Team:Toulouse/Notebook/project-monitoring

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   <div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px;">
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   <p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Calendar</p>  
+
   <div style="margin:0 auto; width:960px;">
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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>
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    </div>
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       <div class="centering" style="padding-top: 85px; padding-bottom:40px;">
       <div class="centering" style="padding-top: 85px; padding-bottom:40px;">
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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>
 +
 +
<div class="technology">Binding module</div>
 +
<div class="thelanguage">
 +
 +
<p class="title2">1. Amplification of Binding Module (pEX-K4) into <i>E. coli</i></p>
 +
<p class="title3">Transformation of Binding module (pEX-K4) into <I>E. coli</i></p>
 +
<p class="texte">Gene "Binding module" synthesized by Eurofins, resuspended in 20μL Tris 10mM
 +
<br><b>Result:</b>  We obtained distinct colonies on plates LB + Kanamycin (50 µg/mL)</p>
 +
 +
<p class="title3">Liquid culture of two clones: 1 et 2 (pEX-K4) transformed into <i>E. coli</i></p>
 +
<p class="texte"><b>Date:</b> 01/08/2014</p>
 +
 +
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit: two clones of Binding Module (pEX-K4) into <i>E. coli</i></p>
 +
<p class="texte"><b>Date:</b> 04/08/2014
 +
<br><b>Result:</b> Binding Module (pEX-K4) obtained</p>
 +
 +
<p class="title3">Digestion of Binding Module (pEX-K4) with EcoRI and PstI</p>
 +
<p class="texte"><b>Date:</b> 04/08/2014
 +
<br><b>Result:</b>
 +
<br>- 3 bands : 1500bp (vector with Kanamycin resistance), 1300bp (Module Binding) and 1000bp (vector with pUC Ori)
 +
<br>- The two Binding Module clones are ok</p>
 +
 +
 +
<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>
 +
<p class="title3">Digestion Binding Module on pEX-K4 and <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a></p>
 +
<p class="texte"><b>Date:</b> 23/08/2014
 +
<br><b>Expected bands after digestion:</b>
 +
<br>- <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a>: 860 bp for RFP and 2100 bp for vector  pSB1C3
 +
<br>- Binding Module: 1400 bp for Binding Module and 1500bp + 1000bp for vector pEX_K4
 +
<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>
 +
 +
<p class="title3">Ligation Binding Module on pSB1C3</p>
 +
<p class="texte"><b>Date:</b> 04/08/2014</p>
 +
 +
<p class="title3">Transformation of Binding Module on pSB1C3 into E. coli</p>
 +
<p class="texte"><b>Date:</b> 04/08/2014
 +
<br>Transformation with 10 µL of the ligation mix and plate on Chloremphenicol LA plate
 +
<br><b>Result:</b> many wrong clones</p>
 +
 +
<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>
 +
<p class="title3">Digestion Binding Module on pEX-K4 and <a href="http://parts.igem.org/Part:BBa_K823003">BBa_K823003</a></p>
 +
<p class="texte"><b>Date:</b> 04/08/2014
 +
<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>Gel Electrophoresis
 +
<br><b>Result:</b>
 +
<br>- Binding Module : 1400 bp for Binding Module and 1500bp + 1000bp for vector pEX_K47
 +
<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>
 +
 +
<p class="title3">Ligation Binding Module on pVeg with pSB1C3</p>
 +
<p class="texte"><b>Date:</b> 04/08/2014
 +
<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>
 +
 +
<p class="title3">Transformation of Binding Module on pVeg into <i>E. coli</i></p>
 +
<p class="texte"><b>Date:</b> 04/08/2014
 +
<br><b>Result:</b> Many good clones (check on 06/08/2014)</p>
 +
 +
<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>
 +
 +
<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>
 +
<p class="texte"><b>Date:</b> 07/08/2014
 +
<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><b>Result:</b>
 +
<br>- Binding Module with Pveg 1600 bp for Binding Module and 2100bp for vector pSB1C3</p>
 +
 +
<p class="title3">Ligation Binding Module with Pveg on pSBbs4S</p>
 +
<p class="texte"><b>Date:</b> 07/08/2014
 +
<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>Ligation
 +
<br><b>Result:</b> Ligation between Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S</p>
 +
 +
<p class="title3">Transformation of Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S into <i>E. coli</i></p>
 +
<p class="texte"><b>Date:</b> 04/08/2014
 +
<br>Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S
 +
<br>Plate on Ampicillin LA plate
 +
<br><b>Result:</b> Many good clones (check on 13/08/2014)</p>
 +
 +
<p class="title3">Transformation of Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S into <i>B. subtilis</i></p>
 +
<p class="texte"><b>Date:</b> 04/08/2014
 +
<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><b>Result:</b> One good clone : PCR (check on 09/09/2014) and threonine test (check on 09/09/2014)</p>
 +
 +
<p class="title2">5. Binding Test</p>
 +
<p class="texte">See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results#select2">here</a></p>
 +
 +
 +
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p>
 +
</div>
 +
 +
 +
<div class="technology">Chemotaxis</div>
 +
<div class="thelanguage">
 +
 +
<p class="title2">1. Transformation of chemotaxis (Puc-57) into <i>E. coli</i></p>
 +
<p class="texte">Gene chemotaxis synthesized by Eurofins, resuspended in 20μL Tris 10mM
 +
<br><b>Date:</b> 01/08/2014
 +
<br><b>Result:</b> We obtained distinct colonies on LA + Ampicillin (100 µg/mL) plates</p>
 +
 +
<p class="title3">Culture of 4 clones: A, B, C, D of chemotaxis (Puc57) transformed into <i>E. coli</i></p>
 +
<p class="texte"><b>Date:</b> 04/08/2014</p>
 +
 +
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit Using a Microcentrifuge: 4 clones of chemotaxis (Puc57) into <i>E. coli</i></p>
 +
<p class="texte"><b>Date:</b> 05/08/2014
 +
<br><b>Result:</b> 4*50µL of chemotaxis (Puc57) obtained</p>
 +
 +
<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>
 +
 +
<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>
 +
<p class="texte"><b>Date:</b> 07/08/2014
 +
<br><b>Result:</b> Expected band after digestion for <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>: 2300 bp
 +
<br><b>Problem:</b> We can't distinguish the vector band (2500 bp)</p>
 +
 +
<p class="title3">Gel extraction of <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a></p>
 +
<p class="texte"><b>Date:</b> 07/08/2014</p>
 +
 +
<p class="title3">Ligation <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> and digested PSB1C3 with EcoRI and PstI</p>
 +
<p class="texte"><b>Date:</b> 08/08/2014</p>
 +
 +
<p class="title3">Transformation <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> in <i>E.coli</i></p>
 +
<p class="texte"><b>Date:</b> 08/08/2014
 +
<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>
 +
 +
<p class="title3">Test of sensibility on Ampicillin</p>
 +
<p class="texte"><b>Date:</b> 10/08/2014
 +
<br><b>Result:</b> we can determine which colonies are sensible for ampicillin and know which bacterium is carrying the chemotaxis gene.</p>
 +
 +
<p class="title3">PCR</p>
 +
<p class="texte"><b>Date:</b> 11/08/2014</p>
