K1364000 on pSB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 11/08/2014<br />
<br><b>Result:</b> There is one colony which presents the right construction.</p><br />
<br />
<br />
<br />
<p class="title2">3. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested BBa_823003 (Pveg) on pSB1C3 with SpeI and PstI into <i>E. coli</i></p><br />
<p class="title3">Ligation BBa_K1364000 and digested BBa_823003 on PsB1C3 with SpeI and PstI</p><br />
<p class="texte"><b>Date</b>: 08/08/2014</p><br />
<p class="title3">Transformation BBa_1364004 in E.coli</p><br />
<p class="texte"><b>Date:</b> 8/08/2014</p><br />
<p class="title3">Test of sensibility on Ampicillin</p><br />
<p class="texte"><b>Date:</b> 10/08/2014<br />
<br><b>Result:</b> We can determine which colony is sensible for ampicillin and know which bacterium is carrying the chemotaxis gene. There is one colony which resists on ampicillin.</p><br />
<br />
<p class="title3">Digestion BBa_K1364004 on pSBC3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 12/08/2014<br />
<br><b>Result:</b> We did not see any colony with chemotaxis insert.</p><br />
<br />
<p class="title2">4. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested BBa_823003 (Pveg) on pSB1C3 with SpeI and PstI into <i>E. coli</i></p><br />
<p class="title3">Ligation BBa_K1364000 and digested BBa_823003 on PsB1C3 with SpeI and PstI</p><br />
<p class="texte"><b>Date:</b> 11/08/2014</p><br />
<p class="title3">Transformation BBa_1364004 in E.coli</p><br />
<p class="texte"><b>Date:</b> 11/08/2014</p><br />
<p class="title3">Test of sensibility on Ampicillin</p><br />
<p class="texte"><b>Date:</b> 14/08/2014<br />
<br><b>Result:</b> we obtained 4 colonies sensible at Ampicilline</p><br />
<br />
<p class="title3">Digestion BBa_K1364004 on PsB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 18/08/2014</p><br />
<p class="title3">Gel extraction of BBa_K1364000</p><br />
<p class="texte"><b>Date:</b> 19/08/2014</p><br />
<br />
<p class="title2">5. Cloning chemotaxis BBa_K1364004 with digested pSBBS4S with EcorI and PstI into <i>E. coli</i></p><br />
<p class="title3">Ligation BBa_K1364004 digested EcorI and PstI and digested PsB1C3 with EcoRI and PstI</p><br />
<p class="texte"><b>Date:</b> 19/08/2014</p><br />
<p class="title3">Transformation BBa_1364004 in pSBBS4S in E.coli</p><br />
<p class="texte"><b>Date:</b> 19/08/2014 <br />
<br><b>Result:</b> We obtained one colony and resuspended it in LB+ Amp</p><br />
<br />
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<div class="technology">Fungicides</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">D4E1</p><br />
<br />
<p class="title2">1. Amplification of synthetic gene (D4E1 on pEX-A2)</p><br />
<p class="title3">Transformation of D4E1 (pEX-A2) into <i>E. coli</i></p><br />
<p class="texte">Gene D4E1 synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br>Concentration of D4E1: 115ng/µL<br />
<br><b>Date:</b> 07/21//2014<br />
<br><b>Result:</b> We obtained distinct colonies on LA + Ampicillin (100 µg/mL)</p><br />
<br />
<p class="title3">Culture of 4 clones: A, B, C, D of D4E1 (pEX-A2) transformed into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/22/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of D4E1 (pEX-A2) into E. coli</p><br />
<p class="texte">Buffer EB at 50-55°C<br />
<br><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up</p><br />
<p class="texte"><b>Date:</b> 07/23/2014<br />
<br><b>Result:</b> 4*20µL D4E1 digested with EcoRI and PstI all the clones seem to have the right D4E1 gene.</p><br />
<br />
<p class="title2">2. Cloning D4E1 in pSB1C3</p><br />
<p class="title3">Digestion of D4E1 on pEX-A2 and pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<p class="title3">Ligation of D4E1 in pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<p class="title3">Transformation in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of D4E1 (pSB1C3) transformed into <i>E. coli</i></p><br />
<p class="texte">Processing details: 5mL of LB + chloramphenicol (15µg/mL) , using sterile plastic loop to culture colonies<br />
<br><b>Date:</b> 07/24/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<p class="title3">Digestion of D4E1 (pSB1C3) A, B, C, D, E, F with EcoRI and PstI - PCR kit Clean up + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014<br />
<br><b>Result:</b> clones C, D have the expected construction</p><br />
<p class="title3">PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014<br />
<br><b>Result:</b> clones C, D have the expected construction, and placed in cryopreservation.</p><br />
<br />
<p class="title2">3. Cloning Pveg+D4E1 on Pveg plasmid (pSB1C3)</p><br />
<br />
<p class="title3">Digestion of D4E1 on pEX-A2 and K823003 (Pveg on pSB1C3)</p><br />
<p class="texte"><b>Dated:</b></b> 07/24/2014<br />
<br><b>Result:</b> 20µL digestion of D4E1 on pEX-A2 and of K823003</p><br />
<br />
<p class="title3">Ligation of D4E1 in K823003 (Pveg on pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/24/2014<br />
<br><b>Result:</b> 20µL ligation of D4E1 in K823003</p><br />
<br />
<p class="title3">Transformation of ligation products in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 07/24/2014<br />
<br><b>Result:</b> E.coli transformed by D4E1+K823003</p><br />
<br />
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of transformed <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/26/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Dated:</b> 07/28/2014<br />
<br><b>Result:</b> clones E, F seem to have the expected construction</p><br />
<p class="title3">Digestion of clones E, F of D4E1+K823003 (Pveg on pSB1C3) with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 28/07/2014<br />
<br><b>Result:</b> clones C, D have the expected construction and are placed in cryopreservation.</p><br />
<br />
<p class="title2">4. Cloning Pveg+D4E1 on pSBBS4S (K823022) </p><br />
<p class="texte"><b>Date:</b> 08/13/2014</p><br />
<br />
<p class="title2">5. Cloning Pveg + D4E1 on pSBBS1C lacZ (23)</p><br />
<br />
<p class="title2">GAFP1</p><br />
<p class="title2">1. Amplification of synthetic gene (GAFP1 on pEX-A2)</p><br />
<br />
<p class="title3">Transformation of GAFP1 (pEX-A2) into E.coli</p><br />
<p class="texte">Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM<br />
<br>Concentration of GAFP1 : 145ng/µL<br />
<br><b>Date:</b> 07/21/2014<br />
<br><b>Result:</b> We obtained distinct colonies on plates LB + Ampicillin (100 µg/mL)</p><br />
<br />
<p class="title3">Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/22/2014<br />
<br><b>Result:</b> Culture of 4 clones ok</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel Electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/23/2014 </p><br />
<br />
<p class="title2">2. Cloning GAFP1 gene on PSB1C3 = K1364002 in E. coli</p><br />
<br />
<p class="title3">Digestion GAFP1_pEX-A2 and BBa_K606013 (RFP_pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/23/2014<br />
<br><b>Result:</b> BBa_K606013 : 860 bp<br />
<br>We decide to conserve the miniprep B for BBa_K606013</p><br />
<br />
<p class="title3">Ligation GAFP1 and BBa_K606013</p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Tranformation of ligation products into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/23/2014</p><br />
<br />
<p class="title3">Culture of x clones of GAFP1+K606013 in <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/24/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge: 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) into <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<br />
<p class="title3">PCR on 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014<br />
<br><b>Result:</b> clones A, C, D, F seem to have the right construction</p><br />
<br />
<p class="title3">Digestion of 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) by EcoR1 and Pst1 + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<br />
<br />
<br />
<p class="title2">3. Cloning GAFP1+terminator(B0015) = K1364007 (pSB1C3)</p><br />
<br />
<p class="title3">Digestion of GAFP1 on pEX-A2 and B0015 (terminator on pSB1C3)<br />
<p class="texte"><b>Date:</b> 07/24/2014<br />
<br />
<p class="title3">Ligation of GAFP1 in B0015 (terminator on pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/24/2014</p><br />
<br />
<p class="title3">Transformation of ligation products in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 07/24/2014</p><br />
<br />
<p class="title3">Culture of 6 clones: A, B, C, D, E, F transformed in <i>E. coli</i></p><br />
<p class="texte"><b>Date:</b> 07/26/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">PCR of GAFP1+B0015 = K1364007 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/28/2014<br />
<br><b>Result:</b> clones B, C, D, E, F seem to have the expected construction.</p><br />
<br />
<p class="title3">Digestion of clones E, F of GAFP1+Ter B0015 = K1364007 with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/28/2014<br />
<br><b>Result:</b> clones B, C, D, E, F have the expected construction.</p><br />
<br />
<br />
<br />
<p class="title2">4. Cloning GAFP1+terminator with Pveg on pSB1C3 (K1364008) in <i>E. coli</i></p><br />
<br />
<p class="title3">Digestion of GAFP1+ter = K1364007 on pSB1C3 and K823003 (terminator on pSB1C3) + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/29/2014</p><br />
<br />
<p class="title3">Gel extraction of K1364007 (extraction of GAFP1+ter gene)</p><br />
<p class="texte"><b>Date:</b> 07/29/2014</p><br />
<br />
<p class="title3">Ligation of GAFP1+ter in K823003 (Pveg on pSB1C3)</p><br />
<p class="texte"><b>Date:</b> 07/29/2014<br />
<br><b>Result:</b> 20µL ligation of GAFP1+ter in K823003</p><br />
<br />
<p class="title3">Transformation of ligation products in E.coli</p><br />
<p class="texte"><b>Date:</b> 07/29/2014</p><br />
<br />
<p class="title3">Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli</p><br />
<p class="texte"><b>Date:</b> 07/30/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 07/31/2014</p><br />
<br />
<p class="title3">PCR of GAFP1+B0015 + K823003 = K1364008 (pSB1C3) A, B, C, D, E, F, G, H + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 31/07/2014<br />
<br><b>Result:</b> clones A, B, C, D, E, G, H seem to have the expected construction</p><br />
<br />
<p class="title3">Digestion of clones A, B, C, D, E, G, H of Pveg+K1364007 = K1364008 with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 31/07/2014<br />
<br><b>Result:</b> clones A, B, C, D, E, G, H have the expected construction</p><br />
<br />
<br />
<br />
<p class="title2">5. Cloning Pveg+GAFP1+Ter B0015 (BBa_K1364008) on pSBBS4S (BBa_K823022)</p><br />
<br />
<p class="title3">Digestion of Pveg+GAFP1+ ter B0015 (K1364008) on pSB1C3 and PsBBs4S (K823022)</p><br />
<p class="texte"><b>Date:</b> 08/01/2014</p><br />
<br />
<p class="title3">Ligation of K134008 (Pveg+GAFP1+ter fragment) and K823022 (PsBBs4S)</p><br />
<p class="texte"><b>Date:</b> 08/01/2014</p><br />
<br />
<p class="title3">Transformation of ligation products in E.coli</p><br />
<p class="texte"><b>Date:</b> 08/01/2014</p><br />
<br />
<p class="title3">Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli</p><br />
<p class="texte"><b>Date:</b> 08/02/2014</p><br />
<br />
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p><br />
<p class="texte"><b>Date:</b> 08/04/2014</p><br />
<br />
<p class="title3">Digestion of clones A, B, C, D, E, G, H of K1364008+K823022 with EcoRI and PstI + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 08/04/2014<br />
<br><b>Result:</b> clones A, E, F have the right construction</p><br />
<br />
<br />
<br />
<br />
<p class="title2">EcAMP</p><br />
<br />
<p class="texte">The EcAMP part was sent by the Utah iGEM team. It was on a pUC plasmid (ampicilin resistance) with biobrick suffix and prefix.</p><br />
<br />
<p class="title2">1. Transformation of EcAMP in Escherichia coli MC 1061</p><br />
<p class="texte"><b>Date:</b> 07/25/2014</p><br />
<br />
<p class="title2">2. Spreading of coli cells transformed with pUC + Utah </p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title2">3. Liquid culture + Miniprep + Test of the miniprep</p><br />
<p class="texte"><b>Date:</b> 07/30/2014</p><br />
<br />
<br />
<br />
<p class="title2">4. Cloning 1: EcAMP + Pveg + RBS</p><br />
<br />
<p class="title3">Digestion of EcAMP (INSERT) by XbaI and PstI</p><br />
<p class="texte"><b>Date:</b> 07/31/2014</p><br />
<br />
<p class="title3">Digestion of Pveg + RBS (VECTOR)</p><br />
<p class="texte"><b>Date:</b> 07/31/2014</p><br />
<br />
<p class="title3">Ligation and transformation</p><br />
<p class="texte"><b>Date:</b> 08/04/2014</p><br />
<br />
<p class="title3">PCR test</p><br />
<p class="texte"><b>Date:</b> 08/05/2014</p><br />
<br />
<p class="title3">Analytical digestion</p><br />
<p class="texte"><b>Date:</b> 08/05/2014<br />
<br />
<br>The first cloning show a lack of DNA in the Miniprep EcAMP. The bands are hardly visible on the gel for the linearization of EcAMP + Pveg + RBS. The problem came from the PCR cleanup step: the fragment size of EcAMP is lower than 200bp.</p><br />
<br />
<br />
<br />
<p class="title2">5. Cloning 2: EcAMP + Pveg + RBS </p><br />
<p class="texte"><b>Date:</b> 06/08/2014</p><br />
<p class="title3">Digestion of EcAMP (INSERT) by XbaI and PstI</p><br />
<p class="title3">Digestion of Pveg + RBS (VECTOR)</p><br />
<p class="title3">Heat inactivation of the enzymes</p><br />
<p class="title3">Ligation and transformation</p><br />
<p class="title3">PCR test</p><br />
<p class="texte"><b>Date:</b> 07/08/2014</p><br />
<br />
<br />
<p class="title3">Striation on a petri dish to purify the clone</p><br />
<p class="texte">Purpose: to isolate a clone with vector+insert<br />
<br><b>Date:</b> 08/072014</p><br />
<br />
<p class="title3">Miniprep of Pveg+SpoVG+EcAMP and analytic digestion</p><br />
<p class="texte"><b>Date:</b> 08/08/2014</p><br />
<br />
<p class="title3">Ligation of Pveg SpoVG EcAMP with double terminateur B0015 +Transformation and liquid culture</p><br />
<p class="texte"><b>Date:</b> 08/11/2014</p><br />
<br />
<p class="title3">Miniprep of Pveg SpoVG EcAMP + analytic digestion</p><br />
<p class="texte"><b>Date:</b> 08/13/2014</p><br />
<br />
<p class="title3">Cloning K1364011 (EcAMP+Pveg +SpoVG + B0015) + K823022 (psBbs4S) : Digestion, ligation, transformation</p><br />
<p class="texte"><b>Date:</b> 08/19/2014</p><br />
<br />
<p class="title3">Cloning K1364011 + K823023 (pSBBS1C) : Digestion, ligation, transformation</p><br />
<p class="texte"><b>Date:</b> 08/18/2014</p><br />
<br />
<p class="title3">Verification of the insertion of K1364011 + K823022 (pSBBS4S) into the subtilis genome by threonine test </p><br />
<p class="texte"><b>Date:</b> 08/21/2014<br />
<br />
<br><br />
<br>Assembling the fungicides module : </p><br />
<br />
<br />
<br />
<p class="title2">D4E1 + GAFP1 </p><br />
<p class="title2">1. Cloning GAFP1+D4E1 on pSB1C3: BBa_K1364012 </p><br />
<br />
<p class="title3">Digestion of D4E1 on pEX-A2 and GAFP1 on pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/28/2014<br />
<br><b>Result:</b> We obtained 40µL digestion of D4E1 on pEX-A2 and of GAFP1 on pSB1C3.</p><br />
<br />
<p class="title3">Ligation of digestions of D4E1 on pEX-A2 and of GAFP1 on pSB1C3</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">Transformation of ligation of GAFP1 and D4E1 on pSB1C3 into E. coli</p><br />
<p class="texte"><b>Date:</b> 07/28/2014</p><br />
<br />
<p class="title3">PCR of GAFP1+D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/29/2014<br />
<br><b>Result:</b> clones A, C, F, G seem to have the expected construction.</p><br />
<br />
<p class="title3">Digestion of GAFP1+D4E1 (pSB1C3) A, C, F, G + electrophoresis</p><br />
<p class="texte"><b>Date:</b> 07/30/2014<br />
<br><b>Result:</b> clone F (BBa_K1364012) has the expected construction and is placed on cryopreservation.</p><br />
<br />
<br />
<br />
<p class="title2">2. Cloning GAFP1+D4E1 (BBa_K1364012) on Pveg plasmid (BBa_K823003): BBa_K1364013</p><br />
<p class="title2">3. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS4S (K823022)</p><br />
<p class="title2">4. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS1C lacZ (K823023)</p><br />
<p class="title2">5. Fungicides tests</p><br />
<br />
<br />
<p class="title2">Cloning D4E1-GAFP1-EcAMP: BBa_K1364014</p><br />
<br />
<p class="title2">1. Construction of BBa_K1364014 in PsB1C3, in E. coli</p><br />
<br />
<p class="title3">Digestion of K1364010 and K1364012</p><br />
<p class="texte">Digestion of K13664010 by Spe1 and Pst1, and digestion of K1364012 by XbaI and Pst1.<br />
<br><b>Date:</b> 08/11/2014<br />
<br><b>Result:</b> We obtained digested fragments of K1364012 and ~500 bp fragment of K1364010</p><br />
<br />
<p class="title3">Ligation of ~500 bp fragment from K1364010 and K1364012</p><br />
<p class="texte"><b>Date:</b> 08/11/2014<br />
<br><b>Result:</b> We obtained ligation of K1364012 and ~500 bp fragment of K1364010</p><br />
<br />
<p class="title3">Transformation of ligation of ~500 bp fragment from K1364010 and K1364012 = K1364014 in <i>E.coli</i></p><br />
<p class="texte"><b>Date:</b> 08/12/2014</p><br />
<br />
<br />
<p class="title3">Testing 8 E.coli+K1364014 clones</p><br />
<p class="texte"><b>Date:</b> 08/14/2014 </p><br />
<br />
<p class="title3">Testing 15 E.coli+K1364014 clones</p><br />
<p class="texte"><b>Date:</b> 08/18/2014<br />
<br><b>Result:</b> clones H, J, O, Q, U, V might have the right K1364014 construction</p><br />
<br />
<br />
<p class="title2">2. BBa_K1364014 in PsBs4s (K823022) and PsBs1C (K823023)</p><br />
<p class="title3">Digestion of K1364014, K823022 and K823023</p><br />
<p class="texte"><b>Date:</b> 08/22/2014<br />
<br><b>Result</b>: digestion of K13664014, K823022 and K823023 by EcoR1 and Pst1 were well performed.</p><br />
<br />
<p class="title3">Ligation of K1364014 with K823022 and K1364014 with K823023</p><br />
<p class="texte"><b>Date:</b> 08/22/2014<br />
<br><b>Result:</b> We obtained 20µL ligation of K1364014 with K823022 and of K1364014 with K823023.<br />
</p><br />
<p class="title3">Transformation of ligations in E.coli</p><br />
<p class="texte">E. coli transformed by K1364014+K823022 spread on LB+Amp 100µg/mL agar plate</p><br />
<p class="texte">E. coli transformed by K1364014+K823023 spread on LB+Amp 100µg/mL agar plate<br />
<br><b>Date:</b> 08/25/2014</p><br />
<br />
<p class="title3">Testing E.coli+K1364014+K823022 and E.coli+K1364014+K823023 clones</p><br />
<p class="texte"><b>Date:</b> 08/27/2014</p><br />
<br />
<br />
<br />
<p class="title2">3. Transformation of K1364014+K823022 and K1364014+K823023 in <i>B. subtilis</i></p><br />
<p class="texte"><i>B. subtilis</i> transformed by 10µL K1364014+K823022 spread on LB+Spec 75µg/mL agar plate<br />
<br><i>B. subtilis</i> transformed by 10µL K1364014+K823023 spread on LB+Cm 15µg/mL agar plate<br />
<br><b>Date:</b> 08/28/2014</p><br />
<br />
<p class="title2">Fungicides tests</p><br />
<p class="title2">1. D4E1-GAFP1</p><br />
<p class="title3">Transformation in bacillus Pveg-D4E1-GAFP1 on pSBBS4S </p><br />
<p class="texte"><b>Date:</b> 08/12/2014</p> <br />
<p class="title3">Integration threonine test + fungicide test </p><br />
<p class="texte"><b>Date:</b> 08/13/2014 </p><br />
<br />
<p class="title2"2. D4E1</p><br />
<p class="title3"><br />
Cloning D4E1 into pSBBS1C + fungicide test</p><br />
<p class="texte"><b>Date:</b>08/15/2014</p><br />
<p class="title3"><br />
D4E1 on pSB1C3 + fungicide test</p><br />
<p class="texte"><b>Date:</b>08/19/2014</p><br />
<br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Calendar</p> <br />
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<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
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<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a> | <a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a></p><br />
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<br />
<div class="technology">December 2013 – January 2014: Team selection</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- iGEM competition presentation and explanations<br/><br />
- Constitution of the team by interviewing different students from Université Paul Sabatier and INSA<br/><br />
</p><br />
<p class="texte" style="text-align:center"><B> The adventure begins for the Toulouse iGEM Team 2014! </B><br />
</p><br />
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<div class="technology">January – June 2014: Projects brainstorming</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- Thanks to weekly meetings, our team was able to discuss about new project ideas and publications.<br />
<br/><br />
- Many ideas were approached and debated regarding the feasibility thanks to the literature and supervisors including teachers and researchers<br />
<br/><br />
This is a list of the main projects: <br />
<br/><br />
- Let's save our trees with SubtiTree: the purpose is to use <i>Bacillus subtilis</i> as an optimized bacterium to detect, target a fungi and secrete fungicides to destroy it<br />
<br/><br />
- <i>E. coli</i> cancer: the concept was to create a bacterium able to colonize specifically the hypoxic regions of the tumor. The oxygen-sensitive bacterium would not be able to develop in the healthy zones<br />
<br/><br />
- Reduction of ruminants’ methane production: the goal is to reduce the production of methane by ingesting a microorganism in the animal’s rumen. This bacterium would be capable of reducing the quantity of metabolic hydrogen and eliminating the methanogenic Archaes population<br />
<br/><br />
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<div class="technology">June 2014: Choice of SubtiTree project</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">The discussion has been long to estimate the practicability of our final project. By the end of June, most of the ideas were eliminated but two of them remained: controlling the cancer or saving the trees. It became easily clear that SubtiTree was successfully completed whether regarding the scientific part or the ethical aspect. Indeed, our knowledge in medicine was not sufficient enough to evaluate the consequences of a bacterium injection in the human body for <i>E. coli</i> cancer.</br><br />
Therefore, the whole team agreed on focusing on a local issue as the preservation of the Canal du Midi plane trees. However, the final goal was based on the idea to allow our optimized bacteria to be efficient for all fungi infections in the environment.</br><br />
By this period, we evaluated our budget to approximately <B>40 k€</B>.<br />
</p> <br />
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<div class="technology">Week 1(16-22 June)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Bibliography researches about our subject<br/><br />
- Cleaning the whole lab to allow good manipulations and avoid any contamination<br/><br />
- Inventory of necessary lab equipment and biobricks<br/><br />
- Summarize the iGEM protocols<br/><br />
- Prepare all growing media <br/><br />
- Preparing competent cells by optimizing protocols<br/><br />
- Transformation of RFP plasmid to practice<br />
</p><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Increase in our communication strategy to find more sponsors specialized in environment and ecological matters <br/><br />
- Support of Adisseo, Toulouse White Biotechnology, LISBP, INSA Toulouse<br/><br />
- Organization of a weekly meeting each Friday with our supervisors to tackle any problem encountered<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/20/2014)</p><br />
<p class="texte"><br />
- Distribution of the non-scientific tasks regarding the wiki, communication, sponsorship, ethical problems, safety but also the lab work<br/><br />
- Discussion about the different construction parts of the project<br/><br />
- Need to check the absence of stop codon in fungicides sequences<br />
</p> <br />
