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;Project monitoring</p>  
+
   <div style="margin:0 auto; width:960px;">
 +
  <p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Project monitoring</p>
 +
    </div>
   </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;">
-
      <p class="texte">Binding module
+
<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>
-
1. Amplification of Binding Module (pEX-K4) into E.coli
+
<div class="technology">Binding module</div>
-
• Transformation of Binding module (pEX-K4) into E. coli
+
<div class="thelanguage">
-
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 2 clones : 1 et 2 (pEX-K4) transformed into E. coli
+
<p class="title2">1. Amplification of Binding Module (pEX-K4) into <i>E. coli</i></p>
-
Date: 01/08/2014
+
<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>
-
• Miniprep with QIAprep Spin Miniprep Kit: 2 clones of Binding Module (pEX-K4) into E. coli
+
<p class="title3">Liquid culture of two clones: 1 et 2 (pEX-K4) transformed into <i>E. coli</i></p>
-
Date: 04/08/2014
+
<p class="texte"><b>Date:</b> 01/08/2014</p>
-
Result : Binding Module (pEX-K4) obtained
+
-
• Digestion of Binding Module (pEX-K4) with EcoRI and PstI
+
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit: two clones of Binding Module (pEX-K4) into <i>E. coli</i></p>
-
Date: 04/08/2014
+
<p class="texte"><b>Date:</b> 04/08/2014
-
Result :  
+
<br><b>Result:</b> Binding Module (pEX-K4) obtained</p>
-
- 3 bands : 1500 bp (vector with Kanamycin resistance), 1300bp (Module Binding) and 1000p bp (vector with pUC Ori)
+
-
- The two Binding Module clones are ok
+
 +
<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>
-
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
 
-
Result :
 
-
Expected bands after digestion for :
 