 +
 +
<p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> on pSB1C3 with EcoRI and PstI</p>
 +
<p class="texte"><b>Date:</b> 11/08/2014
 +
<br><b>Result:</b> There is one colony which presents the right construction.</p>
 +
 +
 +
 +
<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>
 +
<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>
 +
<p class="texte"><b>Date</b>: 08/08/2014</p>
 +
<p class="title3">Transformation <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_1364004</a> in <i>E.coli</i></p>
 +
<p class="texte"><b>Date:</b> 8/08/2014</p>
 +
<p class="title3">Test of sensibility on Ampicillin</p>
 +
<p class="texte"><b>Date:</b> 10/08/2014
 +
<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>
 +
 +
<p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_1364004</a> on pSBC3 with EcoRI and PstI</p>
 +
<p class="texte"><b>Date:</b> 12/08/2014
 +
<br><b>Result:</b> We did not see any colony with chemotaxis insert.</p>
 +
 +
<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>
 +
<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>
 +
<p class="texte"><b>Date:</b> 11/08/2014</p>
 +
<p class="title3">Transformation <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_1364004</a> in <i>E.coli</i></p>
 +
<p class="texte"><b>Date:</b> 11/08/2014</p>
 +
<p class="title3">Test of sensibility on Ampicillin</p>
 +
<p class="texte"><b>Date:</b> 14/08/2014
 +
<br><b>Result:</b> we obtained 4 colonies sensible at Ampicilline</p>
 +
 +
<p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_1364004</a> on PsB1C3 with EcoRI and PstI</p>
 +
<p class="texte"><b>Date:</b> 18/08/2014</p>
 +
<p class="title3">Gel extraction of <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_1364000</a></p>
 +
<p class="texte"><b>Date:</b> 19/08/2014</p>
 +
 +
<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>
 +
<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>
 +
<p class="texte"><b>Date:</b> 19/08/2014</p>
 +
<p class="title3">Transformation <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a> in pSBBS4S in <i>E.coli</i></p>
 +
<p class="texte"><b>Date:</b> 19/08/2014
 +
<br><b>Result:</b> We obtained one colony and resuspended it in LB+ Amp</p>
 +
 +
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 1); return false">Collapse</a></p>
 +
</div>
 +
 +
 +
<div class="technology">Fungicides</div>
 +
<div class="thelanguage">
 +
 +
<div class="technology2">D4E1</div>
 +
<div class="thelanguage2">
 +
 +
<p class="title2">1. Amplification of synthetic gene (D4E1 on pEX-A2)</p>
 +
<p class="title3">Transformation of D4E1 (pEX-A2) into <i>E. coli</i></p>
 +
<p class="texte">Gene D4E1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
 +
<br>Concentration of D4E1: 115ng/µL
 +
<br><b>Date:</b> 07/21//2014
 +
<br><b>Result:</b> We obtained distinct colonies on LA + Ampicillin (100 µg/mL)</p>
 +
 +
<p class="title3">Culture of 4 clones: A, B, C, D of D4E1 (pEX-A2) transformed into E. coli</p>
 +
<p class="texte"><b>Date:</b> 07/22/2014
 +
<br><b>Result:</b> Culture of 4 clones ok</p>
 +
 +
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of D4E1 (pEX-A2) into E. coli</p>
 +
<p class="texte">Buffer EB at 50-55°C
 +
<br><b>Date:</b> 07/23/2014</p>
 +
 +
<p class="title3">Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up</p>
 +
<p class="texte"><b>Date:</b> 07/23/2014
 +
<br><b>Result:</b> 4*20µL D4E1 digested with EcoRI and PstI  all the clones seem to have the right D4E1 gene.</p>
 +
 +
<p class="title2">2. Cloning D4E1 in pSB1C3</p>
 +
<p class="title3">Digestion of D4E1 on pEX-A2 and pSB1C3</p>
 +
<p class="texte"><b>Date:</b> 07/23/2014</p>
 +
<p class="title3">Ligation of D4E1 in pSB1C3</p>
 +
<p class="texte"><b>Date:</b> 07/23/2014</p>
 +
<p class="title3">Transformation in <i>E.coli</i></p>
 +
<p class="texte"><b>Date:</b> 07/23/2014</p>
 +
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of D4E1 (pSB1C3) transformed into <i>E. coli</i></p>
 +
<p class="texte">Processing details: 5mL of LB + chloramphenicol (15µg/mL) , using sterile plastic loop to culture colonies
 +
<br><b>Date:</b> 07/24/2014
 +
<br><b>Result:</b> Culture of 4 clones ok</p>
 +
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
 +
<p class="texte"><b>Date:</b> 07/25/2014</p>
 +
<p class="title3">Digestion of D4E1 (pSB1C3) A, B, C, D, E, F with EcoRI and PstI - PCR kit Clean up + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 07/25/2014
 +
<br><b>Result:</b> clones C, D have the expected construction</p>
 +
<p class="title3">PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 07/25/2014
 +
<br><b>Result:</b> clones C, D have the expected construction, and placed in cryopreservation.</p>
 +
 +
<p class="title2">3. Cloning Pveg+D4E1 on Pveg plasmid (pSB1C3)</p>
 +
 +
<p class="title3">Digestion of D4E1 on pEX-A2 and K823003 (Pveg on pSB1C3)</p>
 +
<p class="texte"><b>Dated:</b></b> 07/24/2014
 +
<br><b>Result:</b> 20µL digestion of D4E1 on pEX-A2 and of K823003</p>
 +
 +
<p class="title3">Ligation of D4E1 in K823003 (Pveg on pSB1C3)</p>
 +
<p class="texte"><b>Date:</b> 07/24/2014
 +
<br><b>Result:</b> 20µL ligation of D4E1 in K823003</p>
 +
 +
<p class="title3">Transformation of ligation products in <i>E.coli</i></p>
 +
<p class="texte"><b>Date:</b> 07/24/2014
 +
<br><b>Result:</b> <i>E.coli</i> transformed by D4E1+K823003</p>
 +
 +
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of transformed <i>E. coli</i></p>
 +
<p class="texte"><b>Date:</b> 07/26/2014
 +
<br><b>Result:</b> Culture of 4 clones ok</p>
 +
 +
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
 +
<p class="texte"><b>Date:</b> 07/28/2014</p>
 +
 +
<p class="title3">PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p>
 +
<p class="texte"><b>Dated:</b> 07/28/2014
 +
<br><b>Result:</b> clones E, F seem to have the expected construction</p>
 +
<p class="title3">Digestion of clones E, F of D4E1+K823003 (Pveg on pSB1C3) with EcoRI and PstI + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 28/07/2014
 +
<br><b>Result:</b> clones C, D have the expected construction and are placed in cryopreservation.</p>
 +
 +
<p class="title2">4. Cloning P<sub>veg</sub> + D4E1 on pSB<sub>BS</sub>4S (K823022) </p>
 +
<p class="texte"><b>Date:</b> 08/13/2014</p>
 +
 +
<p class="title2">5. Cloning P<sub>veg</sub> + D4E1 on pSB<sub>BS</sub>1C lacZ (23)</p>
 +
 +
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p>
 +
</div>
 +
 +
<div class="technology2">GAFP1</div>
 +
<div class="thelanguage2">
 +
 +
<p class="title2">1. Amplification of synthetic gene (GAFP1 on pEX-A2)</p>
 +
 +
<p class="title3">Transformation of GAFP1 (pEX-A2) into <i>E.coli</i></p>
 +
<p class="texte">Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
 +
<br>Concentration of GAFP1 : 145ng/µL
 +
<br><b>Date:</b> 07/21/2014
 +
<br><b>Result:</b> We obtained distinct colonies on plates LB + Ampicillin (100 µg/mL)</p>
 +
 +
<p class="title3">Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli</p>
 +
<p class="texte"><b>Date:</b> 07/22/2014
 +
<br><b>Result:</b> Culture of 4 clones ok</p>
 +
 +
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli</p>
 +
<p class="texte"><b>Date:</b> 07/23/2014</p>
 +
 +
<p class="title3">Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel Electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 07/23/2014 </p>