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<div class="technology">Week 2 (23-29 June)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Ordering the list of necessary products for the laboratory and biobricks<br/><br />
- Checking and validation of the genes sequences<br/><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- BioSynSyS conference work: powerpoint, logo in order to present our SubtiTree project to a whole audience of scientific and researchers (http://biosynsys2014.sciencesconf.org/)<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- E. coli transformation with BBA_J004450 (pSB1C3)<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/27/2014)</p><br />
<p class="texte"><br />
- Concentration of antibiotics in media must be checked<br/><br />
- The transformation rate for pUC19 must be evaluated<br/><br />
- The transformation efficiency must be calculated regarding the quantity of DNA<br/><br />
- Possibility to contact the cities halls for the sponsorship<br/><br />
- Check the mechanism of action of each fungicide to complete the ethical part<br />
</p><br />
<br />
<p class="title1" id="select4">July 2014</p><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Beginning of the laboratory work to create our optimized bacterium <br/><br />
- Research of sponsors and the communication thanks to the press<br />
</p> <br />
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<br />
<div class="technology">Week 3 (30 June -6 July) and Week 4 (7-13 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the BioSynSys conferences: presentation of SubtiTree<br/><br />
- We have put in place a newsletter system the first day of each month to keep all our sponsors and people who support us updated about the project, our financial state.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Design of all the constructions needed with cloning and restriction enzymes to save some time and allow a better comprehension of our bacterium system </br><br />
Problem: A problem was reported in the use of the CYP. Indeed, the biobrick is not expressed properly in <i>Bacillus subtilis</i> strain. Therefore we had to look at the literature to find a new fungicide called GAFP-1.<br/><br />
- A bioreactor was conceived to reproduce the composition of the sap in the plane trees. The main goal was to identify the behavior of Bacillus strain in a medium composed of many different carbon sources.<br/><br />
- Competent cells and transformation practice using GFP and RFP.<br />
</p><br />
<br />
<p class="title3">Weekly meetings with the instructors (07/04/2014) and (07/11/2014)</p><br />
<p class="texte"><br />
- iGEM efficiency kit does not work, we shall not use it anymore<br/><br />
- Organization of a timetable to check the stored and sterile equipment everyday</br><br />
- Try a transformation in <i>Bacillus subtilis</i><br />
</p> <br />
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<div class="technology">Week 5 (14-20 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Our team was interviewed by many local newspapers such as 20minutes, Metronews, La Voix du Midi, La Dépêche but also by the local television channel France3 Midi-Pyrénées to present our draft to the community. Moreover, SubtiTree was approached in some radio programs like Virgin Radio Toulouse and France info.</br><br />
- Support of ThermoFisher, New Englands BioLabs, Qiagen, Eurofins but also GenoToul, Labex Tulip, L’Université Paul Sabatier, CROUS.<br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- GFP and RFP digestion amplified by miniprep and gel electrophoresis <br/><br />
Problem: the digested GFP doesn’t appear on the gel contrary to RFP plasmid. We assumed a problem with the GFP miniprep. We tried the miniprep from the INSA Toulouse team from 2013 and the GFP plasmid was fully digested.<br/><br />
- Transformation and cryopreservation of biobricks BBa_K823002 (PlepA), BBa_K823003 (Pveg), BBa_K606061 (RBS SpoVG) and BBa_B0015 (double terminator), BBa_K823023 (pSBBS1C), BBa_K733013 (Pveg+ RBS) in <i>E. coli</i>.<br/><br />
- Transformation of the Munich B. subtilis backbones<br/><br />
- Cloning of BBa_K606061 (RBS SpoVG) with BBa_K823003 (PvEG) and BBa_K823002 (PlepA)<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (07/18/2014)</p><br />
<p class="texte"><br />
- Transformation of the Eurofins genes<br/><br />
- Start assembling the biobricks for the fungicides<br/><br />
- Check all the cloning<br />
</p><br />
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<br />
<div class="technology">Week 6 (21 July-27 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Description of SubtiTree project and determination of the goodies for a crowdfunding campaign on Ulule website<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Transformation of BBA_1364002 (GAFP1) and BBA_1364003 (D4E1) fungicides genes<br/><br />
- Subculture of the clones for each gene<br/><br />
- PCR and migration on electrophoresis gel<br/><br />
- Transformation BBA_1364003 (D4E1) on pSB1C3 and BBA_1364002 (GAFP1) on pSB1C3<br/><br />
- Cloning BBA_1364002 (GAFP1) with BBa_B0015 (double terminator) and BBA_1364003 (D4E1) with BBa_K823003 (Pveg)<br/><br />
</p><br />
<br />
<p class="title3">Others:</p><br />
<p class="texte"><br />
- Research for plane tickets and hotels in Boston for the Giant Jamboree<br/><br />
- New idea: analyze the plane tree sap to determine the composition<br/><br />
</p><br />
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<div class="technology">August 2014 </div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finishing the cloning for the different parts of the bacterium<br/><br />
- Putting in place the fungicides, binding and chemotaxis tests<br/><br />
</p> <br />
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<div class="technology">Week 7 (28 July-3 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Financial supports of the Ministery (Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt) and Voies Navigables de France<br/><br />
- Interview with Johan Langot from Sciences Animation for a possible presentation at the Toulouse Festival of Science<br/><br />
- Launch of the crowdfunding campaign on Ulule<br />
</p><br />
<br />
<p class="title3">Lab work: </p><br />
<p class="texte"><br />
- Cloning BBa_K823003 (Pveg) + RFP and BBa_K823002 (PlepA) + RFP in pSBBs1C<br/><br />
- Test of every pSBBs vector : BBa_K823021 (pSBBS1C-lacZ), BBa_K823022 (pSBBS4S), BBa_K823023 (pSBBS1C), minipreps and cryopreservation of the best clones<br/><br />
- Checking of BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) transformants (biobrick BBa_K1364007) and BBA_1364003 (D4E1) + BBa_K823003 (Pveg) biobrick BBa_K1364009<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) + pSBBS4S (BBa_K823022) and subculture of the colonies<br/><br />
- Cloning of BBa_K823003 (Pveg) + BBA_1364003 (D4E1) with BBa_B0015 (double terminator)<br/><br />
- Cloning of BBa_K823003 (Pveg) + RFP (BBa_K1364017) in pSBBS4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_K823002 (PlepA) + RFP (BBa_K1364016) in pSBBs4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_1364018 (Strong Promotor + RFP)<br/><br />
- Cloning of BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) + Promotor BBa_K823003 (Pveg) biobirck BBa_K1364012 with gel extraction for BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) and a PCR cleanup for BBa_K823003 (Pveg), subculture<br/><br />
- Cloning BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBS4S in <i>E. coli</i><br/><br />
- Transformation of binding gene with <i>E. coli</i> competent cells<br/><br />
- Transformation of BBa_K1364000, the chemotaxis gene in <i>E. coli</i><br/><br />
Problem: the transformation worked but a cell layer appeared. A new subculture of the colonies was made as well as a new spreading on petri dishes.<br/><br />
- Spreading of BBa_K1162001 (EcAMP), miniprep and digestion<br/><br />
Problem: the digestion did not work => Issue with the quantity of DNA in the miniprep is assumed<br/><br />
- First experience of chemotaxis with a glucose chemo-attractant<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/01/2014)</p><br />
<p class="texte"><br />
- Possible problem with the restriction enzymes regarding the digestion: new recommendations<br />
<br/><br />
- Discussion about EcAMP cloning which presents some issues<br />
<br/><br />
- Discussion about the transfer of pSB1C3 from E. coli to Bacillus strain<br />
</p> <br />
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<br />
<div class="technology">Week 8 (04-10 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the “Passion Jeune” competition organized by a French bank foundation named Fondation Crédit Agricole Toulouse<br />
</p><br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Check the cloning of BBa_K823003 (Pveg)+BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBs4S and cryopreservation<br />
<br/><br />
- Transformation of BBa_K823003 (Pveg) +BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) (biobrick BBa_ K1364008) in BBa_K823022 (pSBBS4S) <br />
<br/><br />
- Cloning of BBa_K1364001, the binding gene + BBa_K823003 (Pveg) on pSB1C3 and pSBBS 4S<br />
<br/><br />
Problem: the band is at 1500 bp instead of 1300 bp on the gel<br />
<br/><br />
- Cloning of BBa_K1162001 (EcAMP) + BBa_K823003 (Pveg) + BBa_K606061 (RBS SpoVG), miniprep, PCR and digestion <br />
<br/><br />
Problem: the fragment of DNA is too small (149 bp) to be seen on the gel and was probably lost during the PCR cleanup. Instead of that, we used the heat enzymes inactivation at 95°C for 15 minutes. <br />
<br/><br />
- Cloning of Promotor + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) in pSB1C3 and pSBBS4S and cryopreservation<br />
<br/><br />
- Elaboration of an efficient fungicide test protocol <br />
<br/><br />
- Test of the fungus growth on PDA medium + test of growth for Bacillus subtilis liquid culture on PDA<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/08/2014)</p><br />
<p class="texte"><br />
- Ask iGEM headquarters if all the biobricks must be on pSB1C3 plasmid<br />
<br/><br />
- Use another strain of E. coli (DH5-1 instead of DH5 alpha) to have a faster growth<br />
<br/><br />
- Check which quantity of Bacillus subtilis is necessary to naturally destroy the fungus<br />
</p><br />
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<div class="technology">Week 9 (11-17 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Publication of more articles about our project in Labiotech, Développement durable (Networkvisio), Midi Libre, l’Indépendant, DigiSchool, La voix du Midi Lauragais, LURIO Addl.<br />
</p><br />
<br />
<p class="title2">Lab work:</p><br />
<p class="texte"><br />
- Evaluation of the bacterial decrease rate from 10°C to 4°C with a sporuling strain of Bacillus subtilis.<br />
<br/><br />
- Chemotaxis test with different concentration of glucose on a 0.3% agar.<br />
<br/><br />
- Miniprep of pSBbs4S with binding gene and transformation in <i>Bacillus subtilis</i>.<br />
<br/><br />
Problem: no digestion was visible on the gel => the cloning failed<br />
<br/><br />
- Transformation of BBa_K1374010 (Pveg + RBS SpoVG + EcAMP) + BBa_K1364012 (RBS + Gafp1 + D4E1 + double terminator), subculture<br />
<br/><br />
- Subculture of Bacillus subtilis clones transformed by BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) in pSBBs4S, cryopreservation<br />
<br/><br />
- Fungicide test<br />
</p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology">Week 10 (18-24 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Ethics: Interview with a specialist in ethical sciences, Vincent Grégoire Delory to complete our reflexion about the ethical issues in SubtiTree project.<br />
<br/><br />
- Wiki: hiring a web designer, Adrien Nicod, to improve the aesthetic effect and the logo.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Subculture and transformation in Bacillus subtilis of BBa_K1364011 in pSBBS4S (BBa_K823022) and cloning of BBa_K1364011 on pSBBS1C (BBa_K823023).<br />
<br/><br />
Problem: no clone had the insert on pSBBS1C => the cloning had to be done again<br />
- Miniprep of BBa_K1364011 in BBa_K823023 (pSBBS1C) + Transformation in <i>B. subtilis</i>.<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) , BBA_1364002 (GAFP1) , BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) on BBa_K823023 (pSBBS1C) <br />
<br/><br />
- Transformation BBa_1364004 (in pSBBS4S in E.coli <br />
<br/><br />
- Cloning of BBa_K1364014 (Pveg + RBS SpoVG + EcAMP + GAFP1 +D4E1 + double terminator) in K823022 (pSBBS4S) and in BBa_K823023 (pSBBS1C), miniprep and digestion by restriction enzymes to see the size of the insert on a 1% gel<br />
<br/><br />
Problem: no clone had the insert on pSBBS1C => the cloning had to be done again<br />
</p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology">Week 11 (25- 31 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Beginning of the creation and redaction of the different parts of the wiki by the Toulouse iGEM Toulouse<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Cloning of BBa_K1364014 in BBa_K823022 and in BBa_K823023 <br />
<br/><br />
- Cloning GAFP1 + BBa_K823023 (pSBBS1C) in Bacillus subtilis with a change in the protocol: pre-induction of the culture with 0.125 µg/ml of chloramphenicol one hour after the addition of DNA, and let grow for another hour.<br />
<br/><br />
Problem: no colony <br />
<br/><br />
- Liquid culture of Bacillus + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) for future fungicide tests<br />
<br/><br />
- Miniprep of BBa_ K1364011 in BBa_ K823023 (pSBBs1C) and transformation in Bacillus subtilis strain. <br />
<br/><br />
- Fungicide test of BBa_K1364011 in pSBBs4S, Promotor + BBA_1364002 (GAFP1) + Terminator (biobrick BBa_K1364008) in pSBBS4S, Promotor + BBA_1364003 (D4E1) + Terminator (biobrick BBa_K1364009) in pSBBS4S, Promotor + BBA_1364002 (GAFP1) + BA_1364003 (D4E1) + Terminator ( biobrick BBa_K1364013) in pSBBS4S<br />
<br/><br />
- Transformation of a new vector PKL 190 (threonine integrative) in Bacillus strain <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/29/2014)</p><br />
<p class="texte"><br />
- Boston accommodation must be booked<br />
<br/><br />
- Discussion about the contamination seen on the fungicide tests : the issue seems to come from the sterile water which contained contaminants<br />
<br/><br />
- Objectives : chemotaxis, binding and fungicides tests have to be performed again<br />
</p> <br />
<br />
<p class="title1" id="select6"> September 2014</p><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finish the tests of the different modules<br />
<br/><br />
- Establish the final editorial parts for the wiki<br />
<br/><br />
- Design of the wiki<br />
<br/><br />
- Sequencing the different assembled parts of our bacterium<br />
</p> <br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology">Week 12 (1-7 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Different parts of the wiki by the Toulouse iGEM Toulouse<br/><br />
- Reservation of Boston accommodation<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Chemotaxis test and modelling<br />
<br/><br />
- Fungicides tests on EcAMP-A, EcAMP-B, EcAMP-C, EcAMP-D, MaxiOpA, MaxiOpB, MaxiOpC, MaxiOpD from 2ml of overnight cultures. Only with 25000 conidia with both fungi.<br />
<br/><br />
- PCR on pSBBS4S + BBa_K1162001 (EcAMP) <br />
<br/><br />
Problem: failed on both diluted plasmid and colony<br />
<br/><br />
- Spreading of BBa_K1364014 + BBa_ K823022 in threonine medium and in medium without threonine <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/05/2014)</p> <br />
<p class="texte"><br />
- The first sequencing was not successful => new sequencing with one primer/tube<br/><br />
- Chemotaxis test: increase the concentration in bacterium, try a new protocol by capillarity<br/><br />
- End of the binding test: IT WORKS PERFECTLY! SubtiTree has the good phenotype and presents a better binding property than the wild type Bacillus subtilis strain.<br/><br />
- Fungicides test: contaminants with the fungus. Need to ask for another culture of our fungi.<br />
</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology">Week 13 (8- 14 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Telephone interview for an article in a French national review named Fluvial which will be published at the end of September.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- PCR on D4E1 colony with different primers<br />
<br/>- Culture and miniprep of EcAMP to get more DNA + analytic digestion <br />
<br/>- Transformation of K1364014+K1364006 in E. coli <br />
<br/>- Transformation of GAFP1 + D4E1, GAFP1 and EcAMP+GAFP1+D4E1 on Bacillus subtilis + colony PCR + threonine test<br />
<br/>NB: good results for the maxi operon and GAFP1 but not for GAFP1 + D4E1 CA <br />
<br/>- Cloning of K606013+pEX_K4, 823022+PlepA_RFP, 823022+ Pveg_RFP<br />
<br/>- Chemotaxis tests: different times of LB + agar addition in the LB medium (after 3 minutes, 5 minutes, 8 minutes, 10 minutes…) are performed => No difference of the results after spreading on the plates.<br />
<br/>- New test of chemotaxis: capillary between two Eppendorf tubes<br />
<br/>- Spreading of the fungi on a medium which mimics the sap<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/11/2014)</p> <br />
<p class="texte"><br />
- Try to use the GFP or RFP to follow the chemotaxis test.<br />
<br/>- Make a new glass process for the chemotaxis capillary test.<br />
<br/>- Possibility of contacting an INRA laboratory to get pictures of the binding test with confocal microscope.<br />
<br/>- Sequencing the binding construction.<br />
<br/>- Try different temperatures for the fungicide tests: 20°C, 25°C, and 37°C to reduce the fungus growth.<br />
</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology">Week 14 (15 - 21 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Support of two French city halls: Mairie de Montréal, Mairie de Ventenac en Minervois.<br />
<br/>- First interview by the French national radio France Inter which will be aired on October 12th.<br />
<br/>- Beginning of the organization of the iGEM Toulouse 2014 acknowledgement day: the purpose is to present the project to the students and give a feedback on the competition to the sponsors.<br />
<br/>- Final design of the wiki<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Threonine tests for the BBa_K1364014 maxi operon (EcAMP1 – GAFP1 – D4E1) and mini operon BBa_K1364013 (GAFP1-D4E1)<br />
<br/>- Colony PCR on the mini operon BBa_K1364013 (GAFP1 - D4E1)<br />
<br/>- Colony PCR on BBa_K1364011 on BBa_K823022 (pSBBS4S)<br />
<br/>- Chemotaxis test with wild type <i>Bacillus subtilis</i> strain using Imperial College protocol<br />
<br/>- Tansformation of BBa_K1364011 + BBa_K823022 (pSBBS4S) in Bacillus subtilis<br />
<br/>- Culture of fungi with fungicides<br />
<br/>- Sequencing of plasmids containing the fungicides<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/18/2014)</p> <br />
<p class="texte"><br />
- Sequencing worked for D4E1 and GAFP1 and the mini operon BBa_K1364013 (GAFP1-D4E1).<br />
<br/>- The next step is to sequence the maxi operon (EcAMP1 – GAFP1 – D4E1).<br />
<br/>- Try different approaches for the chemotaxis:<br />
<br/>- New protocol with the capillary assay with a new system made by a glassblower.<br />
<br/>- Make the Imperial College test again by testing 3 different solutions: chitin, N-acetylglucosamine and another carbon source.<br />
<br/>- Decrease the number of conidia and temperature for the fungicide test.<br />
</p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology">Week 15 (22 - 28 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- New support of Sallèles d’Aude city hall.<br />
<br/>- Payment of the plane tickets.<br />
<br/>- Acknowledgement day (4th December): contact with catering service, reservation of the amphitheater, list of guests…<br />
<br/>- Order of the T shirt for the Jamboree! We look awesome with them!<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Last experiments regarding chemotaxis tests.<br />
</p><br />
<br />
<br />
<p class="title3">Weekly meeting with the instructors(09/26/2014)</p> <br />
<p class="texte"><br />
- Focus on the wiki design and writing.<br />
<br/>- Start making the power point presentation for Boston and the poster.<br />
<br/>- Division of responsibilities for all the non-scientific work.<br />
</p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology">October 2014</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">Main activites</p><br />
<p class="texte"><br />
- Preparation of the wiki every day… all day long…. so hard to work on this even at night!<br />
<br/>- Preparation of the poster and the presentation for the Jamboree with our instructors <br />
<br/><br />
- Daily presentations in front of the laboratory teachers to repeat again and again and again… let’s get ready for the Jamboree!<br />
</p> <br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
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contentclass: "thelanguage", //Shared CSS class name of contents group<br />
revealtype: "click", //Reveal content when user clicks or onmouseover the header? Valid value: "click", "clickgo", or "mouseover"<br />
mouseoverdelay: 200, //if revealtype="mouseover", set delay in milliseconds before header expands onMouseover<br />
collapseprev: false, //Collapse previous content (so only one open at any time)? true/false <br />
defaultexpanded: [], //index of content(s) open by default [index1, index2, etc]. [] denotes no content.<br />
onemustopen: false, //Specify whether at least one header should be open always (so never all headers closed)<br />
animatedefault: false, //Should contents open by default be animated into view?<br />
persiststate: false, //persist state of opened contents within browser session?<br />
toggleclass: ["closedlanguage", "openlanguage"], //Two CSS classes to be applied to the header when it's collapsed and expanded, respectively ["class1", "class2"]<br />
togglehtml: ["suffix", " <small><small>[Expand]</small></small>", " <small><small>[Collapse]</small></small>"], //Additional HTML added to the header when it's collapsed and expanded, respectively ["position", "html1", "html2"] (see docs)<br />
animatespeed: "fast", //speed of animation: integer in milliseconds (ie: 200), or keywords "fast", "normal", or "slow"<br />
oninit:function(expandedindices){ //custom code to run when headers have initalized<br />
//do nothing<br />
},<br />
onopenclose:function(header, index, state, isuseractivated){ //custom code to run whenever a header is opened or closed<br />
//do nothing<br />
}<br />
})<br />
<br />
</script><br />
</head><br />
</html></div>Dbbyhttp://2014.igem.org/Team:Toulouse/template/AccordionTeam:Toulouse/template/Accordion2014-10-15T22:19:02Z<p>Dbby: </p>
<hr />
<div><html><br />
<head><br />
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<br />
<br />
<script type="text/javascript" src="https://2011.igem.org/Team:Imperial_College_London/ddaccordion?action=raw&ctype=text/js"><br />
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/***********************************************<br />
* Accordion Content script- (c) Dynamic Drive DHTML code library (www.dynamicdrive.com)<br />
* Visit http://www.dynamicDrive.com for hundreds of DHTML scripts<br />
* This notice must stay intact for legal use<br />
***********************************************/<br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
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<script type='text/javascript' src='http://ajax.googleapis.com/ajax/libs/jquery/1.9.0/jquery.min.js'><br />
<br />
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<br />
<style type="text/css"><br />
<br />
.technology{ /*header of 2nd demo*/<br />
cursor: hand;<br />
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<br />
font-family:'Open Sans';<br />
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font-weight:600;<br />
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}<br />