-
- 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
+
<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>
-
Date : 04/08/2014
+
<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>
-
• Transformation of Binding Module on pSB1C3 into E. coli
+
<p class="title3">Ligation Binding Module on pSB1C3</p>
-
Date: 04/08/2014
+
<p class="texte"><b>Date:</b> 04/08/2014</p>
-
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
+
<p class="title3">Transformation of Binding Module on pSB1C3 into E. coli</p>
-
• Digestion Binding Module on pEX-K4 and BBa_K823003
+
<p class="texte"><b>Date:</b> 04/08/2014
-
Date : 04/08/2014
+
<br>Transformation with 10 µL of the ligation mix and plate on Chloremphenicol LA plate
-
BBa_K823003 digested by XbaI and PstI and Binding Module digested by SpeIand PstI
+
<br><b>Result:</b> many wrong clones</p>
-
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
+
<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>
-
Date: 04/08/2014
+
<p class="title3">Digestion Binding Module on pEX-K4 and <a href="http://parts.igem.org/Part:BBa_K823003">BBa_K823003</a></p>
-
BBa_K823003 digested by XbaI and PstI and Binding Module digested by SpeIand PstI and ligation
+
<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>
-
• Transformation of Binding Module on pVeg into E. coli
+
<p class="title3">Ligation Binding Module on pVeg with pSB1C3</p>
-
Date: 04/08/2014
+
<p class="texte"><b>Date:</b> 04/08/2014
-
Result: Many good clones (check on 06/08/2014)
+
<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>
-
4. Cloning Binding Module with Pveg (BBa-K1364005) on pSBBS4S (BBa-K823022) into E. coli
+
<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>
-
• Digestion Binding Module with Pveg (BBa-K1364005) and pSBBS4S (BBa-K823022)
+
<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>
-
Date: 07/08/2014
+
-
BBa_K823022 and Binding Module with Pveg (BBa_K1364005) digested by EcoRIand PstIGel Electrophoresis
+
-
Result :
+
-
- Binding Module with Pveg 1600 bp for Binding Module and 2100bp for vector pSB1C3
+
-
• Ligation Binding Module with Pveg on pSBbs4S
+
<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>
-
Date: 07/08/2014
+
<p class="texte"><b>Date:</b> 07/08/2014
-
BBa_K823022 and Binding Module with Pveg (BBa_K1364005) digested by EcoRIand PstI
+
<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
-
Ligation
+
<br><b>Result:</b>
-
Result : Ligation between Binding Module with Pveg on pSBBS4S
+
<br>- Binding Module with Pveg 1600 bp for Binding Module and 2100bp for vector pSB1C3</p>
-
• Transformation of Binding Module with Pveg on pSBBS4S into E. coli
+
<p class="title3">Ligation Binding Module with Pveg on pSBbs4S</p>
-
Date: 04/08/2014
+
<p class="texte"><b>Date:</b> 07/08/2014
-
Binding Module with Pveg on pSBbs4S
+
<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
-
Plate on Ampicillin LA plate
+
<br>Ligation
-
Result: Many good clones (check on 13/08/2014)
+
<br><b>Result:</b> Ligation between Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S</p>
-
• Transformation of Binding Module with Pveg on pSBBS4S into B. subtilis
+
<p class="title3">Transformation of Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S into <i>E. coli</i></p>
-
Date: 04/08/2014
+
<p class="texte"><b>Date:</b> 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.
+
<br>Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S
-
Result : One good clone : PCR (check on 09/09/2014) and threonine test (check on 09/09/2014)
+
<br>Plate on Ampicillin LA plate
 +
<br><b>Result:</b> Many good clones (check on 13/08/2014)</p>
-
5. BindingTest
+
<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>
-
Chemotaxis
+
<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>
-
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
+
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p>
-
  Date: 04/08/2014
+
</div>
-
• 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
+
<div class="technology">Chemotaxis</div>
 +
<div class="thelanguage">
-
• Digestion BBa_K1364000 (chemotaxis_Puc57) with EcoRI and PstI - PCR kit Clean up
+
<p class="title2">1. Transformation of chemotaxis (Puc-57) into <i>E. coli</i></p>
-
Date: 07/08/2014
+
<p class="texte">Gene chemotaxis synthesized by Eurofins, resuspended in 20μL Tris 10mM
-
Result: Expected band after digestion for BBa_K1364000 : 2300 bp
+
<br><b>Date:</b> 01/08/2014
-
Problem: We can't distinguish the vector band (2500 bp)
+
<br><b>Result:</b> We obtained distinct colonies on LA + Ampicillin (100 µg/mL) plates</p>
-
•   Gel extraction of BBa_K1364000
+
<p class="title3">Culture of 4 clones: A, B, C, D of chemotaxis (Puc57) transformed into <i>E. coli</i></p>
-
Date: 07/08/2014
+
<p class="texte"><b>Date:</b> 04/08/2014</p>
-
• Ligation BBa_K1364000 and digested PsB1C3 with EcoRI and PstI
+
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit Using a Microcentrifuge: 4 clones of chemotaxis (Puc57) into <i>E. coli</i></p>
-
Date: 08/08/2014
+
<p class="texte"><b>Date:</b> 05/08/2014
 +
<br><b>Result:</b> 4*50µL of chemotaxis (Puc57) obtained</p>
-
• Transformation BBa_K1364000 in E.coli
+
<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>
-
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
+
<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>
-
Date: 10/08/2014
+
<p class="texte"><b>Date:</b> 07/08/2014
-
Result: we can determine which colonies are sensible for ampicillin and know which bacterium is carrying the chemotaxis gene.
+
<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>
-
• PCR
+
<p class="title3">Gel extraction of <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a></p>
-
Date: 11/08/2014
+
<p class="texte"><b>Date:</b> 07/08/2014</p>
-
• Digestion BBa_K1364000 on pSB1C3 with EcoRI and PstI
+
<p class="title3">Ligation <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> and digested PSB1C3 with EcoRI and PstI</p>
-
Date: 11/08/2014
+
<p class="texte"><b>Date:</b> 08/08/2014</p>
-
Result : There is one colony which presents the right construction.
+
 +
<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>
-
3. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested BBa_823003 (Pveg) on pSB1C3 with SpeI and PstI into E. coli
+
<p class="title3">PCR</p>
-
• Ligation BBa_K1364000 and digested BBa_823003 on PsB1C3 with SpeI and PstI
+
<p class="texte"><b>Date:</b> 11/08/2014</p>
-
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_K1364004 on pSBC3 with EcoRI and PstI
+
<p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> on pSB1C3 with EcoRI and PstI</p>
-
Date: 12/08/2014
+
<p class="texte"><b>Date:</b> 11/08/2014
-
Result: We did not see any colony with chemotaxis insert.
+
<br><b>Result:</b> There is one colony which presents the right construction.</p>
-
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_K1364004 on PsB1C3 with EcoRI and PstI
 