 +
 +
<p class="title2">2. Cloning GAFP1 gene on PSB1C3 = K1364002 in E. coli</p>
 +
 +
<p class="title3">Digestion GAFP1_pEX-A2 and <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a> (RFP_pSB1C3)</p>
 +
<p class="texte"><b>Date:</b> 07/23/2014
 +
<br><b>Result:</b> <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a> : 860 bp
 +
<br>We decide to conserve the miniprep B for <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a></p>
 +
 +
<p class="title3">Ligation GAFP1 and <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a></p>
 +
<p class="texte"><b>Date:</b> 07/23/2014</p>
 +
 +
<p class="title3">Tranformation of ligation products into <i>E. coli</i></p>
 +
<p class="texte"><b>Date:</b> 07/23/2014</p>
 +
 +
<p class="title3">Culture of x clones of GAFP1+K606013 in <i>E. coli</i></p>
 +
<p class="texte"><b>Date:</b> 07/24/2014</p>
 +
 +
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge: 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) into <i>E. coli</i></p>
 +
<p class="texte"><b>Date:</b> 07/25/2014</p>
 +
 +
<p class="title3">PCR on 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 07/25/2014
 +
<br><b>Result:</b> clones A, C, D, F seem to have the right construction</p>
 +
 +
<p class="title3">Digestion of 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) by EcoR1 and Pst1 + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 07/25/2014</p>
 +
 +
 +
 +
<p class="title2">3.  Cloning GAFP1+terminator(B0015) = K1364007 (pSB1C3)</p>
 +
 +
<p class="title3">Digestion of GAFP1 on pEX-A2 and B0015 (terminator on pSB1C3)
 +
<p class="texte"><b>Date:</b> 07/24/2014
 +
 +
<p class="title3">Ligation of GAFP1 in B0015 (terminator on pSB1C3)</p>
 +
<p class="texte"><b>Date:</b> 07/24/2014</p>
 +
 +
<p class="title3">Transformation of ligation products in <i>E.coli</i></p>
 +
<p class="texte"><b>Date:</b> 07/24/2014</p>
 +
 +
<p class="title3">Culture of 6 clones: A, B, C, D, E, F transformed in <i>E. coli</i></p>
 +
<p class="texte"><b>Date:</b> 07/26/2014</p>
 +
 +
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
 +
<p class="texte"><b>Date:</b> 07/28/2014</p>
 +
 +
<p class="title3">PCR of GAFP1+B0015 = K1364007 (pSB1C3) A, B, C, D, E, F + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 07/28/2014
 +
<br><b>Result:</b> clones B, C, D, E, F seem to have the expected construction.</p>
 +
 +
<p class="title3">Digestion of clones E, F of GAFP1+Ter B0015 = K1364007 with EcoRI and PstI + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 07/28/2014
 +
<br><b>Result:</b> clones B, C, D, E, F have the expected construction.</p>
 +
 +
 +
 +
<p class="title2">4. Cloning GAFP1+terminator with Pveg on pSB1C3 (K1364008) in <i>E. coli</i></p>
 +
 +
<p class="title3">Digestion of GAFP1+ter = K1364007 on pSB1C3 and K823003 (terminator on pSB1C3) + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 07/29/2014</p>
 +
 +
<p class="title3">Gel extraction of K1364007 (extraction of GAFP1+ter gene)</p>
 +
<p class="texte"><b>Date:</b> 07/29/2014</p>
 +
 +
<p class="title3">Ligation of GAFP1+ter in K823003 (Pveg on pSB1C3)</p>
 +
<p class="texte"><b>Date:</b> 07/29/2014
 +
<br><b>Result:</b> 20µL ligation of GAFP1+ter in K823003</p>
 +
 +
<p class="title3">Transformation of ligation products in <i>E.coli</i></p>
 +
<p class="texte"><b>Date:</b> 07/29/2014</p>
 +
 +
<p class="title3">Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli</p>
 +
<p class="texte"><b>Date:</b> 07/30/2014</p>
 +
 +
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
 +
<p class="texte"><b>Date:</b> 07/31/2014</p>
 +
 +
<p class="title3">PCR of GAFP1+B0015 + K823003 = K1364008 (pSB1C3) A, B, C, D, E, F, G, H + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 31/07/2014
 +
<br><b>Result:</b> clones A, B, C, D, E, G, H seem to have the expected construction</p>
 +
 +
<p class="title3">Digestion of clones A, B, C, D, E, G, H of Pveg+K1364007 = K1364008 with EcoRI and PstI + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 31/07/2014
 +
<br><b>Result:</b> clones A, B, C, D, E, G, H have the expected construction</p>
 +
 +
 +
 +
<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>
 +
 +
<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>
 +
<p class="texte"><b>Date:</b> 08/01/2014</p>
 +
 +
<p class="title3">Ligation of K134008 (Pveg+GAFP1+ter fragment) and K823022 (PsBBs4S)</p>
 +
<p class="texte"><b>Date:</b> 08/01/2014</p>
 +
 +
<p class="title3">Transformation of ligation products in <i>E.coli</i></p>
 +
<p class="texte"><b>Date:</b> 08/01/2014</p>
 +
 +
<p class="title3">Culture of  8 clones: A, B, C, D, E, F, G, H of transformed E. coli</p>
 +
<p class="texte"><b>Date:</b> 08/02/2014</p>
 +
 +
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
 +
<p class="texte"><b>Date:</b> 08/04/2014</p>
 +
 +
<p class="title3">Digestion of clones A, B, C, D, E, G, H of K1364008+K823022 with EcoRI and PstI + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 08/04/2014
 +
<br><b>Result:</b> clones A, E, F have the right construction</p>
 +
 +
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p>
 +
</div>
 +
 +
<div class="technology2">EcAMP</div>
 +
<div class="thelanguage2">
 +
 +
<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>
 +
 +
<p class="title2">1. Transformation of EcAMP in <i>Escherichia coli</i> MC 1061</p>
 +
<p class="texte"><b>Date:</b> 07/25/2014</p>
 +
 +
<p class="title2">2. Spreading of coli cells transformed with EcAMP (plasmid pUC) </p>
 +
<p class="texte"><b>Date:</b> 07/28/2014</p>
 +
 +
<p class="title2">3. Liquid culture, Miniprep and Test of the miniprep</p>
 +
<p class="texte"><b>Date:</b> 07/30/2014</p>
 +
 +
 +
 +
<p class="title2">4. Cloning 1: EcAMP + P<sub>veg</sub> + RBS</p>
 +
 +
<p class="title3">Digestion of EcAMP (insert) by XbaI and PstI</p>
 +
<p class="texte"><b>Date:</b> 07/31/2014</p>
 +
 +
<p class="title3">Digestion of P<sup>veg</sup> + RBS (VECTOR)</p>
 +
<p class="texte"><b>Date:</b> 07/31/2014</p>
 +
 +
<p class="title3">Ligation and transformation</p>
 +
<p class="texte"><b>Date:</b> 08/04/2014</p>
 +
 +
<p class="title3">PCR test</p>
 +
<p class="texte"><b>Date:</b> 08/05/2014</p>
 +
 +
<p class="title3">Analytical digestion</p>
 +
<p class="texte"><b>Date:</b> 08/05/2014
 +
 +
<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>
 +
 +
 +
 +
<p class="title2">5. Cloning 2: EcAMP + Pveg + RBS </p>
 +
<p class="texte"><b>Date:</b> 08/06/2014</p>
 +
<p class="title3">Digestion of EcAMP (insert) by XbaI and PstI</p>
 +
<p class="texte"><b>Date:</b> 08/06/2014</p>
 +
<p class="title3">Digestion of P<sub>veg</sub> + RBS (vector)</p>
 +
<p class="texte"><b>Date:</b> 08/06/2014</p>
 +
<p class="title3">Heat inactivation of the enzymes</p>
 +
<p class="texte"><b>Date:</b> 08/06/2014</p>
 +
<p class="title3">Ligation and transformation</p>
 +
<p class="texte"><b>Date:</b> 08/06/2014</p>
 +
<p class="title3">PCR test</p>
 +
<p class="texte"><b>Date:</b> 08/07/2014</p>
 +
 +
 +
<p class="title3">Striation on a petri dish to purify the clone</p>
 +
<p class="texte"><b>Purpose:</b> to isolate a clone with vector+insert
 +
<br><b>Date:</b> 08/072014</p>
 +
 +
<p class="title3">Miniprep of P<sub>veg</sub> + SpoVG + EcAMP and analytic digestion</p>