<br />
.technology2{ /*header of 2nd demo title 2*/<br />
cursor: hand;<br />
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font-family:'Open Sans';<br />
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color: #225e22;<br />
}<br />
<br />
</style><br />
<br />
<script type="text/javascript"><br />
<br />
//Initialize 2nd demo:<br />
ddaccordion.init({<br />
headerclass: "technology", //Shared CSS class name of headers group<br />
contentclass: "thelanguage", //Shared CSS class name of contents group<br />
revealtype: "click", //Reveal content when user clicks or onmouseover the header? Valid value: "click", "clickgo", or "mouseover"<br />
mouseoverdelay: 200, //if revealtype="mouseover", set delay in milliseconds before header expands onMouseover<br />
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onemustopen: false, //Specify whether at least one header should be open always (so never all headers closed)<br />
animatedefault: false, //Should contents open by default be animated into view?<br />
persiststate: false, //persist state of opened contents within browser session?<br />
toggleclass: ["closedlanguage", "openlanguage"], //Two CSS classes to be applied to the header when it's collapsed and expanded, respectively ["class1", "class2"]<br />
togglehtml: ["suffix", " <small><small>[Expand]</small></small>", " <small><small>[Collapse]</small></small>"], //Additional HTML added to the header when it's collapsed and expanded, respectively ["position", "html1", "html2"] (see docs)<br />
animatespeed: "fast", //speed of animation: integer in milliseconds (ie: 200), or keywords "fast", "normal", or "slow"<br />
oninit:function(expandedindices){ //custom code to run when headers have initalized<br />
//do nothing<br />
},<br />
onopenclose:function(header, index, state, isuseractivated){ //custom code to run whenever a header is opened or closed<br />
//do nothing<br />
}<br />
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<br />
<br />
<br />
<br />
<br />
//Initialize 2nd demo:<br />
ddaccordion.init({<br />
headerclass: "technology2", //Shared CSS class name of headers group<br />
contentclass: "thelanguage", //Shared CSS class name of contents group<br />
revealtype: "click", //Reveal content when user clicks or onmouseover the header? Valid value: "click", "clickgo", or "mouseover"<br />
mouseoverdelay: 200, //if revealtype="mouseover", set delay in milliseconds before header expands onMouseover<br />
collapseprev: false, //Collapse previous content (so only one open at any time)? true/false <br />
defaultexpanded: [], //index of content(s) open by default [index1, index2, etc]. [] denotes no content.<br />
onemustopen: false, //Specify whether at least one header should be open always (so never all headers closed)<br />
animatedefault: false, //Should contents open by default be animated into view?<br />
persiststate: false, //persist state of opened contents within browser session?<br />
toggleclass: ["closedlanguage", "openlanguage"], //Two CSS classes to be applied to the header when it's collapsed and expanded, respectively ["class1", "class2"]<br />
togglehtml: ["suffix", " <small><small>[Expand]</small></small>", " <small><small>[Collapse]</small></small>"], //Additional HTML added to the header when it's collapsed and expanded, respectively ["position", "html1", "html2"] (see docs)<br />
animatespeed: "fast", //speed of animation: integer in milliseconds (ie: 200), or keywords "fast", "normal", or "slow"<br />
oninit:function(expandedindices){ //custom code to run when headers have initalized<br />
//do nothing<br />
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onopenclose:function(header, index, state, isuseractivated){ //custom code to run whenever a header is opened or closed<br />
//do nothing<br />
}<br />
})<br />
<br />
</script><br />
</head><br />
</html></div>Dbbyhttp://2014.igem.org/Team:Toulouse/template/AccordionTeam:Toulouse/template/Accordion2014-10-15T22:17:15Z<p>Dbby: </p>
<hr />
<div><html><br />
<head><br />
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Strict//EN" "http://www.w3.org/TR/xhtml2/DTD/xhtml1-strict.dtd"><br />
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en"><br />
<br />
<br />
<script type="text/javascript" src="https://2011.igem.org/Team:Imperial_College_London/ddaccordion?action=raw&ctype=text/js"><br />
<br />
/***********************************************<br />
* Accordion Content script- (c) Dynamic Drive DHTML code library (www.dynamicdrive.com)<br />
* Visit http://www.dynamicDrive.com for hundreds of DHTML scripts<br />
* This notice must stay intact for legal use<br />
***********************************************/<br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<script type='text/javascript' src='http://ajax.googleapis.com/ajax/libs/jquery/1.9.0/jquery.min.js'><br />
<br />
<br />
<br />
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<br />
<style type="text/css"><br />
<br />
.technology{ /*header of 2nd demo*/<br />
cursor: hand;<br />
cursor: pointer;<br />
border-bottom:3px solid #BDCBBD;<br />
<br />
font-family:'Open Sans';<br />
color: green;<br />
font-weight:600;<br />
font-size:24px;<br />
margin:0 0 33px 0;<br />
border:none;<br />
<br />
}<br />
<br />
.technology2{ /*header of 2nd demo title 2*/<br />
cursor: hand;<br />
cursor: pointer;<br />
border-bottom:3px solid #BDCBBD;<br />
font-family:'Open Sans';<br />
color:#5a6060; <br />
font-weight:600; <br />
font-size:18px; <br />
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<br />
.openlanguage{ /*class added to contents of 2nd demo when they are open*/<br />
color: green;<br />
}<br />
<br />
.closedlanguage{ /*class added to contents of 2nd demo when they are closed*/<br />
color: #225e22;<br />
}<br />
<br />
</style><br />
<br />
<script type="text/javascript"><br />
<br />
//Initialize 2nd demo:<br />
ddaccordion.init({<br />
headerclass: "technology", "technology2", //Shared CSS class name of headers group<br />
contentclass: "thelanguage", //Shared CSS class name of contents group<br />
revealtype: "click", //Reveal content when user clicks or onmouseover the header? Valid value: "click", "clickgo", or "mouseover"<br />
mouseoverdelay: 200, //if revealtype="mouseover", set delay in milliseconds before header expands onMouseover<br />
collapseprev: false, //Collapse previous content (so only one open at any time)? true/false <br />
defaultexpanded: [], //index of content(s) open by default [index1, index2, etc]. [] denotes no content.<br />
onemustopen: false, //Specify whether at least one header should be open always (so never all headers closed)<br />
animatedefault: false, //Should contents open by default be animated into view?<br />
persiststate: false, //persist state of opened contents within browser session?<br />
toggleclass: ["closedlanguage", "openlanguage"], //Two CSS classes to be applied to the header when it's collapsed and expanded, respectively ["class1", "class2"]<br />
togglehtml: ["suffix", " <small><small>[Expand]</small></small>", " <small><small>[Collapse]</small></small>"], //Additional HTML added to the header when it's collapsed and expanded, respectively ["position", "html1", "html2"] (see docs)<br />
animatespeed: "fast", //speed of animation: integer in milliseconds (ie: 200), or keywords "fast", "normal", or "slow"<br />
oninit:function(expandedindices){ //custom code to run when headers have initalized<br />
//do nothing<br />
},<br />
onopenclose:function(header, index, state, isuseractivated){ //custom code to run whenever a header is opened or closed<br />
//do nothing<br />
}<br />
})<br />
<br />
</script><br />
</head><br />
</html></div>Dbbyhttp://2014.igem.org/Team:Toulouse/template/AccordionTeam:Toulouse/template/Accordion2014-10-15T22:14:57Z<p>Dbby: </p>
<hr />
<div><html><br />
<head><br />
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Strict//EN" "http://www.w3.org/TR/xhtml2/DTD/xhtml1-strict.dtd"><br />
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en"><br />
<br />
<br />
<script type="text/javascript" src="https://2011.igem.org/Team:Imperial_College_London/ddaccordion?action=raw&ctype=text/js"><br />
<br />
/***********************************************<br />
* Accordion Content script- (c) Dynamic Drive DHTML code library (www.dynamicdrive.com)<br />
* Visit http://www.dynamicDrive.com for hundreds of DHTML scripts<br />
* This notice must stay intact for legal use<br />
***********************************************/<br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<script type='text/javascript' src='http://ajax.googleapis.com/ajax/libs/jquery/1.9.0/jquery.min.js'><br />
<br />
<br />
<br />
</script><br />
<br />
<br />
<style type="text/css"><br />
<br />
.technology{ /*header of 2nd demo*/<br />
cursor: hand;<br />
cursor: pointer;<br />
border-bottom:3px solid #BDCBBD;<br />
<br />
font-family:'Open Sans';<br />
color: green;<br />
font-weight:600;<br />
font-size:24px;<br />
margin:0 0 33px 0;<br />
border:none;<br />
<br />
}<br />
<br />
.technology2{ /*header of 2nd demo title 2*/<br />
cursor: hand;<br />
cursor: pointer;<br />
border-bottom:3px solid #BDCBBD;<br />
font-family:'Open Sans';<br />
color:#5a6060; <br />
font-weight:600; <br />
font-size:18px; <br />
margin:0 0 30px 0; <br />
border:none;<br />
}<br />
<br />
.openlanguage{ /*class added to contents of 2nd demo when they are open*/<br />
color: green;<br />
}<br />
<br />
.closedlanguage{ /*class added to contents of 2nd demo when they are closed*/<br />
color: #225e22;<br />
}<br />
<br />
</style><br />
<br />
<script type="text/javascript"><br />
<br />
//Initialize 2nd demo:<br />
ddaccordion.init({<br />
headerclass: "technology", //Shared CSS class name of headers group<br />
contentclass: "thelanguage", //Shared CSS class name of contents group<br />
revealtype: "click", //Reveal content when user clicks or onmouseover the header? Valid value: "click", "clickgo", or "mouseover"<br />
mouseoverdelay: 200, //if revealtype="mouseover", set delay in milliseconds before header expands onMouseover<br />
collapseprev: false, //Collapse previous content (so only one open at any time)? true/false <br />
defaultexpanded: [], //index of content(s) open by default [index1, index2, etc]. [] denotes no content.<br />
onemustopen: false, //Specify whether at least one header should be open always (so never all headers closed)<br />
animatedefault: false, //Should contents open by default be animated into view?<br />
persiststate: false, //persist state of opened contents within browser session?<br />
toggleclass: ["closedlanguage", "openlanguage"], //Two CSS classes to be applied to the header when it's collapsed and expanded, respectively ["class1", "class2"]<br />
togglehtml: ["suffix", " <small><small>[Expand]</small></small>", " <small><small>[Collapse]</small></small>"], //Additional HTML added to the header when it's collapsed and expanded, respectively ["position", "html1", "html2"] (see docs)<br />
animatespeed: "fast", //speed of animation: integer in milliseconds (ie: 200), or keywords "fast", "normal", or "slow"<br />
oninit:function(expandedindices){ //custom code to run when headers have initalized<br />
//do nothing<br />
},<br />
onopenclose:function(header, index, state, isuseractivated){ //custom code to run whenever a header is opened or closed<br />
//do nothing<br />
}<br />
})<br />
<br />
</script><br />
</head><br />
</html></div>Dbbyhttp://2014.igem.org/Team:Toulouse/Notebook/CalendarTeam:Toulouse/Notebook/Calendar2014-10-15T22:11:15Z<p>Dbby: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
{{:Team:Toulouse/template/Accordion}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : font Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<script type='text/javascript' src='http://ajax.googleapis.com/ajax/libs/jquery/1.9.0/jquery.min.js'></script><br />
<br />
<script type='text/javascript'> $(function(){ <br />
$(window).scroll(function () {<br />
if ($(this).scrollTop() > 250) {<br />
$('#column-left').addClass("fixNavigation"); <br />
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<br />
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<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.nomsd{color:#5a6060; font-family:'Open Sans'; font-size:14px; font-weight:bold; margin:0 30px 50px 30px; line-height:24px; text-align: center;}<br />
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<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/d0/Template-igem2014-slide1.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
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<br />
.banniere-content h2{<br />
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.Title{<br />
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color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
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font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
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}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
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a.Link:hover{<br />
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<br />
#column-left{<br />
float:left;<br />
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float: left;<br />
padding: 15px 10px 15px 15px;<br />
border: 1px solid #ccc;<br />
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background-color: #eee;<br />
}<br />
<br />
.fixNavigation{ <br />
z-index: 9999;<br />
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<br />
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padding-bottom: 10px;<br />
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/*Overview*/<br />
.CropImg{<br />
position : absolute;<br />
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margin-left:-350px;<br />
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<br />
</style><br />
<br />
<br />
<body><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px; position:relative;"><br />
<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Calendar</p> <br />
</div><br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<br />
<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a> | <a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a></p><br />
<br />
<div class="technology">December 2013 – January 2014: Team selection</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- iGEM competition presentation and explanations<br/><br />
- Constitution of the team by interviewing different students from Université Paul Sabatier and INSA<br/><br />
</p><br />
<p class="texte" style="text-align:center"><B> The adventure begins for the Toulouse iGEM Team 2014! </B><br />
</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology">January – June 2014: Projects brainstorming</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- Thanks to weekly meetings, our team was able to discuss about new project ideas and publications.<br />
<br/><br />
- Many ideas were approached and debated regarding the feasibility thanks to the literature and supervisors including teachers and researchers<br />
<br/><br />
This is a list of the main projects: <br />
<br/><br />
- Let's save our trees with SubtiTree: the purpose is to use <i>Bacillus subtilis</i> as an optimized bacterium to detect, target a fungi and secrete fungicides to destroy it<br />
<br/><br />
- <i>E. coli</i> cancer: the concept was to create a bacterium able to colonize specifically the hypoxic regions of the tumor. The oxygen-sensitive bacterium would not be able to develop in the healthy zones<br />
<br/><br />
- Reduction of ruminants’ methane production: the goal is to reduce the production of methane by ingesting a microorganism in the animal’s rumen. This bacterium would be capable of reducing the quantity of metabolic hydrogen and eliminating the methanogenic Archaes population<br />
<br/><br />
</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p><br />
</div><br />
<br />
<br />
<div class="technology">June 2014: Choice of SubtiTree project</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">The discussion has been long to estimate the practicability of our final project. By the end of June, most of the ideas were eliminated but two of them remained: controlling the cancer or saving the trees. It became easily clear that SubtiTree was successfully completed whether regarding the scientific part or the ethical aspect. Indeed, our knowledge in medicine was not sufficient enough to evaluate the consequences of a bacterium injection in the human body for <i>E. coli</i> cancer.</br><br />
Therefore, the whole team agreed on focusing on a local issue as the preservation of the Canal du Midi plane trees. However, the final goal was based on the idea to allow our optimized bacteria to be efficient for all fungi infections in the environment.</br><br />
By this period, we evaluated our budget to approximately <B>40 k€</B>.<br />
</p> <br />
<br />
<br />
<br />
<br />
<br />
<br />
<div class="technology">Week 1(16-22 June)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Bibliography researches about our subject<br/><br />
- Cleaning the whole lab to allow good manipulations and avoid any contamination<br/><br />
- Inventory of necessary lab equipment and biobricks<br/><br />
- Summarize the iGEM protocols<br/><br />
- Prepare all growing media <br/><br />
- Preparing competent cells by optimizing protocols<br/><br />
- Transformation of RFP plasmid to practice<br />
</p><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Increase in our communication strategy to find more sponsors specialized in environment and ecological matters <br/><br />
- Support of Adisseo, Toulouse White Biotechnology, LISBP, INSA Toulouse<br/><br />
- Organization of a weekly meeting each Friday with our supervisors to tackle any problem encountered<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/20/2014)</p><br />
<p class="texte"><br />
- Distribution of the non-scientific tasks regarding the wiki, communication, sponsorship, ethical problems, safety but also the lab work<br/><br />
- Discussion about the different construction parts of the project<br/><br />
- Need to check the absence of stop codon in fungicides sequences<br />
</p> <br />
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<br></br><br />
<br />
<div class="technology">Week 2 (23-29 June)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Ordering the list of necessary products for the laboratory and biobricks<br/><br />
- Checking and validation of the genes sequences<br/><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- BioSynSyS conference work: powerpoint, logo in order to present our SubtiTree project to a whole audience of scientific and researchers (http://biosynsys2014.sciencesconf.org/)<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- E. coli transformation with BBA_J004450 (pSB1C3)<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/27/2014)</p><br />
<p class="texte"><br />
- Concentration of antibiotics in media must be checked<br/><br />
- The transformation rate for pUC19 must be evaluated<br/><br />
- The transformation efficiency must be calculated regarding the quantity of DNA<br/><br />
- Possibility to contact the cities halls for the sponsorship<br/><br />
- Check the mechanism of action of each fungicide to complete the ethical part<br />
</p><br />
<br />
<p class="title1" id="select4">July 2014</p><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Beginning of the laboratory work to create our optimized bacterium <br/><br />
- Research of sponsors and the communication thanks to the press<br />
</p> <br />
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<br />
<div class="technology">Week 3 (30 June -6 July) and Week 4 (7-13 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the BioSynSys conferences: presentation of SubtiTree<br/><br />
- We have put in place a newsletter system the first day of each month to keep all our sponsors and people who support us updated about the project, our financial state.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Design of all the constructions needed with cloning and restriction enzymes to save some time and allow a better comprehension of our bacterium system </br><br />
Problem: A problem was reported in the use of the CYP. Indeed, the biobrick is not expressed properly in <i>Bacillus subtilis</i> strain. Therefore we had to look at the literature to find a new fungicide called GAFP-1.<br/><br />
- A bioreactor was conceived to reproduce the composition of the sap in the plane trees. The main goal was to identify the behavior of Bacillus strain in a medium composed of many different carbon sources.<br/><br />
- Competent cells and transformation practice using GFP and RFP.<br />
</p><br />
<br />
<p class="title3">Weekly meetings with the instructors (07/04/2014) and (07/11/2014)</p><br />
<p class="texte"><br />
- iGEM efficiency kit does not work, we shall not use it anymore<br/><br />
- Organization of a timetable to check the stored and sterile equipment everyday</br><br />
- Try a transformation in <i>Bacillus subtilis</i><br />
</p> <br />
<br />
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<br />
<div class="technology">Week 5 (14-20 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Our team was interviewed by many local newspapers such as 20minutes, Metronews, La Voix du Midi, La Dépêche but also by the local television channel France3 Midi-Pyrénées to present our draft to the community. Moreover, SubtiTree was approached in some radio programs like Virgin Radio Toulouse and France info.</br><br />
- Support of ThermoFisher, New Englands BioLabs, Qiagen, Eurofins but also GenoToul, Labex Tulip, L’Université Paul Sabatier, CROUS.<br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- GFP and RFP digestion amplified by miniprep and gel electrophoresis <br/><br />
Problem: the digested GFP doesn’t appear on the gel contrary to RFP plasmid. We assumed a problem with the GFP miniprep. We tried the miniprep from the INSA Toulouse team from 2013 and the GFP plasmid was fully digested.<br/><br />
- Transformation and cryopreservation of biobricks BBa_K823002 (PlepA), BBa_K823003 (Pveg), BBa_K606061 (RBS SpoVG) and BBa_B0015 (double terminator), BBa_K823023 (pSBBS1C), BBa_K733013 (Pveg+ RBS) in <i>E. coli</i>.<br/><br />
- Transformation of the Munich B. subtilis backbones<br/><br />
- Cloning of BBa_K606061 (RBS SpoVG) with BBa_K823003 (PvEG) and BBa_K823002 (PlepA)<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (07/18/2014)</p><br />
<p class="texte"><br />
- Transformation of the Eurofins genes<br/><br />
- Start assembling the biobricks for the fungicides<br/><br />
- Check all the cloning<br />
</p><br />
<br />
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<br />
<div class="technology">Week 6 (21 July-27 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Description of SubtiTree project and determination of the goodies for a crowdfunding campaign on Ulule website<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Transformation of BBA_1364002 (GAFP1) and BBA_1364003 (D4E1) fungicides genes<br/><br />
- Subculture of the clones for each gene<br/><br />
- PCR and migration on electrophoresis gel<br/><br />
- Transformation BBA_1364003 (D4E1) on pSB1C3 and BBA_1364002 (GAFP1) on pSB1C3<br/><br />
- Cloning BBA_1364002 (GAFP1) with BBa_B0015 (double terminator) and BBA_1364003 (D4E1) with BBa_K823003 (Pveg)<br/><br />
</p><br />
<br />
<p class="title3">Others:</p><br />
<p class="texte"><br />
- Research for plane tickets and hotels in Boston for the Giant Jamboree<br/><br />
- New idea: analyze the plane tree sap to determine the composition<br/><br />
</p><br />
<br />
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<br />
<div class="technology">August 2014 </div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finishing the cloning for the different parts of the bacterium<br/><br />
- Putting in place the fungicides, binding and chemotaxis tests<br/><br />
</p> <br />
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<br />