-
Date: 18/08/2014
 
-
• Gel extraction of BBa_K1364000
 
-
Date: 19/08/2014
 
-
5. Cloning chemotaxis BBa_K1364004 with digested pSBBS4S with EcorI and PstI into E. coli
+
<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>
-
• Ligation BBa_K1364004 digested EcorI and PstI and digested PsB1C3 with EcoRI and PstI
+
<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>
-
Date: 19/08/2014
+
<p class="texte"><b>Date</b>: 08/08/2014</p>
-
• Transformation BBa_1364004 in pSBBS4S in E.coli
+
<p class="title3">Transformation <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_1364004</a> in <i>E.coli</i></p>
-
  Date: 19/08/2014  
+
<p class="texte"><b>Date:</b> 8/08/2014</p>
-
Result : We obtained one colony and resuspended it in LB+ Amp
+
<p class="title3">Test of sensibility on Ampicillin</p>
-
A COMPLETER
+
<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>
-
Fungicides
 
-
D4E1
+
<div class="technology">Fungicides</div>
 +
<div class="thelanguage">
-
1. Amplification of synthetic gene (D4E1 on pEX-A2)
+
<div class="technology2">D4E1</div>
-
• Transformation of D4E1 (pEX-A2) into E. coli
+
<div class="thelanguage2">
-
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
+
<p class="title2">1. Amplification of synthetic gene (D4E1 on pEX-A2)</p>
-
Date: 07/22/2014
+
<p class="title3">Transformation of D4E1 (pEX-A2) into <i>E. coli</i></p>
-
Result : Culture of 4 clones ok
+
<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>
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of D4E1 (pEX-A2) into E. coli
+
<p class="title3">Culture of 4 clones: A, B, C, D of D4E1 (pEX-A2) transformed into E. coli</p>
-
Buffer EB at 50-55°C
+
<p class="texte"><b>Date:</b> 07/22/2014
-
Date: 07/23/2014
+
<br><b>Result:</b> Culture of 4 clones ok</p>
-
• Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up
+
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of D4E1 (pEX-A2) into E. coli</p>
-
Date: 07/23/2014
+
<p class="texte">Buffer EB at 50-55°C
-
Result : 4*20µL D4E1 digested with EcoRI and PstI  all the clones seem to have the right D4E1 gene.
+
<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>
   