 +
<p class="texte"><b>Date:</b> 08/08/2014</p>
 +
 +
<p class="title3">Ligation of Pveg SpoVG EcAMP with double terminateur B0015 + transformation and liquid culture</p>
 +
<p class="texte"><b>Date:</b> 08/11/2014</p>
 +
 +
<p class="title3">Miniprep of P<sub>veg</sub> SpoVG EcAMP + analytic digestion</p>
 +
<p class="texte"><b>Date:</b> 08/13/2014</p>
 +
 +
<p class="title3">Cloning K1364011 (EcAMP + P<sub>veg</sub> +SpoVG + B0015) + K823022 (pSB<sub>BS</sub>4S): Digestion, ligation, transformation</p>
 +
<p class="texte"><b>Date:</b> 08/19/2014</p>
 +
 +
<p class="title3">Cloning K1364011 + K823023 (pSB<sub>BS</sub>1C) : Digestion, ligation, transformation</p>
 +
<p class="texte"><b>Date:</b> 08/18/2014</p>
 +
 +
<p class="title3">Verification of the insertion of K1364011 + K823022 (pSB<sub>BS</sub>4S) into the subtilis genome by threonine test </p>
 +
<p class="texte"><b>Date:</b> 08/21/2014</p>
 +
 +
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p>
 +
</div>
 +
 +
<div class="technology2">Assembling the fungicides module: D4E1 + GAFP1</div>
 +
<div class="thelanguage2">
 +
 +
<p class="title2">1.  Cloning GAFP1+D4E1 on pSB1C3: <a href="http://parts.igem.org/Part:BBa_K1364012">BBa_K1364012</a></p>
 +
 +
<p class="title3">Digestion of D4E1 on pEX-A2 and GAFP1 on pSB1C3</p>
 +
<p class="texte"><b>Date:</b> 07/28/2014
 +
<br><b>Result:</b> We obtained 40µL digestion of D4E1 on pEX-A2 and of GAFP1 on pSB1C3.</p>
 +
 +
<p class="title3">Ligation of digestions of D4E1 on pEX-A2 and of GAFP1 on pSB1C3</p>
 +
<p class="texte"><b>Date:</b> 07/28/2014</p>
 +
 +
<p class="title3">Transformation of ligation of GAFP1 and D4E1 on pSB1C3 into E. coli</p>
 +
<p class="texte"><b>Date:</b> 07/28/2014</p>
 +
 +
<p class="title3">PCR of GAFP1 + D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 07/29/2014
 +
<br><b>Result:</b> clones A, C, F, G seem to have the expected construction.</p>
 +
 +
<p class="title3">Digestion of GAFP1 + D4E1 (pSB1C3) A, C, F, G + electrophoresis</p>
 +
<p class="texte"><b>Date:</b> 07/30/2014
 +
<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>
 +
 +
 +
 +
<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>
 +
<p class="texte"><b>Date:</b> 08/04/2014
 +
 +
<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>
 +
<p class="texte"><b>Date:</b> 08/06/2014
 +
 +
<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>
 +
<p class="texte"><b>Date:</b> 08/11/2014
 +
 +
<p class="title2">5. Fungicides tests</p>
 +
<p class="texte"><b>Date:</b> 08/15/2014
 +
 +
<p class="title2">Cloning D4E1-GAFP1-EcAMP: <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a></p>
 +
 +
<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>
 +
 +
<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>
 +
<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><b>Date:</b> 08/11/2014
 +
<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>
 +
 +
<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>
 +
<p class="texte"><b>Date:</b> 08/11/2014
 +
<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>
 +
 +
<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>
 +
<p class="texte"><b>Date:</b> 08/12/2014</p>
 +
 +
 +
<p class="title3">Testing 8 <i>E.coli</i> + <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> clones</p>
 +
<p class="texte"><b>Date:</b> 08/14/2014 </p>
 +
 +
<p class="title3">Testing 15 <i>E.coli</i> + <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> clones</p>
 +
<p class="texte"><b>Date:</b> 08/18/2014
 +
<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>
 +
 +
 +
<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>
 +
<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>
 +
<p class="texte"><b>Date:</b> 08/22/2014
 +
<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>
 +
 +
<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>
 +
<p class="texte"><b>Date:</b> 08/22/2014
 +
<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>.
 +
</p>
 +
<p class="title3">Transformation of ligations in <i>E. coli</i></p>
 +
<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>
 +
<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><b>Date:</b> 08/25/2014</p>
 +
 +
<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>
 +
<p class="texte"><b>Date:</b> 08/27/2014</p>
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 +
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<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>
 +
<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><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
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<br><b>Date:</b> 08/28/2014</p>
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<div class="Title">Project-monitoring</div>  
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<p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nam id blandit velit. Morbi ac condimentum massa. Vestibulum sagittis metus nec ultrices lobortis. Aliquam ac congue libero. Vestibulum varius eget felis sed pellentesque. Cras feugiat nisi in ante faucibus eleifend. Nullam mattis nisi dolor, eget sagittis urna vulputate vel. Vestibulum nec sollicitudin ex. Suspendisse vel orci sit amet erat maximus malesuada. Vivamus consequat pretium nunc ut imperdiet. Cras laoreet auctor lectus at accumsan. Sed rutrum augue dolor, ac pulvinar erat placerat nec.
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<div class="technology2">Fungicides tests</div>
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<div class="thelanguage2">
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Quisque ac metus lobortis, ultrices elit quis, lacinia justo. Vivamus pulvinar, quam ac semper scelerisque, mi erat posuere lacus, ac pretium arcu mi a lorem. Duis egestas, massa vel vulputate varius, odio est suscipit lacus, a lacinia nibh nisi eu dui. Nunc lacinia velit cursus mi auctor bibendum. Etiam at diam tempor, dictum nisi ut, blandit diam. Morbi pellentesque dolor eu orci congue mattis eget a velit. Morbi vitae est sit amet dolor pulvinar imperdiet at et odio. Ut maximus elit eget leo cursus tristique. Phasellus sit amet arcu at dolor faucibus aliquet eu et libero. Pellentesque vel ultrices eros. Integer at gravida velit.</p>
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<p class="title2">1. D4E1-GAFP1</p>
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<p class="title3">Transformation of P<sub>veg</sub> - D4E1 - GAFP1 in <i>Bacillus subtilis</i>  on pSB<sub>BS</sub>4S </p>
 +
<p class="texte"><b>Date:</b> 08/12/2014</p>
 +
<p class="title3">Integration threonine test + fungicide test </p>
 +
<p class="texte"><b>Date:</b> 08/13/2014 </p>
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<p class="title2"2. D4E1</p>
 +
<p class="title3">
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Cloning D4E1 into pSB<sub>BS</sub>1C + fungicide test</p>
 +
<p class="texte"><b>Date:</b>08/15/2014</p>
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<p class="title3">
 +
D4E1 on pSB1C3 + fungicide test</p>
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<p class="texte"><b>Date:</b>08/19/2014</p>
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Latest revision as of 01:22, 18 October 2014