<div class="technology">Week 7 (28 July-3 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Financial supports of the Ministery (Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt) and Voies Navigables de France<br/><br />
- Interview with Johan Langot from Sciences Animation for a possible presentation at the Toulouse Festival of Science<br/><br />
- Launch of the crowdfunding campaign on Ulule<br />
</p><br />
<br />
<p class="title3">Lab work: </p><br />
<p class="texte"><br />
- Cloning BBa_K823003 (Pveg) + RFP and BBa_K823002 (PlepA) + RFP in pSBBs1C<br/><br />
- Test of every pSBBs vector : BBa_K823021 (pSBBS1C-lacZ), BBa_K823022 (pSBBS4S), BBa_K823023 (pSBBS1C), minipreps and cryopreservation of the best clones<br/><br />
- Checking of BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) transformants (biobrick BBa_K1364007) and BBA_1364003 (D4E1) + BBa_K823003 (Pveg) biobrick BBa_K1364009<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) + pSBBS4S (BBa_K823022) and subculture of the colonies<br/><br />
- Cloning of BBa_K823003 (Pveg) + BBA_1364003 (D4E1) with BBa_B0015 (double terminator)<br/><br />
- Cloning of BBa_K823003 (Pveg) + RFP (BBa_K1364017) in pSBBS4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_K823002 (PlepA) + RFP (BBa_K1364016) in pSBBs4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_1364018 (Strong Promotor + RFP)<br/><br />
- Cloning of BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) + Promotor BBa_K823003 (Pveg) biobirck BBa_K1364012 with gel extraction for BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) and a PCR cleanup for BBa_K823003 (Pveg), subculture<br/><br />
- Cloning BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBS4S in <i>E. coli</i><br/><br />
- Transformation of binding gene with <i>E. coli</i> competent cells<br/><br />
- Transformation of BBa_K1364000, the chemotaxis gene in <i>E. coli</i><br/><br />
Problem: the transformation worked but a cell layer appeared. A new subculture of the colonies was made as well as a new spreading on petri dishes.<br/><br />
- Spreading of BBa_K1162001 (EcAMP), miniprep and digestion<br/><br />
Problem: the digestion did not work => Issue with the quantity of DNA in the miniprep is assumed<br/><br />
- First experience of chemotaxis with a glucose chemo-attractant<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/01/2014)</p><br />
<p class="texte"><br />
- Possible problem with the restriction enzymes regarding the digestion: new recommendations<br />
<br/><br />
- Discussion about EcAMP cloning which presents some issues<br />
<br/><br />
- Discussion about the transfer of pSB1C3 from E. coli to Bacillus strain<br />
</p> <br />
<br />
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<br />
<div class="technology">Week 8 (04-10 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the “Passion Jeune” competition organized by a French bank foundation named Fondation Crédit Agricole Toulouse<br />
</p><br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Check the cloning of BBa_K823003 (Pveg)+BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBs4S and cryopreservation<br />
<br/><br />
- Transformation of BBa_K823003 (Pveg) +BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) (biobrick BBa_ K1364008) in BBa_K823022 (pSBBS4S) <br />
<br/><br />
- Cloning of BBa_K1364001, the binding gene + BBa_K823003 (Pveg) on pSB1C3 and pSBBS 4S<br />
<br/><br />
Problem: the band is at 1500 bp instead of 1300 bp on the gel<br />
<br/><br />
- Cloning of BBa_K1162001 (EcAMP) + BBa_K823003 (Pveg) + BBa_K606061 (RBS SpoVG), miniprep, PCR and digestion <br />
<br/><br />
Problem: the fragment of DNA is too small (149 bp) to be seen on the gel and was probably lost during the PCR cleanup. Instead of that, we used the heat enzymes inactivation at 95°C for 15 minutes. <br />
<br/><br />
- Cloning of Promotor + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) in pSB1C3 and pSBBS4S and cryopreservation<br />
<br/><br />
- Elaboration of an efficient fungicide test protocol <br />
<br/><br />
- Test of the fungus growth on PDA medium + test of growth for Bacillus subtilis liquid culture on PDA<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/08/2014)</p><br />
<p class="texte"><br />
- Ask iGEM headquarters if all the biobricks must be on pSB1C3 plasmid<br />
<br/><br />
- Use another strain of E. coli (DH5-1 instead of DH5 alpha) to have a faster growth<br />
<br/><br />
- Check which quantity of Bacillus subtilis is necessary to naturally destroy the fungus<br />
</p><br />
<br />
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<br />
<div class="technology">Week 9 (11-17 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Publication of more articles about our project in Labiotech, Développement durable (Networkvisio), Midi Libre, l’Indépendant, DigiSchool, La voix du Midi Lauragais, LURIO Addl.<br />
</p><br />
<br />
<p class="title2">Lab work:</p><br />
<p class="texte"><br />
- Evaluation of the bacterial decrease rate from 10°C to 4°C with a sporuling strain of Bacillus subtilis.<br />
<br/><br />
- Chemotaxis test with different concentration of glucose on a 0.3% agar.<br />
<br/><br />
- Miniprep of pSBbs4S with binding gene and transformation in <i>Bacillus subtilis</i>.<br />
<br/><br />
Problem: no digestion was visible on the gel => the cloning failed<br />
<br/><br />
- Transformation of BBa_K1374010 (Pveg + RBS SpoVG + EcAMP) + BBa_K1364012 (RBS + Gafp1 + D4E1 + double terminator), subculture<br />
<br/><br />
- Subculture of Bacillus subtilis clones transformed by BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) in pSBBs4S, cryopreservation<br />
<br/><br />
- Fungicide test<br />
</p><br />
<br />
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<br />
<div class="technology">Week 10 (18-24 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Ethics: Interview with a specialist in ethical sciences, Vincent Grégoire Delory to complete our reflexion about the ethical issues in SubtiTree project.<br />
<br/><br />
- Wiki: hiring a web designer, Adrien Nicod, to improve the aesthetic effect and the logo.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Subculture and transformation in Bacillus subtilis of BBa_K1364011 in pSBBS4S (BBa_K823022) and cloning of BBa_K1364011 on pSBBS1C (BBa_K823023).<br />
<br/><br />
Problem: no clone had the insert on pSBBS1C => the cloning had to be done again<br />
- Miniprep of BBa_K1364011 in BBa_K823023 (pSBBS1C) + Transformation in <i>B. subtilis</i>.<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) , BBA_1364002 (GAFP1) , BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) on BBa_K823023 (pSBBS1C) <br />
<br/><br />
- Transformation BBa_1364004 (in pSBBS4S in E.coli <br />
<br/><br />
- Cloning of BBa_K1364014 (Pveg + RBS SpoVG + EcAMP + GAFP1 +D4E1 + double terminator) in K823022 (pSBBS4S) and in BBa_K823023 (pSBBS1C), miniprep and digestion by restriction enzymes to see the size of the insert on a 1% gel<br />
<br/><br />
Problem: no clone had the insert on pSBBS1C => the cloning had to be done again<br />
</p><br />
<br />
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<br />
<div class="technology">Week 11 (25- 31 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Beginning of the creation and redaction of the different parts of the wiki by the Toulouse iGEM Toulouse<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Cloning of BBa_K1364014 in BBa_K823022 and in BBa_K823023 <br />
<br/><br />
- Cloning GAFP1 + BBa_K823023 (pSBBS1C) in Bacillus subtilis with a change in the protocol: pre-induction of the culture with 0.125 µg/ml of chloramphenicol one hour after the addition of DNA, and let grow for another hour.<br />
<br/><br />
Problem: no colony <br />
<br/><br />
- Liquid culture of Bacillus + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) for future fungicide tests<br />
<br/><br />
- Miniprep of BBa_ K1364011 in BBa_ K823023 (pSBBs1C) and transformation in Bacillus subtilis strain. <br />
<br/><br />
- Fungicide test of BBa_K1364011 in pSBBs4S, Promotor + BBA_1364002 (GAFP1) + Terminator (biobrick BBa_K1364008) in pSBBS4S, Promotor + BBA_1364003 (D4E1) + Terminator (biobrick BBa_K1364009) in pSBBS4S, Promotor + BBA_1364002 (GAFP1) + BA_1364003 (D4E1) + Terminator ( biobrick BBa_K1364013) in pSBBS4S<br />
<br/><br />
- Transformation of a new vector PKL 190 (threonine integrative) in Bacillus strain <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/29/2014)</p><br />
<p class="texte"><br />
- Boston accommodation must be booked<br />
<br/><br />
- Discussion about the contamination seen on the fungicide tests : the issue seems to come from the sterile water which contained contaminants<br />
<br/><br />
- Objectives : chemotaxis, binding and fungicides tests have to be performed again<br />
</p> <br />
<br />
<p class="title1" id="select6"> September 2014</p><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finish the tests of the different modules<br />
<br/><br />
- Establish the final editorial parts for the wiki<br />
<br/><br />
- Design of the wiki<br />
<br/><br />
- Sequencing the different assembled parts of our bacterium<br />
</p> <br />
<br />
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<br />
<div class="technology">Week 12 (1-7 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Different parts of the wiki by the Toulouse iGEM Toulouse<br/><br />
- Reservation of Boston accommodation<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Chemotaxis test and modelling<br />
<br/><br />
- Fungicides tests on EcAMP-A, EcAMP-B, EcAMP-C, EcAMP-D, MaxiOpA, MaxiOpB, MaxiOpC, MaxiOpD from 2ml of overnight cultures. Only with 25000 conidia with both fungi.<br />
<br/><br />
- PCR on pSBBS4S + BBa_K1162001 (EcAMP) <br />
<br/><br />
Problem: failed on both diluted plasmid and colony<br />
<br/><br />
- Spreading of BBa_K1364014 + BBa_ K823022 in threonine medium and in medium without threonine <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/05/2014)</p> <br />
<p class="texte"><br />
- The first sequencing was not successful => new sequencing with one primer/tube<br/><br />
- Chemotaxis test: increase the concentration in bacterium, try a new protocol by capillarity<br/><br />
- End of the binding test: IT WORKS PERFECTLY! SubtiTree has the good phenotype and presents a better binding property than the wild type Bacillus subtilis strain.<br/><br />
- Fungicides test: contaminants with the fungus. Need to ask for another culture of our fungi.<br />
</p><br />
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<br />
<div class="technology">Week 13 (8- 14 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Telephone interview for an article in a French national review named Fluvial which will be published at the end of September.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- PCR on D4E1 colony with different primers<br />
<br/>- Culture and miniprep of EcAMP to get more DNA + analytic digestion <br />
<br/>- Transformation of K1364014+K1364006 in E. coli <br />
<br/>- Transformation of GAFP1 + D4E1, GAFP1 and EcAMP+GAFP1+D4E1 on Bacillus subtilis + colony PCR + threonine test<br />
<br/>NB: good results for the maxi operon and GAFP1 but not for GAFP1 + D4E1 CA <br />
<br/>- Cloning of K606013+pEX_K4, 823022+PlepA_RFP, 823022+ Pveg_RFP<br />
<br/>- Chemotaxis tests: different times of LB + agar addition in the LB medium (after 3 minutes, 5 minutes, 8 minutes, 10 minutes…) are performed => No difference of the results after spreading on the plates.<br />
<br/>- New test of chemotaxis: capillary between two Eppendorf tubes<br />
<br/>- Spreading of the fungi on a medium which mimics the sap<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/11/2014)</p> <br />
<p class="texte"><br />
- Try to use the GFP or RFP to follow the chemotaxis test.<br />
<br/>- Make a new glass process for the chemotaxis capillary test.<br />
<br/>- Possibility of contacting an INRA laboratory to get pictures of the binding test with confocal microscope.<br />
<br/>- Sequencing the binding construction.<br />
<br/>- Try different temperatures for the fungicide tests: 20°C, 25°C, and 37°C to reduce the fungus growth.<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology">Week 14 (15 - 21 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Support of two French city halls: Mairie de Montréal, Mairie de Ventenac en Minervois.<br />
<br/>- First interview by the French national radio France Inter which will be aired on October 12th.<br />
<br/>- Beginning of the organization of the iGEM Toulouse 2014 acknowledgement day: the purpose is to present the project to the students and give a feedback on the competition to the sponsors.<br />
<br/>- Final design of the wiki<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Threonine tests for the BBa_K1364014 maxi operon (EcAMP1 – GAFP1 – D4E1) and mini operon BBa_K1364013 (GAFP1-D4E1)<br />
<br/>- Colony PCR on the mini operon BBa_K1364013 (GAFP1 - D4E1)<br />
<br/>- Colony PCR on BBa_K1364011 on BBa_K823022 (pSBBS4S)<br />
<br/>- Chemotaxis test with wild type <i>Bacillus subtilis</i> strain using Imperial College protocol<br />
<br/>- Tansformation of BBa_K1364011 + BBa_K823022 (pSBBS4S) in Bacillus subtilis<br />
<br/>- Culture of fungi with fungicides<br />
<br/>- Sequencing of plasmids containing the fungicides<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/18/2014)</p> <br />
<p class="texte"><br />
- Sequencing worked for D4E1 and GAFP1 and the mini operon BBa_K1364013 (GAFP1-D4E1).<br />
<br/>- The next step is to sequence the maxi operon (EcAMP1 – GAFP1 – D4E1).<br />
<br/>- Try different approaches for the chemotaxis:<br />
<br/>- New protocol with the capillary assay with a new system made by a glassblower.<br />
<br/>- Make the Imperial College test again by testing 3 different solutions: chitin, N-acetylglucosamine and another carbon source.<br />
<br/>- Decrease the number of conidia and temperature for the fungicide test.<br />
</p><br />
<br />
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<br />
<div class="technology">Week 15 (22 - 28 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- New support of Sallèles d’Aude city hall.<br />
<br/>- Payment of the plane tickets.<br />
<br/>- Acknowledgement day (4th December): contact with catering service, reservation of the amphitheater, list of guests…<br />
<br/>- Order of the T shirt for the Jamboree! We look awesome with them!<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Last experiments regarding chemotaxis tests.<br />
</p><br />
<br />
<br />
<p class="title3">Weekly meeting with the instructors(09/26/2014)</p> <br />
<p class="texte"><br />
- Focus on the wiki design and writing.<br />
<br/>- Start making the power point presentation for Boston and the poster.<br />
<br/>- Division of responsibilities for all the non-scientific work.<br />
</p><br />
<br />
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<div class="technology">October 2014</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">Main activites</p><br />
<p class="texte"><br />
- Preparation of the wiki every day… all day long…. so hard to work on this even at night!<br />
<br/>- Preparation of the poster and the presentation for the Jamboree with our instructors <br />
<br/><br />
- Daily presentations in front of the laboratory teachers to repeat again and again and again… let’s get ready for the Jamboree!<br />
</p> <br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Calendar</p> <br />
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<br />
<div class="technology">December 2013 – January 2014: Team selection</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- iGEM competition presentation and explanations<br/><br />
- Constitution of the team by interviewing different students from Université Paul Sabatier and INSA<br/><br />
</p><br />
<p class="texte" style="text-align:center"><B> The adventure begins for the Toulouse iGEM Team 2014! </B><br />
</p><br />
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<br />
<div class="technology">January – June 2014: Projects brainstorming</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- Thanks to weekly meetings, our team was able to discuss about new project ideas and publications.<br />
<br/><br />
- Many ideas were approached and debated regarding the feasibility thanks to the literature and supervisors including teachers and researchers<br />
<br/><br />
This is a list of the main projects: <br />
<br/><br />
- Let's save our trees with SubtiTree: the purpose is to use <i>Bacillus subtilis</i> as an optimized bacterium to detect, target a fungi and secrete fungicides to destroy it<br />
<br/><br />
- <i>E. coli</i> cancer: the concept was to create a bacterium able to colonize specifically the hypoxic regions of the tumor. The oxygen-sensitive bacterium would not be able to develop in the healthy zones<br />
<br/><br />
- Reduction of ruminants’ methane production: the goal is to reduce the production of methane by ingesting a microorganism in the animal’s rumen. This bacterium would be capable of reducing the quantity of metabolic hydrogen and eliminating the methanogenic Archaes population<br />
<br/><br />
</p><br />
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<br />
<div class="technology">June 2014: Choice of SubtiTree project</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">The discussion has been long to estimate the practicability of our final project. By the end of June, most of the ideas were eliminated but two of them remained: controlling the cancer or saving the trees. It became easily clear that SubtiTree was successfully completed whether regarding the scientific part or the ethical aspect. Indeed, our knowledge in medicine was not sufficient enough to evaluate the consequences of a bacterium injection in the human body for <i>E. coli</i> cancer.</br><br />
Therefore, the whole team agreed on focusing on a local issue as the preservation of the Canal du Midi plane trees. However, the final goal was based on the idea to allow our optimized bacteria to be efficient for all fungi infections in the environment.</br><br />
By this period, we evaluated our budget to approximately <B> 40 k€ </B>.<br />
</p> <br />
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<br />
<div class="technology">Week 1(16-22 June)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Bibliography researches about our subject<br/><br />
- Cleaning the whole lab to allow good manipulations and avoid any contamination<br/><br />
- Inventory of necessary lab equipment and biobricks<br/><br />
- Summarize the iGEM protocols<br/><br />
- Prepare all growing media <br/><br />
- Preparing competent cells by optimizing protocols<br/><br />
- Transformation of RFP plasmid to practice<br />
</p><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Increase in our communication strategy to find more sponsors specialized in environment and ecological matters <br/><br />
- Support of Adisseo, Toulouse White Biotechnology, LISBP, INSA Toulouse<br/><br />
- Organization of a weekly meeting each Friday with our supervisors to tackle any problem encountered<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/20/2014)</p><br />
<p class="texte"><br />
- Distribution of the non-scientific tasks regarding the wiki, communication, sponsorship, ethical problems, safety but also the lab work<br/><br />
- Discussion about the different construction parts of the project<br/><br />
- Need to check the absence of stop codon in fungicides sequences<br />
</p> <br />
<br />
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<br />
<br></br><br />
<br />
<div class="technology">Week 2 (23-29 June)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Ordering the list of necessary products for the laboratory and biobricks<br/><br />
- Checking and validation of the genes sequences<br/><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- BioSynSyS conference work: powerpoint, logo in order to present our SubtiTree project to a whole audience of scientific and researchers (http://biosynsys2014.sciencesconf.org/)<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- E. coli transformation with BBA_J004450 (pSB1C3)<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/27/2014)</p><br />
<p class="texte"><br />
- Concentration of antibiotics in media must be checked<br/><br />
- The transformation rate for pUC19 must be evaluated<br/><br />
- The transformation efficiency must be calculated regarding the quantity of DNA<br/><br />
- Possibility to contact the cities halls for the sponsorship<br/><br />
- Check the mechanism of action of each fungicide to complete the ethical part<br />
</p><br />
<br />
<p class="title1" id="select4">July 2014</p><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Beginning of the laboratory work to create our optimized bacterium <br/><br />
- Research of sponsors and the communication thanks to the press<br />
</p> <br />
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<br />
<div class="technology">Week 3 (30 June -6 July) and Week 4 (7-13 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the BioSynSys conferences: presentation of SubtiTree<br/><br />
- We have put in place a newsletter system the first day of each month to keep all our sponsors and people who support us updated about the project, our financial state.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Design of all the constructions needed with cloning and restriction enzymes to save some time and allow a better comprehension of our bacterium system </br><br />
Problem: A problem was reported in the use of the CYP. Indeed, the biobrick is not expressed properly in <i>Bacillus subtilis</i> strain. Therefore we had to look at the literature to find a new fungicide called GAFP-1.<br/><br />
- A bioreactor was conceived to reproduce the composition of the sap in the plane trees. The main goal was to identify the behavior of Bacillus strain in a medium composed of many different carbon sources.<br/><br />
- Competent cells and transformation practice using GFP and RFP.<br />
</p><br />
<br />
<p class="title3">Weekly meetings with the instructors (07/04/2014) and (07/11/2014)</p><br />
<p class="texte"><br />
- iGEM efficiency kit does not work, we shall not use it anymore<br/><br />
- Organization of a timetable to check the stored and sterile equipment everyday</br><br />
- Try a transformation in <i>Bacillus subtilis</i><br />
</p> <br />
<br />
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<br />
<div class="technology">Week 5 (14-20 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Our team was interviewed by many local newspapers such as 20minutes, Metronews, La Voix du Midi, La Dépêche but also by the local television channel France3 Midi-Pyrénées to present our draft to the community. Moreover, SubtiTree was approached in some radio programs like Virgin Radio Toulouse and France info.</br><br />
- Support of ThermoFisher, New Englands BioLabs, Qiagen, Eurofins but also GenoToul, Labex Tulip, L’Université Paul Sabatier, CROUS.<br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- GFP and RFP digestion amplified by miniprep and gel electrophoresis <br/><br />
Problem: the digested GFP doesn’t appear on the gel contrary to RFP plasmid. We assumed a problem with the GFP miniprep. We tried the miniprep from the INSA Toulouse team from 2013 and the GFP plasmid was fully digested.<br/><br />
- Transformation and cryopreservation of biobricks BBa_K823002 (PlepA), BBa_K823003 (Pveg), BBa_K606061 (RBS SpoVG) and BBa_B0015 (double terminator), BBa_K823023 (pSBBS1C), BBa_K733013 (Pveg+ RBS) in <i>E. coli</i>.<br/><br />
- Transformation of the Munich B. subtilis backbones<br/><br />
- Cloning of BBa_K606061 (RBS SpoVG) with BBa_K823003 (PvEG) and BBa_K823002 (PlepA)<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (07/18/2014)</p><br />
<p class="texte"><br />
- Transformation of the Eurofins genes<br/><br />
- Start assembling the biobricks for the fungicides<br/><br />