   
-
2. Cloning D4E1 in pSB1C3
+
<p class="title2">2. Cloning D4E1 in pSB1C3</p>
-
• Digestion of D4E1 on pEX-A2 and pSB1C3
+
<p class="title3">Digestion of D4E1 on pEX-A2 and pSB1C3</p>
-
Date: 07/23/2014
+
<p class="texte"><b>Date:</b> 07/23/2014</p>
-
• Ligation of D4E1 in pSB1C3
+
<p class="title3">Ligation of D4E1 in pSB1C3</p>
-
Date: 07/23/2014
+
<p class="texte"><b>Date:</b> 07/23/2014</p>
-
• Transformation in E.coli
+
<p class="title3">Transformation in <i>E.coli</i></p>
-
Date : 07/23/2014
+
<p class="texte"><b>Date:</b> 07/23/2014</p>
-
• Culture of 6 clones: A, B, C, D, E, F of D4E1 (pSB1C3) transformed intoE. coli
+
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of D4E1 (pSB1C3) transformed into <i>E. coli</i></p>
-
Processing details: 5mL of LB + chloramphenicol (15µg/mL) , using sterile plastic loop to culture colonies
+
<p class="texte">Processing details: 5mL of LB + chloramphenicol (15µg/mL) , using sterile plastic loop to culture colonies
-
Finished by: Emeline and Diane
+
<br><b>Date:</b> 07/24/2014
-
Dated: 07/24/2014
+
<br><b>Result:</b> Culture of 4 clones ok</p>
-
Result: Culture of 4 clones ok
+
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
+
<p class="texte"><b>Date:</b> 07/25/2014</p>
-
Date: 07/25/2014
+
<p class="title3">Digestion of D4E1 (pSB1C3) A, B, C, D, E, F with EcoRI and PstI - PCR kit Clean up + electrophoresis</p>
-
• Digestion of D4E1 (pSB1C3) A, B, C, D, E, F with EcoRI and PstI - PCR kit Clean up + electrophoresis
+
<p class="texte"><b>Date:</b> 07/25/2014
-
Date: 07/25/2014
+
<br><b>Result:</b> clones C, D have the expected construction</p>
-
Result: clones C, D have the expected construction
+
<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>
-
• PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
+
<p class="title3">Ligation of D4E1 in K823003 (Pveg on pSB1C3)</p>
-
Date: 07/25/2014
+
<p class="texte"><b>Date:</b> 07/24/2014
-
Result: clones C, D have the expected construction, and placed in cryopreservation.
+
<br><b>Result:</b> 20µL ligation of D4E1 in K823003</p>
-
3. Cloning Pveg+D4E1 on Pveg plasmid (pSB1C3)
+
<p class="title3">Transformation of ligation products in <i>E.coli</i></p>
-
• Digestion of D4E1 on pEX-A2 and K823003 (Pveg on pSB1C3)
+
<p class="texte"><b>Date:</b> 07/24/2014
-
Finished by: Emeline and Diane
+
<br><b>Result:</b> <i>E.coli</i> transformed by D4E1+K823003</p>
-
Dated: 07/24/2014
+
 
-
Result: 20µL digestion of D4E1 on pEX-A2 and of K823003
+
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of transformed <i>E. coli</i></p>
-
• Ligation of D4E1 in K823003 (Pveg on pSB1C3)
+
<p class="texte"><b>Date:</b> 07/26/2014
-
Date: 07/24/2014
+
<br><b>Result:</b> Culture of 4 clones ok</p>
-
Result: 20µL ligation of D4E1 in K823003
+
 
-
• Transformation of ligation products in E.coli
+
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
-
Date : 07/24/2014
+
<p class="texte"><b>Date:</b> 07/28/2014</p>
-
Result: E.coli transformed by D4E1+K823003
+
 
-
• Culture of 6 clones: A, B, C, D, E, F of transformed E. coli
+
<p class="title3">PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p>
-
Date: 07/26/2014
+
<p class="texte"><b>Dated:</b> 07/28/2014
-
Result: Culture of 4 clones ok
+
<br><b>Result:</b> clones E, F seem to have the expected construction</p>
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
+
<p class="title3">Digestion of clones E, F of D4E1+K823003 (Pveg on pSB1C3) with EcoRI and PstI + electrophoresis</p>
-
Date: 07/28/2014
+
<p class="texte"><b>Date:</b> 28/07/2014
-
• PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
+
<br><b>Result:</b> clones C, D have the expected construction and are placed in cryopreservation.</p>
-
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)  
+
<p class="title2">4. Cloning P<sub>veg</sub> + D4E1 on pSB<sub>BS</sub>4S (K823022) </p>
-
Date: 08/13/2014
+
<p class="texte"><b>Date:</b> 08/13/2014</p>
   