           
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Notebook   >   Project monitoring

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Binding module

1. Amplification of Binding Module (pEX-K4) into E. coli

Transformation of Binding module (pEX-K4) into E. coli

Gene "Binding module" synthesized by Eurofins, resuspended in 20μL Tris 10mM
Result: We obtained distinct colonies on plates LB + Kanamycin (50 µg/mL)

Liquid culture of two clones: 1 et 2 (pEX-K4) transformed into E. coli

Date: 01/08/2014

Miniprep with QIAprep Spin Miniprep Kit: two clones of Binding Module (pEX-K4) into E. coli

Date: 04/08/2014
Result: Binding Module (pEX-K4) obtained

Digestion of Binding Module (pEX-K4) with EcoRI and PstI

Date: 04/08/2014
Result:
- 3 bands : 1500bp (vector with Kanamycin resistance), 1300bp (Module Binding) and 1000bp (vector with pUC Ori)
- The two Binding Module clones are ok

2. Cloning Binding Module on pEX-K4 with pSB1C3 (BBa_K606013 without RFP) into E. coli

Digestion Binding Module on pEX-K4 and BBa_K606013

Date: 23/08/2014
Expected bands after digestion:
- BBa_K606013: 860 bp for RFP and 2100 bp for vector pSB1C3
- Binding Module: 1400 bp for Binding Module and 1500bp + 1000bp for vector pEX_K4
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.