- Check all the cloning<br />
</p><br />
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<br />
<div class="technology">Week 6 (21 July-27 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Description of SubtiTree project and determination of the goodies for a crowdfunding campaign on Ulule website<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Transformation of BBA_1364002 (GAFP1) and BBA_1364003 (D4E1) fungicides genes<br/><br />
- Subculture of the clones for each gene<br/><br />
- PCR and migration on electrophoresis gel<br/><br />
- Transformation BBA_1364003 (D4E1) on pSB1C3 and BBA_1364002 (GAFP1) on pSB1C3<br/><br />
- Cloning BBA_1364002 (GAFP1) with BBa_B0015 (double terminator) and BBA_1364003 (D4E1) with BBa_K823003 (Pveg)<br/><br />
</p><br />
<br />
<p class="title3">Others:</p><br />
<p class="texte"><br />
- Research for plane tickets and hotels in Boston for the Giant Jamboree<br/><br />
- New idea: analyze the plane tree sap to determine the composition<br/><br />
</p><br />
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<br />
<div class="technology">August 2014 </div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finishing the cloning for the different parts of the bacterium<br/><br />
- Putting in place the fungicides, binding and chemotaxis tests<br/><br />
</p> <br />
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<br />
<div class="technology">Week 7 (28 July-3 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Financial supports of the Ministery (Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt) and Voies Navigables de France<br/><br />
- Interview with Johan Langot from Sciences Animation for a possible presentation at the Toulouse Festival of Science<br/><br />
- Launch of the crowdfunding campaign on Ulule<br />
</p><br />
<br />
<p class="title3">Lab work: </p><br />
<p class="texte"><br />
- Cloning BBa_K823003 (Pveg) + RFP and BBa_K823002 (PlepA) + RFP in pSBBs1C<br/><br />
- Test of every pSBBs vector : BBa_K823021 (pSBBS1C-lacZ), BBa_K823022 (pSBBS4S), BBa_K823023 (pSBBS1C), minipreps and cryopreservation of the best clones<br/><br />
- Checking of BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) transformants (biobrick BBa_K1364007) and BBA_1364003 (D4E1) + BBa_K823003 (Pveg) biobrick BBa_K1364009<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) + pSBBS4S (BBa_K823022) and subculture of the colonies<br/><br />
- Cloning of BBa_K823003 (Pveg) + BBA_1364003 (D4E1) with BBa_B0015 (double terminator)<br/><br />
- Cloning of BBa_K823003 (Pveg) + RFP (BBa_K1364017) in pSBBS4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_K823002 (PlepA) + RFP (BBa_K1364016) in pSBBs4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_1364018 (Strong Promotor + RFP)<br/><br />
- Cloning of BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) + Promotor BBa_K823003 (Pveg) biobirck BBa_K1364012 with gel extraction for BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) and a PCR cleanup for BBa_K823003 (Pveg), subculture<br/><br />
- Cloning BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBS4S in <i>E. coli</i><br/><br />
- Transformation of binding gene with <i>E. coli</i> competent cells<br/><br />
- Transformation of BBa_K1364000, the chemotaxis gene in <i>E. coli</i><br/><br />
Problem: the transformation worked but a cell layer appeared. A new subculture of the colonies was made as well as a new spreading on petri dishes.<br/><br />
- Spreading of BBa_K1162001 (EcAMP), miniprep and digestion<br/><br />
Problem: the digestion did not work => Issue with the quantity of DNA in the miniprep is assumed<br/><br />
- First experience of chemotaxis with a glucose chemo-attractant<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/01/2014)</p><br />
<p class="texte"><br />
- Possible problem with the restriction enzymes regarding the digestion: new recommendations<br />
<br/><br />
- Discussion about EcAMP cloning which presents some issues<br />
<br/><br />
- Discussion about the transfer of pSB1C3 from E. coli to Bacillus strain<br />
</p> <br />
<br />
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<br />
<div class="technology">Week 8 (04-10 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the “Passion Jeune” competition organized by a French bank foundation named Fondation Crédit Agricole Toulouse<br />
</p><br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Check the cloning of BBa_K823003 (Pveg)+BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBs4S and cryopreservation<br />
<br/><br />
- Transformation of BBa_K823003 (Pveg) +BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) (biobrick BBa_ K1364008) in BBa_K823022 (pSBBS4S) <br />
<br/><br />
- Cloning of BBa_K1364001, the binding gene + BBa_K823003 (Pveg) on pSB1C3 and pSBBS 4S<br />
<br/><br />
Problem: the band is at 1500 bp instead of 1300 bp on the gel<br />
<br/><br />
- Cloning of BBa_K1162001 (EcAMP) + BBa_K823003 (Pveg) + BBa_K606061 (RBS SpoVG), miniprep, PCR and digestion <br />
<br/><br />
Problem: the fragment of DNA is too small (149 bp) to be seen on the gel and was probably lost during the PCR cleanup. Instead of that, we used the heat enzymes inactivation at 95°C for 15 minutes. <br />
<br/><br />
- Cloning of Promotor + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) in pSB1C3 and pSBBS4S and cryopreservation<br />
<br/><br />
- Elaboration of an efficient fungicide test protocol <br />
<br/><br />
- Test of the fungus growth on PDA medium + test of growth for Bacillus subtilis liquid culture on PDA<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/08/2014)</p><br />
<p class="texte"><br />
- Ask iGEM headquarters if all the biobricks must be on pSB1C3 plasmid<br />
<br/><br />
- Use another strain of E. coli (DH5-1 instead of DH5 alpha) to have a faster growth<br />
<br/><br />
- Check which quantity of Bacillus subtilis is necessary to naturally destroy the fungus<br />
</p><br />
<br />
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<br />
<div class="technology">Week 9 (11-17 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Publication of more articles about our project in Labiotech, Développement durable (Networkvisio), Midi Libre, l’Indépendant, DigiSchool, La voix du Midi Lauragais, LURIO Addl.<br />
</p><br />
<br />
<p class="title2">Lab work:</p><br />
<p class="texte"><br />
- Evaluation of the bacterial decrease rate from 10°C to 4°C with a sporuling strain of Bacillus subtilis.<br />
<br/><br />
- Chemotaxis test with different concentration of glucose on a 0.3% agar.<br />
<br/><br />
- Miniprep of pSBbs4S with binding gene and transformation in <i>Bacillus subtilis</i>.<br />
<br/><br />
Problem: no digestion was visible on the gel => the cloning failed<br />
<br/><br />
- Transformation of BBa_K1374010 (Pveg + RBS SpoVG + EcAMP) + BBa_K1364012 (RBS + Gafp1 + D4E1 + double terminator), subculture<br />
<br/><br />
- Subculture of Bacillus subtilis clones transformed by BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) in pSBBs4S, cryopreservation<br />
<br/><br />
- Fungicide test<br />
</p><br />
<br />
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<br />
<div class="technology">Week 10 (18-24 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Ethics: Interview with a specialist in ethical sciences, Vincent Grégoire Delory to complete our reflexion about the ethical issues in SubtiTree project.<br />
<br/><br />
- Wiki: hiring a web designer, Adrien Nicod, to improve the aesthetic effect and the logo.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Subculture and transformation in Bacillus subtilis of BBa_K1364011 in pSBBS4S (BBa_K823022) and cloning of BBa_K1364011 on pSBBS1C (BBa_K823023).<br />
<br/><br />
Problem: no clone had the insert on pSBBS1C => the cloning had to be done again<br />
- Miniprep of BBa_K1364011 in BBa_K823023 (pSBBS1C) + Transformation in <i>B. subtilis</i>.<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) , BBA_1364002 (GAFP1) , BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) on BBa_K823023 (pSBBS1C) <br />
<br/><br />
- Transformation BBa_1364004 (in pSBBS4S in E.coli <br />
<br/><br />
- Cloning of BBa_K1364014 (Pveg + RBS SpoVG + EcAMP + GAFP1 +D4E1 + double terminator) in K823022 (pSBBS4S) and in BBa_K823023 (pSBBS1C), miniprep and digestion by restriction enzymes to see the size of the insert on a 1% gel<br />
<br/><br />
Problem: no clone had the insert on pSBBS1C => the cloning had to be done again<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology">Week 11 (25- 31 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Beginning of the creation and redaction of the different parts of the wiki by the Toulouse iGEM Toulouse<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Cloning of BBa_K1364014 in BBa_K823022 and in BBa_K823023 <br />
<br/><br />
- Cloning GAFP1 + BBa_K823023 (pSBBS1C) in Bacillus subtilis with a change in the protocol: pre-induction of the culture with 0.125 µg/ml of chloramphenicol one hour after the addition of DNA, and let grow for another hour.<br />
<br/><br />
Problem: no colony <br />
<br/><br />
- Liquid culture of Bacillus + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) for future fungicide tests<br />
<br/><br />
- Miniprep of BBa_ K1364011 in BBa_ K823023 (pSBBs1C) and transformation in Bacillus subtilis strain. <br />
<br/><br />
- Fungicide test of BBa_K1364011 in pSBBs4S, Promotor + BBA_1364002 (GAFP1) + Terminator (biobrick BBa_K1364008) in pSBBS4S, Promotor + BBA_1364003 (D4E1) + Terminator (biobrick BBa_K1364009) in pSBBS4S, Promotor + BBA_1364002 (GAFP1) + BA_1364003 (D4E1) + Terminator ( biobrick BBa_K1364013) in pSBBS4S<br />
<br/><br />
- Transformation of a new vector PKL 190 (threonine integrative) in Bacillus strain <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/29/2014)</p><br />
<p class="texte"><br />
- Boston accommodation must be booked<br />
<br/><br />
- Discussion about the contamination seen on the fungicide tests : the issue seems to come from the sterile water which contained contaminants<br />
<br/><br />
- Objectives : chemotaxis, binding and fungicides tests have to be performed again<br />
</p> <br />
<br />
<p class="title1" id="select6"> September 2014</p><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finish the tests of the different modules<br />
<br/><br />
- Establish the final editorial parts for the wiki<br />
<br/><br />
- Design of the wiki<br />
<br/><br />
- Sequencing the different assembled parts of our bacterium<br />
</p> <br />
<br />
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</div><br />
<br />
<div class="technology">Week 12 (1-7 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Different parts of the wiki by the Toulouse iGEM Toulouse<br/><br />
- Reservation of Boston accommodation<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Chemotaxis test and modelling<br />
<br/><br />
- Fungicides tests on EcAMP-A, EcAMP-B, EcAMP-C, EcAMP-D, MaxiOpA, MaxiOpB, MaxiOpC, MaxiOpD from 2ml of overnight cultures. Only with 25000 conidia with both fungi.<br />
<br/><br />
- PCR on pSBBS4S + BBa_K1162001 (EcAMP) <br />
<br/><br />
Problem: failed on both diluted plasmid and colony<br />
<br/><br />
- Spreading of BBa_K1364014 + BBa_ K823022 in threonine medium and in medium without threonine <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/05/2014)</p> <br />
<p class="texte"><br />
- The first sequencing was not successful => new sequencing with one primer/tube<br/><br />
- Chemotaxis test: increase the concentration in bacterium, try a new protocol by capillarity<br/><br />
- End of the binding test: IT WORKS PERFECTLY! SubtiTree has the good phenotype and presents a better binding property than the wild type Bacillus subtilis strain.<br/><br />
- Fungicides test: contaminants with the fungus. Need to ask for another culture of our fungi.<br />
</p><br />
<br />
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<br />
<div class="technology">Week 13 (8- 14 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Telephone interview for an article in a French national review named Fluvial which will be published at the end of September.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- PCR on D4E1 colony with different primers<br />
<br/>- Culture and miniprep of EcAMP to get more DNA + analytic digestion <br />
<br/>- Transformation of K1364014+K1364006 in E. coli <br />
<br/>- Transformation of GAFP1 + D4E1, GAFP1 and EcAMP+GAFP1+D4E1 on Bacillus subtilis + colony PCR + threonine test<br />
<br/>NB: good results for the maxi operon and GAFP1 but not for GAFP1 + D4E1 CA <br />
<br/>- Cloning of K606013+pEX_K4, 823022+PlepA_RFP, 823022+ Pveg_RFP<br />
<br/>- Chemotaxis tests: different times of LB + agar addition in the LB medium (after 3 minutes, 5 minutes, 8 minutes, 10 minutes…) are performed => No difference of the results after spreading on the plates.<br />
<br/>- New test of chemotaxis: capillary between two Eppendorf tubes<br />
<br/>- Spreading of the fungi on a medium which mimics the sap<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/11/2014)</p> <br />
<p class="texte"><br />
- Try to use the GFP or RFP to follow the chemotaxis test.<br />
<br/>- Make a new glass process for the chemotaxis capillary test.<br />
<br/>- Possibility of contacting an INRA laboratory to get pictures of the binding test with confocal microscope.<br />
<br/>- Sequencing the binding construction.<br />
<br/>- Try different temperatures for the fungicide tests: 20°C, 25°C, and 37°C to reduce the fungus growth.<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology">Week 14 (15 - 21 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Support of two French city halls: Mairie de Montréal, Mairie de Ventenac en Minervois.<br />
<br/>- First interview by the French national radio France Inter which will be aired on October 12th.<br />
<br/>- Beginning of the organization of the iGEM Toulouse 2014 acknowledgement day: the purpose is to present the project to the students and give a feedback on the competition to the sponsors.<br />
<br/>- Final design of the wiki<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Threonine tests for the BBa_K1364014 maxi operon (EcAMP1 – GAFP1 – D4E1) and mini operon BBa_K1364013 (GAFP1-D4E1)<br />
<br/>- Colony PCR on the mini operon BBa_K1364013 (GAFP1 - D4E1)<br />
<br/>- Colony PCR on BBa_K1364011 on BBa_K823022 (pSBBS4S)<br />
<br/>- Chemotaxis test with wild type <i>Bacillus subtilis</i> strain using Imperial College protocol<br />
<br/>- Tansformation of BBa_K1364011 + BBa_K823022 (pSBBS4S) in Bacillus subtilis<br />
<br/>- Culture of fungi with fungicides<br />
<br/>- Sequencing of plasmids containing the fungicides<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/18/2014)</p> <br />
<p class="texte"><br />
- Sequencing worked for D4E1 and GAFP1 and the mini operon BBa_K1364013 (GAFP1-D4E1).<br />
<br/>- The next step is to sequence the maxi operon (EcAMP1 – GAFP1 – D4E1).<br />
<br/>- Try different approaches for the chemotaxis:<br />
<br/>- New protocol with the capillary assay with a new system made by a glassblower.<br />
<br/>- Make the Imperial College test again by testing 3 different solutions: chitin, N-acetylglucosamine and another carbon source.<br />
<br/>- Decrease the number of conidia and temperature for the fungicide test.<br />
</p><br />
<br />
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<br />
<div class="technology">Week 15 (22 - 28 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- New support of Sallèles d’Aude city hall.<br />
<br/>- Payment of the plane tickets.<br />
<br/>- Acknowledgement day (4th December): contact with catering service, reservation of the amphitheater, list of guests…<br />
<br/>- Order of the T shirt for the Jamboree! We look awesome with them!<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Last experiments regarding chemotaxis tests.<br />
</p><br />
<br />
<br />
<p class="title3">Weekly meeting with the instructors(09/26/2014)</p> <br />
<p class="texte"><br />
- Focus on the wiki design and writing.<br />
<br/>- Start making the power point presentation for Boston and the poster.<br />
<br/>- Division of responsibilities for all the non-scientific work.<br />
</p><br />
<br />
<br />
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<br />
<div class="technology">October 2014</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">Main activites</p><br />
<p class="texte"><br />
- Preparation of the wiki every day… all day long…. so hard to work on this even at night!<br />
<br/>- Preparation of the poster and the presentation for the Jamboree with our instructors <br />
<br/><br />
- Daily presentations in front of the laboratory teachers to repeat again and again and again… let’s get ready for the Jamboree!<br />
</p> <br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Calendar</p> <br />
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<div class="technology">December 2013 – January 2014: Team selection</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- iGEM competition presentation and explanations<br/><br />
- Constitution of the team by interviewing different students from Université Paul Sabatier and INSA<br/><br />
</p><br />
<p class="texte" style="text-align:center"><B> The adventure begins for the Toulouse iGEM Team 2014! </B><br />
</p><br />
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<div class="technology">January – June 2014: Projects brainstorming</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- Thanks to weekly meetings, our team was able to discuss about new project ideas and publications.<br />
<br/><br />
- Many ideas were approached and debated regarding the feasibility thanks to the literature and supervisors including teachers and researchers<br />
<br/><br />
This is a list of the main projects: <br />
<br/><br />
- Let's save our trees with SubtiTree: the purpose is to use <i>Bacillus subtilis</i> as an optimized bacterium to detect, target a fungi and secrete fungicides to destroy it<br />
<br/><br />
- <i>E. coli</i> cancer: the concept was to create a bacterium able to colonize specifically the hypoxic regions of the tumor. The oxygen-sensitive bacterium would not be able to develop in the healthy zones<br />
<br/><br />
- Reduction of ruminants’ methane production: the goal is to reduce the production of methane by ingesting a microorganism in the animal’s rumen. This bacterium would be capable of reducing the quantity of metabolic hydrogen and eliminating the methanogenic Archaes population<br />
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<div class="technology">June 2014: Choice of SubtiTree project</div><br />
<div class="thelanguage"><br />
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<p class="texte">The discussion has been long to estimate the practicability of our final project. By the end of June, most of the ideas were eliminated but two of them remained: controlling the cancer or saving the trees. It became easily clear that SubtiTree was successfully completed whether regarding the scientific part or the ethical aspect. Indeed, our knowledge in medicine was not sufficient enough to evaluate the consequences of a bacterium injection in the human body for <i>E. coli</i> cancer.</br><br />
Therefore, the whole team agreed on focusing on a local issue as the preservation of the Canal du Midi plane trees. However, the final goal was based on the idea to allow our optimized bacteria to be efficient for all fungi infections in the environment.</br><br />
By this period, we evaluated our budget to approximately <B> 40 k€ </B>.<br />
</p> <br />
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<div class="technology">Week 1(16-22 June)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Bibliography researches about our subject<br/><br />
- Cleaning the whole lab to allow good manipulations and avoid any contamination<br/><br />
- Inventory of necessary lab equipment and biobricks<br/><br />
- Summarize the iGEM protocols<br/><br />
- Prepare all growing media <br/><br />
- Preparing competent cells by optimizing protocols<br/><br />
- Transformation of RFP plasmid to practice<br />
</p><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Increase in our communication strategy to find more sponsors specialized in environment and ecological matters <br/><br />
- Support of Adisseo, Toulouse White Biotechnology, LISBP, INSA Toulouse<br/><br />
- Organization of a weekly meeting each Friday with our supervisors to tackle any problem encountered<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/20/2014)</p><br />
<p class="texte"><br />
- Distribution of the non-scientific tasks regarding the wiki, communication, sponsorship, ethical problems, safety but also the lab work<br/><br />
- Discussion about the different construction parts of the project<br/><br />
- Need to check the absence of stop codon in fungicides sequences<br />
</p> <br />
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<div class="technology">Week 2 (23-29 June)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Ordering the list of necessary products for the laboratory and biobricks<br/><br />
- Checking and validation of the genes sequences<br/><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- BioSynSyS conference work: powerpoint, logo in order to present our SubtiTree project to a whole audience of scientific and researchers (http://biosynsys2014.sciencesconf.org/)<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- E. coli transformation with BBA_J004450 (pSB1C3)<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/27/2014)</p><br />
<p class="texte"><br />
- Concentration of antibiotics in media must be checked<br/><br />
- The transformation rate for pUC19 must be evaluated<br/><br />
- The transformation efficiency must be calculated regarding the quantity of DNA<br/><br />
- Possibility to contact the cities halls for the sponsorship<br/><br />
- Check the mechanism of action of each fungicide to complete the ethical part<br />
</p><br />
<br />
<p class="title1" id="select4">July 2014</p><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Beginning of the laboratory work to create our optimized bacterium <br/><br />
- Research of sponsors and the communication thanks to the press<br />
</p> <br />
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<div class="technology">Week 3 (30 June -6 July) and Week 4 (7-13 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the BioSynSys conferences: presentation of SubtiTree<br/><br />
- We have put in place a newsletter system the first day of each month to keep all our sponsors and people who support us updated about the project, our financial state.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Design of all the constructions needed with cloning and restriction enzymes to save some time and allow a better comprehension of our bacterium system </br><br />
Problem: A problem was reported in the use of the CYP. Indeed, the biobrick is not expressed properly in <i>Bacillus subtilis</i> strain. Therefore we had to look at the literature to find a new fungicide called GAFP-1.<br/><br />
- A bioreactor was conceived to reproduce the composition of the sap in the plane trees. The main goal was to identify the behavior of Bacillus strain in a medium composed of many different carbon sources.<br/><br />
- Competent cells and transformation practice using GFP and RFP.<br />
</p><br />
<br />
<p class="title3">Weekly meetings with the instructors (07/04/2014) and (07/11/2014)</p><br />
<p class="texte"><br />
- iGEM efficiency kit does not work, we shall not use it anymore<br/><br />
- Organization of a timetable to check the stored and sterile equipment everyday</br><br />
- Try a transformation in <i>Bacillus subtilis</i><br />
</p> <br />