   
-
5. Cloning Pveg + D4E1 on pSBBS1C lacZ (23)
+
<p class="title2">5. Cloning P<sub>veg</sub> + D4E1 on pSB<sub>BS</sub>1C lacZ (23)</p>
-
COMPLETER !
+
<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>
-
GAFP1
+
<p class="title2">2. Cloning GAFP1 gene on PSB1C3 = K1364002 in E. coli</p>
-
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: 07/23/201
+
-
2. Cloning GAFP1 gene on PSB1C3 = K1364002 in E. coli
+
<p class="title3">Digestion GAFP1_pEX-A2 and <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a> (RFP_pSB1C3)</p>
-
• Digestion GAFP1_pEX-A2 and BBa_K606013 (RFP_pSB1C3)
+
<p class="texte"><b>Date:</b> 07/23/2014
-
Date: 07/23/2014
+
<br><b>Result:</b> <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a> : 860 bp
-
Result : BBa_K606013 : 860 bp
+
<br>We decide to conserve the miniprep B for <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a></p>
-
We decide to conserve the miniprep B for BBa_K606013
+
 
-
• Ligation GAFP1 and BBa_K606013
+
<p class="title3">Ligation GAFP1 and <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a></p>
-
Date : 07/23/2014
+
<p class="texte"><b>Date:</b> 07/23/2014</p>
-
• Tranformation of ligation products into E. coli
+
 
-
Date : 07/23/2014
+
<p class="title3">Tranformation of ligation products into <i>E. coli</i></p>
-
• Culture of x clones of GAFP1+K606013 in E. coli
+
<p class="texte"><b>Date:</b> 07/23/2014</p>
-
Date : 07/24/2014
+
 
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge: 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) into E. coli
+
<p class="title3">Culture of x clones of GAFP1+K606013 in <i>E. coli</i></p>
-
Date: 07/25/2014
+
<p class="texte"><b>Date:</b> 07/24/2014</p>
-
• PCR on 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) + electrophoresis
+
 
-
Date: 07/25/2014
+
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge: 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) into <i>E. coli</i></p>
-
Result : clones A, C, D, F seem to have the right construction
+
<p class="texte"><b>Date:</b> 07/25/2014</p>
-
• Digestion of 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) by EcoR1 and Pst1 + electrophoresis
+
 
-
Date: 07/25/2014
+
<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>
   
   
-
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 (K1364008) on pSB1C3 and PsBBs4S (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
 
-
6. Cloning GAFP1+Ter B0015 on psBBs1C lacZ (23)
+
<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>
   