Ligation Binding Module on pSB1C3

Date: 04/08/2014

Transformation of Binding Module on pSB1C3 into E. coli

Date: 04/08/2014
Transformation with 10 µL of the ligation mix and plate on Chloremphenicol LA plate
Result: many wrong clones

3. Cloning Binding Module on pEX-K4 with pVeg on pSB1C3 (BBa_K823003) into E. coli

Digestion Binding Module on pEX-K4 and BBa_K823003

Date: 04/08/2014
BBa_K823003 digested by XbaI and PstI and Binding Module digested by SpeIand PstI
Gel Electrophoresis
Result:
- Binding Module : 1400 bp for Binding Module and 1500bp + 1000bp for vector pEX_K47
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.

Ligation Binding Module on pVeg with pSB1C3

Date: 04/08/2014
BBa_K823003 digested by XbaI and PstI and Binding Module digested by SpeI and PstI and ligation

Transformation of Binding Module on pVeg into E. coli

Date: 04/08/2014
Result: Many good clones (check on 06/08/2014)

4. Cloning Binding Module with Pveg (BBa_K1364005) on pSBBS4S (BBa_K823022) into E. coli

Digestion Binding Module with Pveg (BBa_K1364005) and pSBBS4S (BBa_K823022)

Date: 07/08/2014
BBa_K823022 and Binding Module with Pveg (BBa_K1364005) digested by EcoRI and PstI Gel Electrophoresis
Result:
- Binding Module with Pveg 1600 bp for Binding Module and 2100bp for vector pSB1C3

Ligation Binding Module with Pveg on pSBbs4S

Date: 07/08/2014
BBa_K823022 and Binding Module with Pveg (BBa_K1364005) digested by EcoRI and PstI
Ligation
Result: Ligation between Binding Module with Pveg on pSBBS4S

Transformation of Binding Module with Pveg on pSBBS4S into E. coli

Date: 04/08/2014
Binding Module with Pveg on pSBBS4S
Plate on Ampicillin LA plate
Result: Many good clones (check on 13/08/2014)

Transformation of Binding Module with Pveg on pSBBS4S into B. subtilis

Date: 04/08/2014
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.
Result: One good clone : PCR (check on 09/09/2014) and threonine test (check on 09/09/2014)

5. Binding Test

See here

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Chemotaxis

1. Transformation of chemotaxis (Puc-57) into E. coli

Gene chemotaxis synthesized by Eurofins, resuspended in 20μL Tris 10mM
Date: 01/08/2014
Result: We obtained distinct colonies on LA + Ampicillin (100 µg/mL) plates

Culture of 4 clones: A, B, C, D of chemotaxis (Puc57) transformed into E. coli

Date: 04/08/2014

Miniprep with QIAprep Spin Miniprep Kit Using a Microcentrifuge: 4 clones of chemotaxis (Puc57) into E. coli

Date: 05/08/2014
Result: 4*50µL of chemotaxis (Puc57) obtained

2. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested pSB1C3 with EcoRI and PstI into E. coli

Digestion BBa_K1364000 (chemotaxis_Puc57) with EcoRI and PstI - PCR kit Clean up

Date: 07/08/2014
Result: Expected band after digestion for BBa_K1364000: 2300 bp
Problem: We can't distinguish the vector band (2500 bp)

Gel extraction of BBa_K1364000

Date: 07/08/2014

Ligation BBa_K1364000 and digested PSB1C3 with EcoRI and PstI

Date: 08/08/2014

Transformation BBa_K1364000 in E.coli

Date: 08/08/2014
Result: We obtained distinct colonies on LA + Cm (15 µg/mL) plates and resuspended 15 colonies in LB+Cm (15 µg/mL)

Test of sensibility on Ampicillin

Date: 10/08/2014
Result: we can determine which colonies are sensible for ampicillin and know which bacterium is carrying the chemotaxis gene.

PCR

Date: 11/08/2014

Digestion BBa_K1364000 on pSB1C3 with EcoRI and PstI

Date: 11/08/2014
Result: There is one colony which presents the right construction.

3. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested BBa_823003 (Pveg) on pSB1C3 with SpeI and PstI into E. coli

Ligation BBa_K1364000 and digested BBa_823003 on PsB1C3 with SpeI and PstI

Date: 08/08/2014

Transformation BBa_1364004 in E.coli

Date: 8/08/2014

Test of sensibility on Ampicillin

Date: 10/08/2014
Result: 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.

Digestion BBa_1364004 on pSBC3 with EcoRI and PstI

Date: 12/08/2014
Result: We did not see any colony with chemotaxis insert.

4. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested BBa_823003 (Pveg) on pSB1C3 with SpeI and PstI into E. coli

Ligation BBa_K1364000 and digested BBa_823003 on PsB1C3 with SpeI and PstI

Date: 11/08/2014

Transformation BBa_1364004 in E.coli

Date: 11/08/2014

Test of sensibility on Ampicillin

Date: 14/08/2014
Result: we obtained 4 colonies sensible at Ampicilline

Digestion BBa_1364004 on PsB1C3 with EcoRI and PstI

Date: 18/08/2014

Gel extraction of BBa_1364000

Date: 19/08/2014

5. Cloning chemotaxis BBa_K1364004 with digested pSBBS4S with EcorI and PstI into E. coli

Ligation BBa_K1364004 digested EcorI and PstI and digested PsB1C3 with EcoRI and PstI

Date: 19/08/2014

Transformation BBa_K1364004 in pSBBS4S in E.coli

Date: 19/08/2014
Result: We obtained one colony and resuspended it in LB+ Amp

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Fungicides
D4E1

1. Amplification of synthetic gene (D4E1 on pEX-A2)

Transformation of D4E1 (pEX-A2) into E. coli

Gene D4E1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
Concentration of D4E1: 115ng/µL
Date: 07/21//2014
Result: We obtained distinct colonies on LA + Ampicillin (100 µg/mL)

Culture of 4 clones: A, B, C, D of D4E1 (pEX-A2) transformed into E. coli

Date: 07/22/2014
Result: Culture of 4 clones ok

QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of D4E1 (pEX-A2) into E. coli

Buffer EB at 50-55°C
Date: 07/23/2014

Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up

Date: 07/23/2014
Result: 4*20µL D4E1 digested with EcoRI and PstI  all the clones seem to have the right D4E1 gene.