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<div class="technology">Week 5 (14-20 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Our team was interviewed by many local newspapers such as 20minutes, Metronews, La Voix du Midi, La Dépêche but also by the local television channel France3 Midi-Pyrénées to present our draft to the community. Moreover, SubtiTree was approached in some radio programs like Virgin Radio Toulouse and France info.</br><br />
- Support of ThermoFisher, New Englands BioLabs, Qiagen, Eurofins but also GenoToul, Labex Tulip, L’Université Paul Sabatier, CROUS.<br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- GFP and RFP digestion amplified by miniprep and gel electrophoresis <br/><br />
Problem: the digested GFP doesn’t appear on the gel contrary to RFP plasmid. We assumed a problem with the GFP miniprep. We tried the miniprep from the INSA Toulouse team from 2013 and the GFP plasmid was fully digested.<br/><br />
- Transformation and cryopreservation of biobricks BBa_K823002 (PlepA), BBa_K823003 (Pveg), BBa_K606061 (RBS SpoVG) and BBa_B0015 (double terminator), BBa_K823023 (pSBBS1C), BBa_K733013 (Pveg+ RBS) in <i>E. coli</i>.<br/><br />
- Transformation of the Munich B. subtilis backbones<br/><br />
- Cloning of BBa_K606061 (RBS SpoVG) with BBa_K823003 (PvEG) and BBa_K823002 (PlepA)<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (07/18/2014)</p><br />
<p class="texte"><br />
- Transformation of the Eurofins genes<br/><br />
- Start assembling the biobricks for the fungicides<br/><br />
- Check all the cloning<br />
</p><br />
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<div class="technology">Week 6 (21 July-27 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Description of SubtiTree project and determination of the goodies for a crowdfunding campaign on Ulule website<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Transformation of BBA_1364002 (GAFP1) and BBA_1364003 (D4E1) fungicides genes<br/><br />
- Subculture of the clones for each gene<br/><br />
- PCR and migration on electrophoresis gel<br/><br />
- Transformation BBA_1364003 (D4E1) on pSB1C3 and BBA_1364002 (GAFP1) on pSB1C3<br/><br />
- Cloning BBA_1364002 (GAFP1) with BBa_B0015 (double terminator) and BBA_1364003 (D4E1) with BBa_K823003 (Pveg)<br/><br />
</p><br />
<br />
<p class="title3">Others:</p><br />
<p class="texte"><br />
- Research for plane tickets and hotels in Boston for the Giant Jamboree<br/><br />
- New idea: analyze the plane tree sap to determine the composition<br/><br />
</p><br />
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<div class="technology">August 2014 </div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finishing the cloning for the different parts of the bacterium<br/><br />
- Putting in place the fungicides, binding and chemotaxis tests<br/><br />
</p> <br />
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<div class="technology">Week 7 (28 July-3 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Financial supports of the Ministery (Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt) and Voies Navigables de France<br/><br />
- Interview with Johan Langot from Sciences Animation for a possible presentation at the Toulouse Festival of Science<br/><br />
- Launch of the crowdfunding campaign on Ulule<br />
</p><br />
<br />
<p class="title3">Lab work: </p><br />
<p class="texte"><br />
- Cloning BBa_K823003 (Pveg) + RFP and BBa_K823002 (PlepA) + RFP in pSBBs1C<br/><br />
- Test of every pSBBs vector : BBa_K823021 (pSBBS1C-lacZ), BBa_K823022 (pSBBS4S), BBa_K823023 (pSBBS1C), minipreps and cryopreservation of the best clones<br/><br />
- Checking of BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) transformants (biobrick BBa_K1364007) and BBA_1364003 (D4E1) + BBa_K823003 (Pveg) biobrick BBa_K1364009<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) + pSBBS4S (BBa_K823022) and subculture of the colonies<br/><br />
- Cloning of BBa_K823003 (Pveg) + BBA_1364003 (D4E1) with BBa_B0015 (double terminator)<br/><br />
- Cloning of BBa_K823003 (Pveg) + RFP (BBa_K1364017) in pSBBS4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_K823002 (PlepA) + RFP (BBa_K1364016) in pSBBs4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_1364018 (Strong Promotor + RFP)<br/><br />
- Cloning of BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) + Promotor BBa_K823003 (Pveg) biobirck BBa_K1364012 with gel extraction for BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) and a PCR cleanup for BBa_K823003 (Pveg), subculture<br/><br />
- Cloning BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBS4S in <i>E. coli</i><br/><br />
- Transformation of binding gene with <i>E. coli</i> competent cells<br/><br />
- Transformation of BBa_K1364000, the chemotaxis gene in <i>E. coli</i><br/><br />
Problem: the transformation worked but a cell layer appeared. A new subculture of the colonies was made as well as a new spreading on petri dishes.<br/><br />
- Spreading of BBa_K1162001 (EcAMP), miniprep and digestion<br/><br />
Problem: the digestion did not work => Issue with the quantity of DNA in the miniprep is assumed<br/><br />
- First experience of chemotaxis with a glucose chemo-attractant<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/01/2014)</p><br />
<p class="texte"><br />
- Possible problem with the restriction enzymes regarding the digestion: new recommendations<br />
<br/><br />
- Discussion about EcAMP cloning which presents some issues<br />
<br/><br />
- Discussion about the transfer of pSB1C3 from E. coli to Bacillus strain<br />
</p> <br />
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<div class="technology">Week 8 (04-10 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the “Passion Jeune” competition organized by a French bank foundation named Fondation Crédit Agricole Toulouse<br />
</p><br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Check the cloning of BBa_K823003 (Pveg)+BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBs4S and cryopreservation<br />
<br/><br />
- Transformation of BBa_K823003 (Pveg) +BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) (biobrick BBa_ K1364008) in BBa_K823022 (pSBBS4S) <br />
<br/><br />
- Cloning of BBa_K1364001, the binding gene + BBa_K823003 (Pveg) on pSB1C3 and pSBBS 4S<br />
<br/><br />
Problem: the band is at 1500 bp instead of 1300 bp on the gel<br />
<br/><br />
- Cloning of BBa_K1162001 (EcAMP) + BBa_K823003 (Pveg) + BBa_K606061 (RBS SpoVG), miniprep, PCR and digestion <br />
<br/><br />
Problem: the fragment of DNA is too small (149 bp) to be seen on the gel and was probably lost during the PCR cleanup. Instead of that, we used the heat enzymes inactivation at 95°C for 15 minutes. <br />
<br/><br />
- Cloning of Promotor + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) in pSB1C3 and pSBBS4S and cryopreservation<br />
<br/><br />
- Elaboration of an efficient fungicide test protocol <br />
<br/><br />
- Test of the fungus growth on PDA medium + test of growth for Bacillus subtilis liquid culture on PDA<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/08/2014)</p><br />
<p class="texte"><br />
- Ask iGEM headquarters if all the biobricks must be on pSB1C3 plasmid<br />
<br/><br />
- Use another strain of E. coli (DH5-1 instead of DH5 alpha) to have a faster growth<br />
<br/><br />
- Check which quantity of Bacillus subtilis is necessary to naturally destroy the fungus<br />
</p><br />
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<div class="technology">Week 9 (11-17 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Publication of more articles about our project in Labiotech, Développement durable (Networkvisio), Midi Libre, l’Indépendant, DigiSchool, La voix du Midi Lauragais, LURIO Addl.<br />
</p><br />
<br />
<p class="title2">Lab work:</p><br />
<p class="texte"><br />
- Evaluation of the bacterial decrease rate from 10°C to 4°C with a sporuling strain of Bacillus subtilis.<br />
<br/><br />
- Chemotaxis test with different concentration of glucose on a 0.3% agar.<br />
<br/><br />
- Miniprep of pSBbs4S with binding gene and transformation in <i>Bacillus subtilis</i>.<br />
<br/><br />
Problem: no digestion was visible on the gel => the cloning failed<br />
<br/><br />
- Transformation of BBa_K1374010 (Pveg + RBS SpoVG + EcAMP) + BBa_K1364012 (RBS + Gafp1 + D4E1 + double terminator), subculture<br />
<br/><br />
- Subculture of Bacillus subtilis clones transformed by BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) in pSBBs4S, cryopreservation<br />
<br/><br />
- Fungicide test<br />
</p><br />
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<div class="technology">Week 10 (18-24 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Ethics: Interview with a specialist in ethical sciences, Vincent Grégoire Delory to complete our reflexion about the ethical issues in SubtiTree project.<br />
<br/><br />
- Wiki: hiring a web designer, Adrien Nicod, to improve the aesthetic effect and the logo.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Subculture and transformation in Bacillus subtilis of BBa_K1364011 in pSBBS4S (BBa_K823022) and cloning of BBa_K1364011 on pSBBS1C (BBa_K823023).<br />
<br/><br />
Problem: no clone had the insert on pSBBS1C => the cloning had to be done again<br />
- Miniprep of BBa_K1364011 in BBa_K823023 (pSBBS1C) + Transformation in <i>B. subtilis</i>.<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) , BBA_1364002 (GAFP1) , BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) on BBa_K823023 (pSBBS1C) <br />
<br/><br />
- Transformation BBa_1364004 (in pSBBS4S in E.coli <br />
<br/><br />
- Cloning of BBa_K1364014 (Pveg + RBS SpoVG + EcAMP + GAFP1 +D4E1 + double terminator) in K823022 (pSBBS4S) and in BBa_K823023 (pSBBS1C), miniprep and digestion by restriction enzymes to see the size of the insert on a 1% gel<br />
<br/><br />
Problem: no clone had the insert on pSBBS1C => the cloning had to be done again<br />
</p><br />
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<div class="technology">Week 11 (25- 31 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Beginning of the creation and redaction of the different parts of the wiki by the Toulouse iGEM Toulouse<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Cloning of BBa_K1364014 in BBa_K823022 and in BBa_K823023 <br />
<br/><br />
- Cloning GAFP1 + BBa_K823023 (pSBBS1C) in Bacillus subtilis with a change in the protocol: pre-induction of the culture with 0.125 µg/ml of chloramphenicol one hour after the addition of DNA, and let grow for another hour.<br />
<br/><br />
Problem: no colony <br />
<br/><br />
- Liquid culture of Bacillus + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) for future fungicide tests<br />
<br/><br />
- Miniprep of BBa_ K1364011 in BBa_ K823023 (pSBBs1C) and transformation in Bacillus subtilis strain. <br />
<br/><br />
- Fungicide test of BBa_K1364011 in pSBBs4S, Promotor + BBA_1364002 (GAFP1) + Terminator (biobrick BBa_K1364008) in pSBBS4S, Promotor + BBA_1364003 (D4E1) + Terminator (biobrick BBa_K1364009) in pSBBS4S, Promotor + BBA_1364002 (GAFP1) + BA_1364003 (D4E1) + Terminator ( biobrick BBa_K1364013) in pSBBS4S<br />
<br/><br />
- Transformation of a new vector PKL 190 (threonine integrative) in Bacillus strain <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/29/2014)</p><br />
<p class="texte"><br />
- Boston accommodation must be booked<br />
<br/><br />
- Discussion about the contamination seen on the fungicide tests : the issue seems to come from the sterile water which contained contaminants<br />
<br/><br />
- Objectives : chemotaxis, binding and fungicides tests have to be performed again<br />
</p> <br />
<br />
<p class="title1" id="select6"> September 2014</p><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finish the tests of the different modules<br />
<br/><br />
- Establish the final editorial parts for the wiki<br />
<br/><br />
- Design of the wiki<br />
<br/><br />
- Sequencing the different assembled parts of our bacterium<br />
</p> <br />
<br />
<br />
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<br />
<div class="technology">Week 12 (1-7 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Different parts of the wiki by the Toulouse iGEM Toulouse<br/><br />
- Reservation of Boston accommodation<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Chemotaxis test and modelling<br />
<br/><br />
- Fungicides tests on EcAMP-A, EcAMP-B, EcAMP-C, EcAMP-D, MaxiOpA, MaxiOpB, MaxiOpC, MaxiOpD from 2ml of overnight cultures. Only with 25000 conidia with both fungi.<br />
<br/><br />
- PCR on pSBBS4S + BBa_K1162001 (EcAMP) <br />
<br/><br />
Problem: failed on both diluted plasmid and colony<br />
<br/><br />
- Spreading of BBa_K1364014 + BBa_ K823022 in threonine medium and in medium without threonine <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/05/2014)</p> <br />
<p class="texte"><br />
- The first sequencing was not successful => new sequencing with one primer/tube<br/><br />
- Chemotaxis test: increase the concentration in bacterium, try a new protocol by capillarity<br/><br />
- End of the binding test: IT WORKS PERFECTLY! SubtiTree has the good phenotype and presents a better binding property than the wild type Bacillus subtilis strain.<br/><br />
- Fungicides test: contaminants with the fungus. Need to ask for another culture of our fungi.<br />
</p><br />
<br />
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<br />
<div class="technology">Week 13 (8- 14 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Telephone interview for an article in a French national review named Fluvial which will be published at the end of September.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- PCR on D4E1 colony with different primers<br />
<br/>- Culture and miniprep of EcAMP to get more DNA + analytic digestion <br />
<br/>- Transformation of K1364014+K1364006 in E. coli <br />
<br/>- Transformation of GAFP1 + D4E1, GAFP1 and EcAMP+GAFP1+D4E1 on Bacillus subtilis + colony PCR + threonine test<br />
<br/>NB: good results for the maxi operon and GAFP1 but not for GAFP1 + D4E1 CA <br />
<br/>- Cloning of K606013+pEX_K4, 823022+PlepA_RFP, 823022+ Pveg_RFP<br />
<br/>- Chemotaxis tests: different times of LB + agar addition in the LB medium (after 3 minutes, 5 minutes, 8 minutes, 10 minutes…) are performed => No difference of the results after spreading on the plates.<br />
<br/>- New test of chemotaxis: capillary between two Eppendorf tubes<br />
<br/>- Spreading of the fungi on a medium which mimics the sap<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/11/2014)</p> <br />
<p class="texte"><br />
- Try to use the GFP or RFP to follow the chemotaxis test.<br />
<br/>- Make a new glass process for the chemotaxis capillary test.<br />
<br/>- Possibility of contacting an INRA laboratory to get pictures of the binding test with confocal microscope.<br />
<br/>- Sequencing the binding construction.<br />
<br/>- Try different temperatures for the fungicide tests: 20°C, 25°C, and 37°C to reduce the fungus growth.<br />
</p><br />
<br />
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<br />
<div class="technology">Week 14 (15 - 21 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Support of two French city halls: Mairie de Montréal, Mairie de Ventenac en Minervois.<br />
<br/>- First interview by the French national radio France Inter which will be aired on October 12th.<br />
<br/>- Beginning of the organization of the iGEM Toulouse 2014 acknowledgement day: the purpose is to present the project to the students and give a feedback on the competition to the sponsors.<br />
<br/>- Final design of the wiki<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Threonine tests for the BBa_K1364014 maxi operon (EcAMP1 – GAFP1 – D4E1) and mini operon BBa_K1364013 (GAFP1-D4E1)<br />
<br/>- Colony PCR on the mini operon BBa_K1364013 (GAFP1 - D4E1)<br />
<br/>- Colony PCR on BBa_K1364011 on BBa_K823022 (pSBBS4S)<br />
<br/>- Chemotaxis test with wild type <i>Bacillus subtilis</i> strain using Imperial College protocol<br />
<br/>- Tansformation of BBa_K1364011 + BBa_K823022 (pSBBS4S) in Bacillus subtilis<br />
<br/>- Culture of fungi with fungicides<br />
<br/>- Sequencing of plasmids containing the fungicides<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/18/2014)</p> <br />
<p class="texte"><br />
- Sequencing worked for D4E1 and GAFP1 and the mini operon BBa_K1364013 (GAFP1-D4E1).<br />
<br/>- The next step is to sequence the maxi operon (EcAMP1 – GAFP1 – D4E1).<br />
<br/>- Try different approaches for the chemotaxis:<br />
<br/>- New protocol with the capillary assay with a new system made by a glassblower.<br />
<br/>- Make the Imperial College test again by testing 3 different solutions: chitin, N-acetylglucosamine and another carbon source.<br />
<br/>- Decrease the number of conidia and temperature for the fungicide test.<br />
</p><br />
<br />
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<br />
<div class="technology">Week 15 (22 - 28 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- New support of Sallèles d’Aude city hall.<br />
<br/>- Payment of the plane tickets.<br />
<br/>- Acknowledgement day (4th December): contact with catering service, reservation of the amphitheater, list of guests…<br />
<br/>- Order of the T shirt for the Jamboree! We look awesome with them!<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Last experiments regarding chemotaxis tests.<br />
</p><br />
<br />
<br />
<p class="title3">Weekly meeting with the instructors(09/26/2014)</p> <br />
<p class="texte"><br />
- Focus on the wiki design and writing.<br />
<br/>- Start making the power point presentation for Boston and the poster.<br />
<br/>- Division of responsibilities for all the non-scientific work.<br />
</p><br />
<br />
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<div class="technology">October 2014</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">Main activites</p><br />
<p class="texte"><br />
- Preparation of the wiki every day… all day long…. so hard to work on this even at night!<br />
<br/>- Preparation of the poster and the presentation for the Jamboree with our instructors <br />
<br/><br />
- Daily presentations in front of the laboratory teachers to repeat again and again and again… let’s get ready for the Jamboree!<br />
</p> <br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Calendar</p> <br />
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<br />
<div class="technology">December 2013 – January 2014: Team selection</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- iGEM competition presentation and explanations<br/><br />
- Constitution of the team by interviewing different students from Université Paul Sabatier and INSA<br/><br />
</p><br />
<p class="texte" style="text-align:center"><B> The adventure begins for the Toulouse iGEM Team 2014! </B><br />
</p><br />
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<br />
<div class="technology">January – June 2014: Projects brainstorming</div><br />
<div class="thelanguage"><br />
<p class="texte"><br />
- Thanks to weekly meetings, our team was able to discuss about new project ideas and publications.<br />
<br/><br />
- Many ideas were approached and debated regarding the feasibility thanks to the literature and supervisors including teachers and researchers<br />
<br/><br />
This is a list of the main projects: <br />
<br/><br />
- Let's save our trees with SubtiTree: the purpose is to use <i>Bacillus subtilis</i> as an optimized bacterium to detect, target a fungi and secrete fungicides to destroy it<br />
<br/><br />
- <i>E. coli</i> cancer: the concept was to create a bacterium able to colonize specifically the hypoxic regions of the tumor. The oxygen-sensitive bacterium would not be able to develop in the healthy zones<br />
<br/><br />
- Reduction of ruminants’ methane production: the goal is to reduce the production of methane by ingesting a microorganism in the animal’s rumen. This bacterium would be capable of reducing the quantity of metabolic hydrogen and eliminating the methanogenic Archaes population<br />
<br/><br />
</p><br />
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<br />
<div class="technology">June 2014: Choice of SubtiTree project</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">The discussion has been long to estimate the practicability of our final project. By the end of June, most of the ideas were eliminated but two of them remained: controlling the cancer or saving the trees. It became easily clear that SubtiTree was successfully completed whether regarding the scientific part or the ethical aspect. Indeed, our knowledge in medicine was not sufficient enough to evaluate the consequences of a bacterium injection in the human body for <i>E. coli</i> cancer.</br><br />
Therefore, the whole team agreed on focusing on a local issue as the preservation of the Canal du Midi plane trees. However, the final goal was based on the idea to allow our optimized bacteria to be efficient for all fungi infections in the environment.</br><br />
By this period, we evaluated our budget to approximately <B> 40 k€ </B>.<br />
</p> <br />
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<br />
<div class="technology">Week 1(16-22 June)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Bibliography researches about our subject<br/><br />
- Cleaning the whole lab to allow good manipulations and avoid any contamination<br/><br />
- Inventory of necessary lab equipment and biobricks<br/><br />
- Summarize the iGEM protocols<br/><br />
- Prepare all growing media <br/><br />
- Preparing competent cells by optimizing protocols<br/><br />
- Transformation of RFP plasmid to practice<br />
</p><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Increase in our communication strategy to find more sponsors specialized in environment and ecological matters <br/><br />
- Support of Adisseo, Toulouse White Biotechnology, LISBP, INSA Toulouse<br/><br />
- Organization of a weekly meeting each Friday with our supervisors to tackle any problem encountered<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/20/2014)</p><br />
<p class="texte"><br />
- Distribution of the non-scientific tasks regarding the wiki, communication, sponsorship, ethical problems, safety but also the lab work<br/><br />
- Discussion about the different construction parts of the project<br/><br />
- Need to check the absence of stop codon in fungicides sequences<br />
</p> <br />
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<br></br><br />
<br />
<div class="technology">Week 2 (23-29 June)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Ordering the list of necessary products for the laboratory and biobricks<br/><br />
- Checking and validation of the genes sequences<br/><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- BioSynSyS conference work: powerpoint, logo in order to present our SubtiTree project to a whole audience of scientific and researchers (http://biosynsys2014.sciencesconf.org/)<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- E. coli transformation with BBA_J004450 (pSB1C3)<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (06/27/2014)</p><br />
<p class="texte"><br />
- Concentration of antibiotics in media must be checked<br/><br />
- The transformation rate for pUC19 must be evaluated<br/><br />
- The transformation efficiency must be calculated regarding the quantity of DNA<br/><br />
- Possibility to contact the cities halls for the sponsorship<br/><br />
- Check the mechanism of action of each fungicide to complete the ethical part<br />
</p><br />
<br />
<p class="title1" id="select4">July 2014</p><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Beginning of the laboratory work to create our optimized bacterium <br/><br />
- Research of sponsors and the communication thanks to the press<br />
</p> <br />
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<br />
<div class="technology">Week 3 (30 June -6 July) and Week 4 (7-13 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the BioSynSys conferences: presentation of SubtiTree<br/><br />