   
-
COMPLETER
+
 
 +
 
 +
<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>
   
   
-
7. Tests
 
-
COMPLETER
 
-
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 pUC + Utah
 
-
Date: 07/28/2014
 
-
3. Liquid culture + Miniprep + Test of the miniprep
+
<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>
-
Date: 07/30/2014
+
-
4. Cloning 1: EcAMP + Pveg + RBS
+
<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>
-
• Digestion of EcAMP (INSERT) by XbaI and PstI
+
<p class="texte"><b>Date:</b> 08/01/2014</p>
-
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
+
-
 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 200pb.
+
<p class="title3">Ligation of K134008 (Pveg+GAFP1+ter fragment) and K823022 (PsBBs4S)</p>
 +
<p class="texte"><b>Date:</b> 08/01/2014</p>
-
5. Cloning 2: EcAMP + Pveg + RBS
+
<p class="title3">Transformation of ligation products in <i>E.coli</i></p>
-
Date: 06/08/2014
+
<p class="texte"><b>Date:</b> 08/01/2014</p>
-
• Digestion of EcAMP (INSERT) by XbaI and PstI
+
-
• Digestion of Pveg + RBS (VECTOR)
+
-
• Heat inactivation of the enzymes
+
-
• Ligation and transformation
+
-
• PCR test
+
-
Date: 07/08/2014
+
 +
<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>
-
• Striation on a petri dish to purify the clone
+
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
-
Purpose: to isolate a clone with vector+insert
+
<p class="texte"><b>Date:</b> 08/04/2014</p>
-
Date: 08/072014
+
-
• Miniprep of Pveg+SpoVG+EcAMP and analytic digestion
+
<p class="title3">Digestion of clones A, B, C, D, E, G, H of K1364008+K823022 with EcoRI and PstI + electrophoresis</p>
-
Date: 08/08/2014
+
<p class="texte"><b>Date:</b> 08/04/2014
 +
<br><b>Result:</b> clones A, E, F have the right construction</p>
-
• Ligation of Pveg SpoVG EcAMP with double terminateur B0015 +Transformation and liquid culture
+
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p>
-
Date: 08/11/2014
+
</div>
-
• Miniprep of Pveg SpoVG EcAMP + analytic digestion
+
<div class="technology2">EcAMP</div>
-
Date : 08/13/2014
+
<div class="thelanguage2">
-
• Cloning K1364011 (EcAMP+Pveg +SpoVG + B0015) + K823022 (psBbs4S) : Digestion, ligation, transformation
+
<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>
-
Date: 08/19/2014
+
-
• Cloning K1364011 + K823023 (pSBBS1C) : Digestion, ligation, transformation
+
<p class="title2">1. Transformation of EcAMP in <i>Escherichia coli</i> MC 1061</p>
-
Date: 08/18/2014
+
<p class="texte"><b>Date:</b> 07/25/2014</p>
-
• Verification of the insertion of K1364011 + K823022 (pSBBS4S) into the subtilis genome by threonine test
+
<p class="title2">2. Spreading of coli cells transformed with EcAMP (plasmid pUC) </p>
-
Date: 08/21/2014
+
<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>
-
Assembling the fungicides module
+
<div class="technology2">Assembling the fungicides module: D4E1 + GAFP1</div>
 +
<div class="thelanguage2">
-
D4E1 + GAFP1
+
<p class="title2">1.  Cloning GAFP1+D4E1 on pSB1C3: <a href="http://parts.igem.org/Part:BBa_K1364012">BBa_K1364012</a></p>
-
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
+
<p class="title3">Digestion of D4E1 on pEX-A2 and GAFP1 on pSB1C3</p>
-
Date: 07/28/2014
+
<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>
-
• Transformation of ligation of GAFP1 and D4E1 on pSB1C3 into E. coli
+
<p class="title3">Ligation of digestions of D4E1 on pEX-A2 and of GAFP1 on pSB1C3</p>
-
Date: 07/28/2014
+
<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>
   
   
-
• PCR of GAFP1+D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
+
<p class="title3">PCR of GAFP1 + D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p>
-
Date: 07/29/2014
+
<p class="texte"><b>Date:</b> 07/29/2014
-
Result: clones A, C, F, G seem to have the expected construction.
+
<br><b>Result:</b> clones A, C, F, G seem to have the expected construction.</p>
   
   
-
• Digestion of GAFP1+D4E1 (pSB1C3) A, C, F, G + electrophoresis
+
<p class="title3">Digestion of GAFP1 + D4E1 (pSB1C3) A, C, F, G + electrophoresis</p>
-
Date: 07/30/2014
+
<p class="texte"><b>Date:</b> 07/30/2014
-
Result: clone  F (BBa_K1364012) has the expected construction and is placed on cryopreservation.
+
<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>
   
   
-
2. Cloning GAFP1+D4E1 (BBa_K1364012) on Pveg plasmid (BBa_K823003): BBa_K1364013
 
-
3. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS4S (K823022)
 
-
4. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS1C lacZ (K823023)
 
-
5. Fungicides tests
 
-
Cloning D4E1-GAFP1-EcAMP: BBa_K1364014
 
-
1.  Construction of BBa_K1364014 in PsB1C3, in E. coli
 
-
• Digestion of K1364010 and K1364012
 
-
Digestion of K13664010 by Spe1 and Pst1, and digestion of K1364012 by XbaI and Pst1.
 