2. Cloning D4E1 in pSB1C3

Digestion of D4E1 on pEX-A2 and pSB1C3

Date: 07/23/2014

Ligation of D4E1 in pSB1C3

Date: 07/23/2014

Transformation in E.coli

Date: 07/23/2014

Culture of 6 clones: A, B, C, D, E, F of D4E1 (pSB1C3) transformed into E. coli

Processing details: 5mL of LB + chloramphenicol (15µg/mL) , using sterile plastic loop to culture colonies
Date: 07/24/2014
Result: Culture of 4 clones ok

QIAprep Spin Miniprep Kit Using a Microcentrifuge

Date: 07/25/2014

Digestion of D4E1 (pSB1C3) A, B, C, D, E, F with EcoRI and PstI - PCR kit Clean up + electrophoresis

Date: 07/25/2014
Result: clones C, D have the expected construction

PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis

Date: 07/25/2014
Result: clones C, D have the expected construction, and placed in cryopreservation.

3. Cloning Pveg+D4E1 on Pveg plasmid (pSB1C3)

Digestion of D4E1 on pEX-A2 and K823003 (Pveg on pSB1C3)

Dated: 07/24/2014
Result: 20µL digestion of D4E1 on pEX-A2 and of K823003

Ligation of D4E1 in K823003 (Pveg on pSB1C3)

Date: 07/24/2014
Result: 20µL ligation of D4E1 in K823003

Transformation of ligation products in E.coli

Date: 07/24/2014
Result: E.coli transformed by D4E1+K823003

Culture of 6 clones: A, B, C, D, E, F of transformed E. coli

Date: 07/26/2014
Result: Culture of 4 clones ok

QIAprep Spin Miniprep Kit Using a Microcentrifuge

Date: 07/28/2014

PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis

Dated: 07/28/2014
Result: clones E, F seem to have the expected construction

Digestion of clones E, F of D4E1+K823003 (Pveg on pSB1C3) with EcoRI and PstI + electrophoresis

Date: 28/07/2014
Result: clones C, D have the expected construction and are placed in cryopreservation.

4. Cloning Pveg + D4E1 on pSBBS4S (K823022)

Date: 08/13/2014

5. Cloning Pveg + D4E1 on pSBBS1C lacZ (23)

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GAFP1

1. Amplification of synthetic gene (GAFP1 on pEX-A2)

Transformation of GAFP1 (pEX-A2) into E.coli

Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
Concentration of GAFP1 : 145ng/µL
Date: 07/21/2014
Result: We obtained distinct colonies on plates LB + Ampicillin (100 µg/mL)

Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli

Date: 07/22/2014
Result: Culture of 4 clones ok

QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli

Date: 07/23/2014

Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel Electrophoresis

Date: 07/23/2014

2. Cloning GAFP1 gene on PSB1C3 = K1364002 in E. coli

Digestion GAFP1_pEX-A2 and BBa_K606013 (RFP_pSB1C3)

Date: 07/23/2014
Result: BBa_K606013 : 860 bp
We decide to conserve the miniprep B for BBa_K606013

Ligation GAFP1 and BBa_K606013

Date: 07/23/2014

Tranformation of ligation products into E. coli

Date: 07/23/2014

Culture of x clones of GAFP1+K606013 in E. coli

Date: 07/24/2014

QIAprep Spin Miniprep Kit Using a Microcentrifuge: 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) into E. coli

Date: 07/25/2014

PCR on 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) + electrophoresis

Date: 07/25/2014
Result: clones A, C, D, F seem to have the right construction

Digestion of 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) by EcoR1 and Pst1 + electrophoresis

Date: 07/25/2014

3. Cloning GAFP1+terminator(B0015) = K1364007 (pSB1C3)

Digestion of GAFP1 on pEX-A2 and B0015 (terminator on pSB1C3)

Date: 07/24/2014

Ligation of GAFP1 in B0015 (terminator on pSB1C3)

Date: 07/24/2014

Transformation of ligation products in E.coli

Date: 07/24/2014

Culture of 6 clones: A, B, C, D, E, F transformed in E. coli

Date: 07/26/2014

QIAprep Spin Miniprep Kit Using a Microcentrifuge

Date: 07/28/2014

PCR of GAFP1+B0015 = K1364007 (pSB1C3) A, B, C, D, E, F + electrophoresis

Date: 07/28/2014
Result: clones B, C, D, E, F seem to have the expected construction.

Digestion of clones E, F of GAFP1+Ter B0015 = K1364007 with EcoRI and PstI + electrophoresis

Date: 07/28/2014
Result: clones B, C, D, E, F have the expected construction.

4. Cloning GAFP1+terminator with Pveg on pSB1C3 (K1364008) in E. coli

Digestion of GAFP1+ter = K1364007 on pSB1C3 and K823003 (terminator on pSB1C3) + electrophoresis

Date: 07/29/2014

Gel extraction of K1364007 (extraction of GAFP1+ter gene)

Date: 07/29/2014

Ligation of GAFP1+ter in K823003 (Pveg on pSB1C3)

Date: 07/29/2014
Result: 20µL ligation of GAFP1+ter in K823003

Transformation of ligation products in E.coli

Date: 07/29/2014

Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli

Date: 07/30/2014

QIAprep Spin Miniprep Kit Using a Microcentrifuge

Date: 07/31/2014

PCR of GAFP1+B0015 + K823003 = K1364008 (pSB1C3) A, B, C, D, E, F, G, H + electrophoresis

Date: 31/07/2014
Result: clones A, B, C, D, E, G, H seem to have the expected construction

Digestion of clones A, B, C, D, E, G, H of Pveg+K1364007 = K1364008 with EcoRI and PstI + electrophoresis

Date: 31/07/2014
Result: clones A, B, C, D, E, G, H have the expected construction

5. Cloning Pveg+GAFP1+Ter B0015 (BBa_K1364008) on pSBBS4S (BBa_K823022)

Digestion of Pveg+GAFP1+ ter B0015 (BBa_K1364008) on pSB1C3 and PsBBs4S (BBa_K823022)

Date: 08/01/2014

Ligation of K134008 (Pveg+GAFP1+ter fragment) and K823022 (PsBBs4S)

Date: 08/01/2014

Transformation of ligation products in E.coli

Date: 08/01/2014

Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli

Date: 08/02/2014

QIAprep Spin Miniprep Kit Using a Microcentrifuge

Date: 08/04/2014

Digestion of clones A, B, C, D, E, G, H of K1364008+K823022 with EcoRI and PstI + electrophoresis

Date: 08/04/2014
Result: clones A, E, F have the right construction

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EcAMP

The EcAMP part was sent by the Utah iGEM team. It was on a pUC plasmid (ampicilin resistance) with BioBrick suffix and prefix.