- We have put in place a newsletter system the first day of each month to keep all our sponsors and people who support us updated about the project, our financial state.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Design of all the constructions needed with cloning and restriction enzymes to save some time and allow a better comprehension of our bacterium system </br><br />
Problem: A problem was reported in the use of the CYP. Indeed, the biobrick is not expressed properly in <i>Bacillus subtilis</i> strain. Therefore we had to look at the literature to find a new fungicide called GAFP-1.<br/><br />
- A bioreactor was conceived to reproduce the composition of the sap in the plane trees. The main goal was to identify the behavior of Bacillus strain in a medium composed of many different carbon sources.<br/><br />
- Competent cells and transformation practice using GFP and RFP.<br />
</p><br />
<br />
<p class="title3">Weekly meetings with the instructors (07/04/2014) and (07/11/2014)</p><br />
<p class="texte"><br />
- iGEM efficiency kit does not work, we shall not use it anymore<br/><br />
- Organization of a timetable to check the stored and sterile equipment everyday</br><br />
- Try a transformation in <i>Bacillus subtilis</i><br />
</p> <br />
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<br />
<div class="technology">Week 5 (14-20 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
- Our team was interviewed by many local newspapers such as 20minutes, Metronews, La Voix du Midi, La Dépêche but also by the local television channel France3 Midi-Pyrénées to present our draft to the community. Moreover, SubtiTree was approached in some radio programs like Virgin Radio Toulouse and France info.</br><br />
- Support of ThermoFisher, New Englands BioLabs, Qiagen, Eurofins but also GenoToul, Labex Tulip, L’Université Paul Sabatier, CROUS.<br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- GFP and RFP digestion amplified by miniprep and gel electrophoresis <br/><br />
Problem: the digested GFP doesn’t appear on the gel contrary to RFP plasmid. We assumed a problem with the GFP miniprep. We tried the miniprep from the INSA Toulouse team from 2013 and the GFP plasmid was fully digested.<br/><br />
- Transformation and cryopreservation of biobricks BBa_K823002 (PlepA), BBa_K823003 (Pveg), BBa_K606061 (RBS SpoVG) and BBa_B0015 (double terminator), BBa_K823023 (pSBBS1C), BBa_K733013 (Pveg+ RBS) in <i>E. coli</i>.<br/><br />
- Transformation of the Munich B. subtilis backbones<br/><br />
- Cloning of BBa_K606061 (RBS SpoVG) with BBa_K823003 (PvEG) and BBa_K823002 (PlepA)<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors (07/18/2014)</p><br />
<p class="texte"><br />
- Transformation of the Eurofins genes<br/><br />
- Start assembling the biobricks for the fungicides<br/><br />
- Check all the cloning<br />
</p><br />
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<div class="technology">Week 6 (21 July-27 July)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Description of SubtiTree project and determination of the goodies for a crowdfunding campaign on Ulule website<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Transformation of BBA_1364002 (GAFP1) and BBA_1364003 (D4E1) fungicides genes<br/><br />
- Subculture of the clones for each gene<br/><br />
- PCR and migration on electrophoresis gel<br/><br />
- Transformation BBA_1364003 (D4E1) on pSB1C3 and BBA_1364002 (GAFP1) on pSB1C3<br/><br />
- Cloning BBA_1364002 (GAFP1) with BBa_B0015 (double terminator) and BBA_1364003 (D4E1) with BBa_K823003 (Pveg)<br/><br />
</p><br />
<br />
<p class="title3">Others:</p><br />
<p class="texte"><br />
- Research for plane tickets and hotels in Boston for the Giant Jamboree<br/><br />
- New idea: analyze the plane tree sap to determine the composition<br/><br />
</p><br />
<br />
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<br />
<div class="technology">August 2014 </div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finishing the cloning for the different parts of the bacterium<br/><br />
- Putting in place the fungicides, binding and chemotaxis tests<br/><br />
</p> <br />
<br />
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<br />
<div class="technology">Week 7 (28 July-3 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Financial supports of the Ministery (Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt) and Voies Navigables de France<br/><br />
- Interview with Johan Langot from Sciences Animation for a possible presentation at the Toulouse Festival of Science<br/><br />
- Launch of the crowdfunding campaign on Ulule<br />
</p><br />
<br />
<p class="title3">Lab work: </p><br />
<p class="texte"><br />
- Cloning BBa_K823003 (Pveg) + RFP and BBa_K823002 (PlepA) + RFP in pSBBs1C<br/><br />
- Test of every pSBBs vector : BBa_K823021 (pSBBS1C-lacZ), BBa_K823022 (pSBBS4S), BBa_K823023 (pSBBS1C), minipreps and cryopreservation of the best clones<br/><br />
- Checking of BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) transformants (biobrick BBa_K1364007) and BBA_1364003 (D4E1) + BBa_K823003 (Pveg) biobrick BBa_K1364009<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) + pSBBS4S (BBa_K823022) and subculture of the colonies<br/><br />
- Cloning of BBa_K823003 (Pveg) + BBA_1364003 (D4E1) with BBa_B0015 (double terminator)<br/><br />
- Cloning of BBa_K823003 (Pveg) + RFP (BBa_K1364017) in pSBBS4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_K823002 (PlepA) + RFP (BBa_K1364016) in pSBBs4S (BBa_K823022), subculture and minipreps<br/><br />
- Cloning of BBa_1364018 (Strong Promotor + RFP)<br/><br />
- Cloning of BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) + Promotor BBa_K823003 (Pveg) biobirck BBa_K1364012 with gel extraction for BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) and a PCR cleanup for BBa_K823003 (Pveg), subculture<br/><br />
- Cloning BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBS4S in <i>E. coli</i><br/><br />
- Transformation of binding gene with <i>E. coli</i> competent cells<br/><br />
- Transformation of BBa_K1364000, the chemotaxis gene in <i>E. coli</i><br/><br />
Problem: the transformation worked but a cell layer appeared. A new subculture of the colonies was made as well as a new spreading on petri dishes.<br/><br />
- Spreading of BBa_K1162001 (EcAMP), miniprep and digestion<br/><br />
Problem: the digestion did not work => Issue with the quantity of DNA in the miniprep is assumed<br/><br />
- First experience of chemotaxis with a glucose chemo-attractant<br/><br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/01/2014)</p><br />
<p class="texte"><br />
- Possible problem with the restriction enzymes regarding the digestion: new recommendations<br />
<br/><br />
- Discussion about EcAMP cloning which presents some issues<br />
<br/><br />
- Discussion about the transfer of pSB1C3 from E. coli to Bacillus strain<br />
</p> <br />
<br />
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<br />
<br />
<div class="technology">Week 8 (04-10 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Participation at the “Passion Jeune” competition organized by a French bank foundation named Fondation Crédit Agricole Toulouse<br />
</p><br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Check the cloning of BBa_K823003 (Pveg)+BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBs4S and cryopreservation<br />
<br/><br />
- Transformation of BBa_K823003 (Pveg) +BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) (biobrick BBa_ K1364008) in BBa_K823022 (pSBBS4S) <br />
<br/><br />
- Cloning of BBa_K1364001, the binding gene + BBa_K823003 (Pveg) on pSB1C3 and pSBBS 4S<br />
<br/><br />
Problem: the band is at 1500 bp instead of 1300 bp on the gel<br />
<br/><br />
- Cloning of BBa_K1162001 (EcAMP) + BBa_K823003 (Pveg) + BBa_K606061 (RBS SpoVG), miniprep, PCR and digestion <br />
<br/><br />
Problem: the fragment of DNA is too small (149 bp) to be seen on the gel and was probably lost during the PCR cleanup. Instead of that, we used the heat enzymes inactivation at 95°C for 15 minutes. <br />
<br/><br />
- Cloning of Promotor + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) in pSB1C3 and pSBBS4S and cryopreservation<br />
<br/><br />
- Elaboration of an efficient fungicide test protocol <br />
<br/><br />
- Test of the fungus growth on PDA medium + test of growth for Bacillus subtilis liquid culture on PDA<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/08/2014)</p><br />
<p class="texte"><br />
- Ask iGEM headquarters if all the biobricks must be on pSB1C3 plasmid<br />
<br/><br />
- Use another strain of E. coli (DH5-1 instead of DH5 alpha) to have a faster growth<br />
<br/><br />
- Check which quantity of Bacillus subtilis is necessary to naturally destroy the fungus<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology">Week 9 (11-17 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Publication of more articles about our project in Labiotech, Développement durable (Networkvisio), Midi Libre, l’Indépendant, DigiSchool, La voix du Midi Lauragais, LURIO Addl.<br />
</p><br />
<br />
<p class="title2">Lab work:</p><br />
<p class="texte"><br />
- Evaluation of the bacterial decrease rate from 10°C to 4°C with a sporuling strain of Bacillus subtilis.<br />
<br/><br />
- Chemotaxis test with different concentration of glucose on a 0.3% agar.<br />
<br/><br />
- Miniprep of pSBbs4S with binding gene and transformation in <i>Bacillus subtilis</i>.<br />
<br/><br />
Problem: no digestion was visible on the gel => the cloning failed<br />
<br/><br />
- Transformation of BBa_K1374010 (Pveg + RBS SpoVG + EcAMP) + BBa_K1364012 (RBS + Gafp1 + D4E1 + double terminator), subculture<br />
<br/><br />
- Subculture of Bacillus subtilis clones transformed by BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) in pSBBs4S, cryopreservation<br />
<br/><br />
- Fungicide test<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology">Week 10 (18-24 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Ethics: Interview with a specialist in ethical sciences, Vincent Grégoire Delory to complete our reflexion about the ethical issues in SubtiTree project.<br />
<br/><br />
- Wiki: hiring a web designer, Adrien Nicod, to improve the aesthetic effect and the logo.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Subculture and transformation in Bacillus subtilis of BBa_K1364011 in pSBBS4S (BBa_K823022) and cloning of BBa_K1364011 on pSBBS1C (BBa_K823023).<br />
<br/><br />
Problem: no clone had the insert on pSBBS1C => the cloning had to be done again<br />
- Miniprep of BBa_K1364011 in BBa_K823023 (pSBBS1C) + Transformation in <i>B. subtilis</i>.<br />
<br/><br />
- Cloning of BBA_1364003 (D4E1) , BBA_1364002 (GAFP1) , BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) on BBa_K823023 (pSBBS1C) <br />
<br/><br />
- Transformation BBa_1364004 (in pSBBS4S in E.coli <br />
<br/><br />
- Cloning of BBa_K1364014 (Pveg + RBS SpoVG + EcAMP + GAFP1 +D4E1 + double terminator) in K823022 (pSBBS4S) and in BBa_K823023 (pSBBS1C), miniprep and digestion by restriction enzymes to see the size of the insert on a 1% gel<br />
<br/><br />
Problem: no clone had the insert on pSBBS1C => the cloning had to be done again<br />
</p><br />
<br />
<br />
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</div><br />
<br />
<div class="technology">Week 11 (25- 31 August)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Beginning of the creation and redaction of the different parts of the wiki by the Toulouse iGEM Toulouse<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Cloning of BBa_K1364014 in BBa_K823022 and in BBa_K823023 <br />
<br/><br />
- Cloning GAFP1 + BBa_K823023 (pSBBS1C) in Bacillus subtilis with a change in the protocol: pre-induction of the culture with 0.125 µg/ml of chloramphenicol one hour after the addition of DNA, and let grow for another hour.<br />
<br/><br />
Problem: no colony <br />
<br/><br />
- Liquid culture of Bacillus + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) for future fungicide tests<br />
<br/><br />
- Miniprep of BBa_ K1364011 in BBa_ K823023 (pSBBs1C) and transformation in Bacillus subtilis strain. <br />
<br/><br />
- Fungicide test of BBa_K1364011 in pSBBs4S, Promotor + BBA_1364002 (GAFP1) + Terminator (biobrick BBa_K1364008) in pSBBS4S, Promotor + BBA_1364003 (D4E1) + Terminator (biobrick BBa_K1364009) in pSBBS4S, Promotor + BBA_1364002 (GAFP1) + BA_1364003 (D4E1) + Terminator ( biobrick BBa_K1364013) in pSBBS4S<br />
<br/><br />
- Transformation of a new vector PKL 190 (threonine integrative) in Bacillus strain <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(08/29/2014)</p><br />
<p class="texte"><br />
- Boston accommodation must be booked<br />
<br/><br />
- Discussion about the contamination seen on the fungicide tests : the issue seems to come from the sterile water which contained contaminants<br />
<br/><br />
- Objectives : chemotaxis, binding and fungicides tests have to be performed again<br />
</p> <br />
<br />
<p class="title1" id="select6"> September 2014</p><br />
<p class="title2">Main activities</p><br />
<p class="texte"><br />
- Finish the tests of the different modules<br />
<br/><br />
- Establish the final editorial parts for the wiki<br />
<br/><br />
- Design of the wiki<br />
<br/><br />
- Sequencing the different assembled parts of our bacterium<br />
</p> <br />
<br />
<br />
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</div><br />
<br />
<div class="technology">Week 12 (1-7 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Different parts of the wiki by the Toulouse iGEM Toulouse<br/><br />
- Reservation of Boston accommodation<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Chemotaxis test and modelling<br />
<br/><br />
- Fungicides tests on EcAMP-A, EcAMP-B, EcAMP-C, EcAMP-D, MaxiOpA, MaxiOpB, MaxiOpC, MaxiOpD from 2ml of overnight cultures. Only with 25000 conidia with both fungi.<br />
<br/><br />
- PCR on pSBBS4S + BBa_K1162001 (EcAMP) <br />
<br/><br />
Problem: failed on both diluted plasmid and colony<br />
<br/><br />
- Spreading of BBa_K1364014 + BBa_ K823022 in threonine medium and in medium without threonine <br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/05/2014)</p> <br />
<p class="texte"><br />
- The first sequencing was not successful => new sequencing with one primer/tube<br/><br />
- Chemotaxis test: increase the concentration in bacterium, try a new protocol by capillarity<br/><br />
- End of the binding test: IT WORKS PERFECTLY! SubtiTree has the good phenotype and presents a better binding property than the wild type Bacillus subtilis strain.<br/><br />
- Fungicides test: contaminants with the fungus. Need to ask for another culture of our fungi.<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology">Week 13 (8- 14 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Telephone interview for an article in a French national review named Fluvial which will be published at the end of September.<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- PCR on D4E1 colony with different primers<br />
<br/>- Culture and miniprep of EcAMP to get more DNA + analytic digestion <br />
<br/>- Transformation of K1364014+K1364006 in E. coli <br />
<br/>- Transformation of GAFP1 + D4E1, GAFP1 and EcAMP+GAFP1+D4E1 on Bacillus subtilis + colony PCR + threonine test<br />
<br/>NB: good results for the maxi operon and GAFP1 but not for GAFP1 + D4E1 CA <br />
<br/>- Cloning of K606013+pEX_K4, 823022+PlepA_RFP, 823022+ Pveg_RFP<br />
<br/>- Chemotaxis tests: different times of LB + agar addition in the LB medium (after 3 minutes, 5 minutes, 8 minutes, 10 minutes…) are performed => No difference of the results after spreading on the plates.<br />
<br/>- New test of chemotaxis: capillary between two Eppendorf tubes<br />
<br/>- Spreading of the fungi on a medium which mimics the sap<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/11/2014)</p> <br />
<p class="texte"><br />
- Try to use the GFP or RFP to follow the chemotaxis test.<br />
<br/>- Make a new glass process for the chemotaxis capillary test.<br />
<br/>- Possibility of contacting an INRA laboratory to get pictures of the binding test with confocal microscope.<br />
<br/>- Sequencing the binding construction.<br />
<br/>- Try different temperatures for the fungicide tests: 20°C, 25°C, and 37°C to reduce the fungus growth.<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology">Week 14 (15 - 21 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- Support of two French city halls: Mairie de Montréal, Mairie de Ventenac en Minervois.<br />
<br/>- First interview by the French national radio France Inter which will be aired on October 12th.<br />
<br/>- Beginning of the organization of the iGEM Toulouse 2014 acknowledgement day: the purpose is to present the project to the students and give a feedback on the competition to the sponsors.<br />
<br/>- Final design of the wiki<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Threonine tests for the BBa_K1364014 maxi operon (EcAMP1 – GAFP1 – D4E1) and mini operon BBa_K1364013 (GAFP1-D4E1)<br />
<br/>- Colony PCR on the mini operon BBa_K1364013 (GAFP1 - D4E1)<br />
<br/>- Colony PCR on BBa_K1364011 on BBa_K823022 (pSBBS4S)<br />
<br/>- Chemotaxis test with wild type <i>Bacillus subtilis</i> strain using Imperial College protocol<br />
<br/>- Tansformation of BBa_K1364011 + BBa_K823022 (pSBBS4S) in Bacillus subtilis<br />
<br/>- Culture of fungi with fungicides<br />
<br/>- Sequencing of plasmids containing the fungicides<br />
</p><br />
<br />
<p class="title3">Weekly meeting with the instructors(09/18/2014)</p> <br />
<p class="texte"><br />
- Sequencing worked for D4E1 and GAFP1 and the mini operon BBa_K1364013 (GAFP1-D4E1).<br />
<br/>- The next step is to sequence the maxi operon (EcAMP1 – GAFP1 – D4E1).<br />
<br/>- Try different approaches for the chemotaxis:<br />
<br/>- New protocol with the capillary assay with a new system made by a glassblower.<br />
<br/>- Make the Imperial College test again by testing 3 different solutions: chitin, N-acetylglucosamine and another carbon source.<br />
<br/>- Decrease the number of conidia and temperature for the fungicide test.<br />
</p><br />
<br />
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</div><br />
<br />
<div class="technology">Week 15 (22 - 28 September)</div><br />
<div class="thelanguage"><br />
<br />
<p class="title3">Communication and financial support:</p><br />
<p class="texte"><br />
- New support of Sallèles d’Aude city hall.<br />
<br/>- Payment of the plane tickets.<br />
<br/>- Acknowledgement day (4th December): contact with catering service, reservation of the amphitheater, list of guests…<br />
<br/>- Order of the T shirt for the Jamboree! We look awesome with them!<br />
</p><br />
<br />
<p class="title3">Lab work:</p><br />
<p class="texte"><br />
- Last experiments regarding chemotaxis tests.<br />
</p><br />
<br />
<br />
<p class="title3">Weekly meeting with the instructors(09/26/2014)</p> <br />
<p class="texte"><br />
- Focus on the wiki design and writing.<br />
<br/>- Start making the power point presentation for Boston and the poster.<br />
<br/>- Division of responsibilities for all the non-scientific work.<br />
</p><br />
<br />
<br />
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<br />
<div class="technology">October 2014</div><br />
<div class="thelanguage"><br />
<br />
<p class="title2">Main activites</p><br />
<p class="texte"><br />
- Preparation of the wiki every day… all day long…. so hard to work on this even at night!<br />
<br/>- Preparation of the poster and the presentation for the Jamboree with our instructors <br />
<br/><br />
- Daily presentations in front of the laboratory teachers to repeat again and again and again… let’s get ready for the Jamboree!<br />
</p> <br />
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<p class="texte">How did we confirm our three different modules and how did we improve our new test? Click on these next titles to see SubtiTree abilities.</p><br />
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<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a> | <a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a></p><br />
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<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">For this module, we performed several tests to prove the existence of chemotaxis in <i>Bacillus subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.<br />
</p><br />
<br />
<p class="texte">We wanted to see chemotaxis on petri dish. We hoped to obtain pictures with bacteria halos directed or around attractive components. Thus we tried different protocols on <i>Bacillus subtilis</i>.<br />
The first one was a protocol from <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>. They put attractive compound on paper disk in the middle of a petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Figure 1).</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Schema showing how cells are filed in the medium. (A) pipetman are used to put cells in the gelose. (B) Bacteria should move to the attractive compound which diffuses.</p><br />
<br />
<p class="texte">We did not have any result with positive test on <i>Bacillus subtilis</i> and with glucose as attractive compound (Figure 2-A). <i>B. sub</i> is attracted by many other glucides and amino-acids so we have diluted glucose in LB medium and used this solution as a target (Figure 2-B).</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with Glucose as attractive compound (A) and Glucose in add to LB medium as attractant (B).</p><br />
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<p class="texte"><We could not notice any difference between the petri dish with or without glucose on paper. With an addition of LB medium to sugar, a large halo around paper disk is observed. This halo may corresponds to cells attracted by solution, or it may be diffusion of the mix.</br><br />
Anyway we did not have enough reproducible and reliable results to be satisfied with this test. Furthermore if we are forced to add LB to sugar to observe something, it is hard to distinguish between attracting and chemotaxis effects.</br><br />
We have started new tries using different protocols</p><br />
<br />
<p class="title2">1. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol on which we worked is taken from a thesis (ref thèse). <i>B.subtilis</i> are grown overnight and if necessary bacteria cells are concentrated by centrifugation. Goal is to obtain a cells density to 8x10⁸ cells/mL. 10mL of bacteria cells are mixed with 15mL of LB medium with 1.5 % agar maintained at 50°C. We obtain a medium with 0.9 % agar at final concentration. We add tetracycline at 25µg/mL thus growth are stopped. Plate are cooled and dried, then well are made with punch or 1mL tips. In well attractive compound are put (Figure 3). After one hour at room temperature, we take a picture of plates and analyzed results.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Schema showing how are made plug-in-pond tests.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells by plate (Figure 4). In wells we put glucose at different concentration and in one of the plate we do not put tetracycline.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
We respect the protocol and after one hour we observe nothing, it's only after 12h than we can observe an halo around well with glucose at 1M in the plate where there are no tetracycline. Tetracycline concentration seems to be too large and inhibit our bacteria. Thereafter we have work with tetracycline at 15µg/mL.<br />
We retry this protocol with less tetracycline. We made two wells by plate (Figure 5) one with attractive compound, Glucose or n-acetyl-glucosamide and one with LB medium. After 1h there are no halos, 12h after we observe something.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>Bacillus subtilis</i> WT. The upper well contain attractive compound and the lower contain medium without attractive compound. </p><br />
<br />
<p class="texte"><br />