-
Date: 08/11/2014
 
-
Result : We obtained digested fragments of K1364012 and ~500 bp fragment of K1364010
 
-
• Ligation of ~500 bp fragment from K1364010 and K1364012
+
<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>
-
Date: 08/11/2014
+
<p class="texte"><b>Date:</b> 08/04/2014
-
Result: We obtained ligation of K1364012 and ~500 bp fragment of K1364010
+
-
• Transformation of ligation of ~500 bp fragment from K1364010 and K1364012 = K1364014 in E.coli
+
<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>
-
Date: 08/12/2014
+
<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
-
• Testing 8 E.coli+K1364014 clones
+
<p class="title2">5. Fungicides tests</p>
-
Date: 08/14/2014  
+
<p class="texte"><b>Date:</b> 08/15/2014
-
• Testing 15 E.coli+K1364014 clones
+
<p class="title2">Cloning D4E1-GAFP1-EcAMP: <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a></p>
-
Date: 08/18/2014
+
-
Result: clones H, J, O, Q, U, V might have the right K1364014 construction
+
-
II. BBa_K1364014 in PsBs4s (K823022) and PsBs1C (K823023)
+
<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>
-
• Digestion of K1364014, K823022 and K823023
+
-
Date: 08/22/2014
+
-
Result: digestion of K13664014, K823022 and K823023 by EcoR1 and Pst1 were well performed.
+
-
• Ligation of K1364014 with K823022 and K1364014 with K823023
+
<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>
-
Date: 08/22/2014
+
<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.
-
Result: We obtained 20µL ligation of K1364014 with K823022 and of K1364014 with K823023.
+
<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>
-
• Transformation of ligations in E.coli
+
<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>
-
E. coli transformed by K1364014+K823022 spread on LB+Amp 100µg/mL agar plate
+
<p class="texte"><b>Date:</b> 08/11/2014
-
E. coli transformed by K1364014+K823023 spread on LB+Amp 100µg/mL agar plate
+
<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>
-
Date: 08/25/2014
+
-
• Testing E.coli+K1364014+K823022 and E.coli+K1364014+K823023 clones
+
-
Date: 08/27/2014
+
-
3.  Transformation of K1364014+K823022 and K1364014+K823023 in B. subtilis
+
<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>
-
B. subtilis transformed by 10µL K1364014+K823022 spread on LB+Spec 75µg/mL agar plate
+
<p class="texte"><b>Date:</b> 08/12/2014</p>
-
B. subtilis transformed by 10µL K1364014+K823023 spread on LB+Cm 15µg/mL agar plate
+
-
Date : 08/28/2014
+
-
Fungicides tests
 
-
1. D4E1-GAFP1
 
-
• 08/12/2014 : Transformation in bacillus  Pveg-D4E1-GAFP1 on pSBBS4S
+
<p class="title3">Testing 8 <i>E.coli</i> + <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> clones</p>
-
• 08/13/2014 :  integration threonine test + fungicide test
+
<p class="texte"><b>Date:</b> 08/14/2014 </p>
-
2. D4E1
+
<p class="title3">Testing 15 <i>E.coli</i> + <a href="http://parts.igem.org/Part:BBa_K1364014">BBa_K1364014</a> clones</p>
-
• 08/15/2014 : Cloning D4E1 into pSBBS1C + fungicide test
+
<p class="texte"><b>Date:</b> 08/18/2014
-
• 08/19/2014 : D4E1 on pSB1C3 + fungicide test
+
<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>
-
+
 
-
COMPLETER
+
 
 +
<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>
 +
<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>
 +
 +
 +
 +
<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
 +
<br><b>Date:</b> 08/28/2014</p>
 +
 +
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p>
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</div>
 +
 +
<div class="technology2">Fungicides tests</div>
 +
<div class="thelanguage2">
 +
 +
<p class="title2">1. D4E1-GAFP1</p>
 +
<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>
 +
 +
<p class="title2"2. D4E1</p>
 +
<p class="title3">
 +
Cloning D4E1 into pSB<sub>BS</sub>1C + fungicide test</p>
 +
<p class="texte"><b>Date:</b>08/15/2014</p>
 +
<p class="title3">
 +
D4E1 on pSB1C3 + fungicide test</p>
 +
<p class="texte"><b>Date:</b>08/19/2014</p>
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<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 1); return false">Collapse</a></p>
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</div>
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<div class="clear"></div>
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</div>
     </div>
     </div>
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   </div>

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"