1. Transformation of EcAMP in Escherichia coli MC 1061

Date: 07/25/2014

2. Spreading of coli cells transformed with EcAMP (plasmid pUC)

Date: 07/28/2014

3. Liquid culture, Miniprep and Test of the miniprep

Date: 07/30/2014

4. Cloning 1: EcAMP + Pveg + RBS

Digestion of EcAMP (insert) by XbaI and PstI

Date: 07/31/2014

Digestion of Pveg + RBS (VECTOR)

Date: 07/31/2014

Ligation and transformation

Date: 08/04/2014

PCR test

Date: 08/05/2014

Analytical digestion

Date: 08/05/2014
Result: 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.

5. Cloning 2: EcAMP + Pveg + RBS

Date: 08/06/2014

Digestion of EcAMP (insert) by XbaI and PstI

Date: 08/06/2014

Digestion of Pveg + RBS (vector)

Date: 08/06/2014

Heat inactivation of the enzymes

Date: 08/06/2014

Ligation and transformation

Date: 08/06/2014

PCR test

Date: 08/07/2014

Striation on a petri dish to purify the clone

Purpose: to isolate a clone with vector+insert
Date: 08/072014

Miniprep of Pveg + SpoVG + EcAMP and analytic digestion

Date: 08/08/2014

Ligation of Pveg SpoVG EcAMP with double terminateur B0015 + transformation and liquid culture

Date: 08/11/2014

Miniprep of Pveg SpoVG EcAMP + analytic digestion

Date: 08/13/2014

Cloning K1364011 (EcAMP + Pveg +SpoVG + B0015) + K823022 (pSBBS4S): Digestion, ligation, transformation

Date: 08/19/2014

Cloning K1364011 + K823023 (pSBBS1C) : Digestion, ligation, transformation

Date: 08/18/2014

Verification of the insertion of K1364011 + K823022 (pSBBS4S) into the subtilis genome by threonine test

Date: 08/21/2014

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Assembling the fungicides module: D4E1 + GAFP1

1. Cloning GAFP1+D4E1 on pSB1C3: BBa_K1364012

Digestion of D4E1 on pEX-A2 and GAFP1 on pSB1C3

Date: 07/28/2014
Result: We obtained 40µL digestion of D4E1 on pEX-A2 and of GAFP1 on pSB1C3.

Ligation of digestions of D4E1 on pEX-A2 and of GAFP1 on pSB1C3

Date: 07/28/2014

Transformation of ligation of GAFP1 and D4E1 on pSB1C3 into E. coli

Date: 07/28/2014

PCR of GAFP1 + D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis

Date: 07/29/2014
Result: clones A, C, F, G seem to have the expected construction.

Digestion of GAFP1 + D4E1 (pSB1C3) A, C, F, G + electrophoresis

Date: 07/30/2014
Result: clone F (BBa_K1364012) has the expected construction and is placed on cryopreservation.

2. Cloning GAFP1 + D4E1 (BBa_K1364012) on Pveg plasmid (BBa_K823003): BBa_K1364013

Date: 08/04/2014

3. Cloning Pveg + GAFP1 + D4E1 (BBa_K1364013) on pSBBS4S (BBa_K823022)

Date: 08/06/2014

4. Cloning Pveg + GAFP1 + D4E1 (BBa_K1364013) on pSBBS1C lacZ (BBa_K823023)

Date: 08/11/2014

5. Fungicides tests

Date: 08/15/2014

Cloning D4E1-GAFP1-EcAMP: BBa_K1364014

1. Construction of BBa_K1364014 in pSB1C3, in E. coli

Digestion of BBa_K1364010 and BBa_K1364012

Digestion of BBa_K1364010 by SpeI and PstI, and digestion of BBa_K1364012 by XbaI and PstI.
Date: 08/11/2014
Result: We obtained digested fragments of BBa_K1364012 and ~500 bp fragment of BBa_K1364010

Ligation of ~500 bp fragment from BBa_K1364010 and BBa_K1364012

Date: 08/11/2014
Result: We obtained ligation of K1364012 and ~500 bp fragment of BBa_K1364010

Transformation of ligation of ~500 bp fragment from BBa_K1364010 and BBa_K1364012 = BBa_K1364014 in E.coli

Date: 08/12/2014

Testing 8 E.coli + BBa_K1364014 clones

Date: 08/14/2014

Testing 15 E.coli + BBa_K1364014 clones

Date: 08/18/2014
Result: clones H, J, O, Q, U, V might have the right BBa_K1364014 construction

2. BBa_K1364014 in pSBBS4S (BBa_K823022) and pSBBS1C (BBa_K823023)

Digestion of BBa_K1364014, BBa_K823022 and BBa_K823023

Date: 08/22/2014
Result: Digestion of BBa_K1364014, BBa_K823022 and BBa_K823023 by EcoRI and PstI were well performed.

Ligation of BBa_K1364014 with BBa_K823022 and K1364014 with K823023

Date: 08/22/2014
Result: We obtained 20µL ligation of K1364014 with BBa_K823022 and of BBa_K1364014 with BBa_K823023.

Transformation of ligations in E. coli

E. coli transformed by BBa_K1364014 + BBa_K823022 spread on LB + Amp 100µg/mL agar plate

E. coli transformed by BBa_K1364014 + BBa_K823023 spread on LB + Amp 100µg/mL agar plate
Date: 08/25/2014

Testing E. coli + BBa_K1364014 + BBa_K823022 and E. coli + BBa_K1364014 + BBa_K823023 clones

Date: 08/27/2014

3. Transformation of BBa_K1364014+BBa_K823022 and BBa_K1364014 + BBa_K823023 in B. subtilis

B. subtilis transformed by 10µL BBa_K1364014+BBa_K823022 spread on LB+Spec 75µg/mL agar plate
B. subtilis transformed by 10µL BBa_K1364014+BBa_K823023 spread on LB+Cm 15µg/mL agar plate
Date: 08/28/2014

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Fungicides tests

1. D4E1-GAFP1

Transformation of Pveg - D4E1 - GAFP1 in Bacillus subtilis on pSBBS4S

Date: 08/12/2014

Integration threonine test + fungicide test

Date: 08/13/2014

Cloning D4E1 into pSBBS1C + fungicide test

Date:08/15/2014

D4E1 on pSB1C3 + fungicide test

Date:08/19/2014

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iGEM Toulouse 2014

Retrieved from "http://2014.igem.org/Team:Toulouse/Notebook/project-monitoring"