Results are not as clear as the first time, but we observe halo around well with glucose at 250mM with and without tetracycline. We have made tries with N-acetyl-glucosamide and we see no halo, this show that our strain <i>B. subtilis</i> 168 is not attracted by N-acetyl-glucosamide.</br><br />
Results are not enough clear and reliable with plug-in-pond. We do not understand why we have to wait 12 hours to see halos. So we tried other protocols.<br />
</p><br />
<br />
<br />
<p class="texte"><br />
<b>References:</b></br><br />
thesis : Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez Escherichia coli et Shewanella oneidensis, 2008, Claudine Baraquet, université de la méditerranée Aix-Marseille II<br />
</p><br />
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<p class="title2">2. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug in pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse iGEM Team asked the help from the glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
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<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of by the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacteria solution of WT Bacillus subtilis in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed of the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
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<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the Imperial college iGEM team 2011: the tips capillary test.</br><br />
</p><br />
<br />
<p class="title2"> 3. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br><br />
<br><br />
First of all, parafilm was used to close the tips:<br><br />
- 15µL of each chemo-attractant was then pipetted. <br><br />
- The tips with the pipette were then put on a piece of parafilm and the pipette was removed from the tip.<br><br />
- The tip was sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the chemo-attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 10.<br><br />
- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plate instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette. The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
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<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium with the chemotaxis gene… That is why we concentrated our efforts on WT <i>Bacillus subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
The experiment was conducted with fuchsin as a negative control and was tested with different positive controls: glucose (25mM) and xylose (25mM).<br><br />
<br><br />
We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology and on our strength! Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
We finally used galactose (25mM) as a negative control. The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
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<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2">1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 16).<br />
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="legend">Figure 16: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).<br />
</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. <br />
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determine the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of bead surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin</p><br />
<br />
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of bacteria after washing <br />
</p><br />
<br />
<p class="texte">Finally, overall results are consistent with the presence of functional binding system.</p><br />
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<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporing halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
<br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<p class="legend"> Figure 21: Results of the preliminary tests</p><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree</p><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1 (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of SubtiTree in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p><br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves' necrosis </b>.<br />
</p><br />
<br />
<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.</p><br />
<br />
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<br />
<p class="texte">How did we confirm our three different modules and how did we improve our new test? Click on these next titles to see SubtiTree abilities.</p><br />
<br />
<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a> | <a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a></p><br />
<br />
<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">For this module, we performed several tests to prove the existence of chemotaxis in <i>Bacillus subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.<br />
</p><br />
<br />
<p class="texte">We wanted to see chemotaxis on petri dish. We hoped to obtain pictures with bacteria halos directed or around attractive components. Thus we tried different protocols on <i>Bacillus subtilis</i>.<br />
The first one was a protocol from <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>. They put attractive compound on paper disk in the middle of a petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Figure 1).</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Schema showing how cells are filed in the medium. (A) pipetman are used to put cells in the gelose. (B) Bacteria should move to the attractive compound which diffuses.</p><br />
<br />
<p class="texte">We did not have any result with positive test on <i>Bacillus subtilis</i> and with glucose as attractive compound (Figure 2-A). <i>B. sub</i> is attracted by many other glucides and amino-acids so we have diluted glucose in LB medium and used this solution as a target (Figure 2-B).</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with Glucose as attractive compound (A) and Glucose in add to LB medium as attractant (B).</p><br />
<br />
<p class="texte"><We could not notice any difference between the petri dish with or without glucose on paper. With an addition of LB medium to sugar, a large halo around paper disk is observed. This halo may corresponds to cells attracted by solution, or it may be diffusion of the mix.</br><br />
Anyway we did not have enough reproducible and reliable results to be satisfied with this test. Furthermore if we are forced to add LB to sugar to observe something, it is hard to distinguish between attracting and chemotaxis effects.</br><br />
We have started new tries using different protocols</p><br />
<br />
<p class="title2">1. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol on which we worked is taken from a thesis (ref thèse). <i>B.subtilis</i> are grown overnight and if necessary bacteria cells are concentrated by centrifugation. Goal is to obtain a cells density to 8x10⁸ cells/mL. 10mL of bacteria cells are mixed with 15mL of LB medium with 1.5 % agar maintained at 50°C. We obtain a medium with 0.9 % agar at final concentration. We add tetracycline at 25µg/mL thus growth are stopped. Plate are cooled and dried, then well are made with punch or 1mL tips. In well attractive compound are put (Figure 3). After one hour at room temperature, we take a picture of plates and analyzed results.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Schema showing how are made plug-in-pond tests.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells by plate (Figure 4). In wells we put glucose at different concentration and in one of the plate we do not put tetracycline.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
We respect the protocol and after one hour we observe nothing, it's only after 12h than we can observe an halo around well with glucose at 1M in the plate where there are no tetracycline. Tetracycline concentration seems to be too large and inhibit our bacteria. Thereafter we have work with tetracycline at 15µg/mL.<br />
We retry this protocol with less tetracycline. We made two wells by plate (Figure 5) one with attractive compound, Glucose or n-acetyl-glucosamide and one with LB medium. After 1h there are no halos, 12h after we observe something.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>Bacillus subtilis</i> WT. The upper well contain attractive compound and the lower contain medium without attractive compound. </p><br />
<br />
<p class="texte"><br />
Results are not as clear as the first time, but we observe halo around well with glucose at 250mM with and without tetracycline. We have made tries with N-acetyl-glucosamide and we see no halo, this show that our strain <i>B. subtilis</i> 168 is not attracted by N-acetyl-glucosamide.</br><br />
Results are not enough clear and reliable with plug-in-pond. We do not understand why we have to wait 12 hours to see halos. So we tried other protocols.<br />
</p><br />
<br />
<br />
<p class="texte"><br />
<b>References:</b></br><br />
thesis : Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez Escherichia coli et Shewanella oneidensis, 2008, Claudine Baraquet, université de la méditerranée Aix-Marseille II<br />
</p><br />
<br />
<br />
<br />
<br />
<p class="title2">2. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug in pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse iGEM Team asked the help from the glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of by the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacteria solution of WT Bacillus subtilis in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed of the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the Imperial college iGEM team 2011: the tips capillary test.</br><br />
</p><br />
<br />
<p class="title2"> 3. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br><br />
<br><br />
First of all, parafilm was used to close the tips:<br><br />
- 15µL of each chemo-attractant was then pipetted. <br><br />
- The tips with the pipette were then put on a piece of parafilm and the pipette was removed from the tip.<br><br />
- The tip was sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the chemo-attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 10.<br><br />
- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plate instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette. The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium with the chemotaxis gene… That is why we concentrated our efforts on WT <i>Bacillus subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
The experiment was conducted with fuchsin as a negative control and was tested with different positive controls: glucose (25mM) and xylose (25mM).<br><br />
<br><br />
We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology and on our strength! Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
We finally used galactose (25mM) as a negative control. The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
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<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2">1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 16).<br />
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="legend">Figure 16: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).<br />
</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. <br />
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determine the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of bead surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin</p><br />
<br />
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of bacteria after washing <br />
</p><br />
<br />
<p class="texte">Finally, overall results are consistent with the presence of functional binding system.</p><br />
<br />
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<br />
<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporing halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
<br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<p class="legend"> Figure 21: Results of the preliminary tests</p><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree</p><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1 (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of SubtiTree in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p><br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves' necrosis </b>.<br />
</p><br />
<br />
<img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg" <p class="legend"> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.</p><br />
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<br />
<p class="texte">How did we confirm our three different modules and how did we improve our new test? Click on these next titles to see SubtiTree abilities.</p><br />
<br />
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<br />
<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">For this module, we performed several tests to prove the existence of chemotaxis in <i>Bacillus subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.<br />
</p><br />
<br />
<p class="texte">We wanted to see chemotaxis on petri dish. We hoped to obtain pictures with bacteria halos directed or around attractive components. Thus we tried different protocols on <i>Bacillus subtilis</i>.<br />
The first one was a protocol from <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>. They put attractive compound on paper disk in the middle of a petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Figure 1).</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Schema showing how cells are filed in the medium. (A) pipetman are used to put cells in the gelose. (B) Bacteria should move to the attractive compound which diffuses.</p><br />
<br />
<p class="texte">We did not have any result with positive test on <i>Bacillus subtilis</i> and with glucose as attractive compound (Figure 2-A). <i>B. sub</i> is attracted by many other glucides and amino-acids so we have diluted glucose in LB medium and used this solution as a target (Figure 2-B).</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with Glucose as attractive compound (A) and Glucose in add to LB medium as attractant (B).</p><br />
<br />
<p class="texte"><We could not notice any difference between the petri dish with or without glucose on paper. With an addition of LB medium to sugar, a large halo around paper disk is observed. This halo may corresponds to cells attracted by solution, or it may be diffusion of the mix.</br><br />
Anyway we did not have enough reproducible and reliable results to be satisfied with this test. Furthermore if we are forced to add LB to sugar to observe something, it is hard to distinguish between attracting and chemotaxis effects.</br><br />
We have started new tries using different protocols</p><br />
<br />
<p class="title2">1. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol on which we worked is taken from a thesis (ref thèse). <i>B.subtilis</i> are grown overnight and if necessary bacteria cells are concentrated by centrifugation. Goal is to obtain a cells density to 8x10⁸ cells/mL. 10mL of bacteria cells are mixed with 15mL of LB medium with 1.5 % agar maintained at 50°C. We obtain a medium with 0.9 % agar at final concentration. We add tetracycline at 25µg/mL thus growth are stopped. Plate are cooled and dried, then well are made with punch or 1mL tips. In well attractive compound are put (Figure 3). After one hour at room temperature, we take a picture of plates and analyzed results.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Schema showing how are made plug-in-pond tests.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells by plate (Figure 4). In wells we put glucose at different concentration and in one of the plate we do not put tetracycline.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
We respect the protocol and after one hour we observe nothing, it's only after 12h than we can observe an halo around well with glucose at 1M in the plate where there are no tetracycline. Tetracycline concentration seems to be too large and inhibit our bacteria. Thereafter we have work with tetracycline at 15µg/mL.<br />
We retry this protocol with less tetracycline. We made two wells by plate (Figure 5) one with attractive compound, Glucose or n-acetyl-glucosamide and one with LB medium. After 1h there are no halos, 12h after we observe something.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>Bacillus subtilis</i> WT. The upper well contain attractive compound and the lower contain medium without attractive compound. </p><br />
<br />
<p class="texte"><br />
Results are not as clear as the first time, but we observe halo around well with glucose at 250mM with and without tetracycline. We have made tries with N-acetyl-glucosamide and we see no halo, this show that our strain <i>B. subtilis</i> 168 is not attracted by N-acetyl-glucosamide.</br><br />
Results are not enough clear and reliable with plug-in-pond. We do not understand why we have to wait 12 hours to see halos. So we tried other protocols.<br />
</p><br />
<br />
<br />
<p class="texte"><br />
<b>References:</b></br><br />
thesis : Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez Escherichia coli et Shewanella oneidensis, 2008, Claudine Baraquet, université de la méditerranée Aix-Marseille II<br />
</p><br />
<br />
<br />
<br />
<br />
<p class="title2">2. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug in pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse iGEM Team asked the help from the glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of by the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacteria solution of WT Bacillus subtilis in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed of the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the Imperial college iGEM team 2011: the tips capillary test.</br><br />
</p><br />
<br />
<p class="title2"> 3. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br><br />
<br><br />
First of all, parafilm was used to close the tips:<br><br />
- 15µL of each chemo-attractant was then pipetted. <br><br />
- The tips with the pipette were then put on a piece of parafilm and the pipette was removed from the tip.<br><br />
- The tip was sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the chemo-attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 10.<br><br />
- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plate instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette. The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium with the chemotaxis gene… That is why we concentrated our efforts on WT <i>Bacillus subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
The experiment was conducted with fuchsin as a negative control and was tested with different positive controls: glucose (25mM) and xylose (25mM).<br><br />
<br><br />
We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology and on our strength! Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
We finally used galactose (25mM) as a negative control. The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
<br />
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<br />
<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2">1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 16).<br />
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="legend">Figure 16: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).<br />
</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. <br />
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determine the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of bead surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin</p><br />
<br />
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of bacteria after washing <br />
</p><br />
<br />
<p class="texte">Finally, overall results are consistent with the presence of functional binding system.</p><br />
<br />
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<br />
<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporing halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
<br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<p class="legend"> Figure 21: Results of the preliminary tests</p><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree<p/><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1 (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/><br />
<p class="legend"> Figure 23: Injection of SubtiTree in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p><br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves' necrosis </b>.<br />
</p><br />
<br />
<img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg" <p class="legend"> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.</p><br />
<br />
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<script type="text/javascript" src="https://2011.igem.org/Team:Imperial_College_London/ddaccordion?action=raw&ctype=text/js"><br />
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/***********************************************<br />
* Accordion Content script- (c) Dynamic Drive DHTML code library (www.dynamicdrive.com)<br />
* Visit http://www.dynamicDrive.com for hundreds of DHTML scripts<br />
* This notice must stay intact for legal use<br />
***********************************************/<br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<script type='text/javascript' src='http://ajax.googleapis.com/ajax/libs/jquery/1.9.0/jquery.min.js'><br />
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<br />
</script><br />
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<br />
<style type="text/css"><br />
<br />
.technology{ /*header of 2nd demo*/<br />
cursor: hand;<br />
cursor: pointer;<br />
border-bottom:3px solid #BDCBBD;<br />
<br />
font-family:'Open Sans';<br />
color: green;<br />
font-weight:600;<br />
font-size:24px;<br />
margin:0 0 33px 0;<br />
border:none;<br />
<br />
}<br />
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<br />
.openlanguage{ /*class added to contents of 2nd demo when they are open*/<br />
color: green;<br />
}<br />
<br />
.closedlanguage{ /*class added to contents of 2nd demo when they are closed*/<br />
color: #225e22;<br />
}<br />
<br />
</style><br />
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<script type="text/javascript"><br />
<br />
//Initialize 2nd demo:<br />
ddaccordion.init({<br />
headerclass: "technology", //Shared CSS class name of headers group<br />
contentclass: "thelanguage", //Shared CSS class name of contents group<br />
revealtype: "click", //Reveal content when user clicks or onmouseover the header? Valid value: "click", "clickgo", or "mouseover"<br />
mouseoverdelay: 200, //if revealtype="mouseover", set delay in milliseconds before header expands onMouseover<br />
collapseprev: false, //Collapse previous content (so only one open at any time)? true/false <br />
defaultexpanded: [], //index of content(s) open by default [index1, index2, etc]. [] denotes no content.<br />
onemustopen: false, //Specify whether at least one header should be open always (so never all headers closed)<br />
animatedefault: false, //Should contents open by default be animated into view?<br />
persiststate: false, //persist state of opened contents within browser session?<br />
toggleclass: ["closedlanguage", "openlanguage"], //Two CSS classes to be applied to the header when it's collapsed and expanded, respectively ["class1", "class2"]<br />
togglehtml: ["suffix", " <small><small>[Expand]</small></small>", " <small><small>[Collapse]</small></small>"], //Additional HTML added to the header when it's collapsed and expanded, respectively ["position", "html1", "html2"] (see docs)<br />
animatespeed: "fast", //speed of animation: integer in milliseconds (ie: 200), or keywords "fast", "normal", or "slow"<br />
oninit:function(expandedindices){ //custom code to run when headers have initalized<br />
//do nothing<br />
},<br />
onopenclose:function(header, index, state, isuseractivated){ //custom code to run whenever a header is opened or closed<br />
//do nothing<br />
}<br />
})<br />
<br />
</script><br />
</head><br />
</html></div>Dbby