Team:Toulouse/Notebook/project-monitoring

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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;">
 +
  <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 class="centering" style="padding-top: 85px; padding-bottom:40px;">
       <div class="centering" style="padding-top: 85px; padding-bottom:40px;">
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<br/>
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<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p>
-
<div class="Sub_title"> December 2013 – January 2014: Team selection</div>
+
 
-
<table width="100%"><tr><td bgColor="#097F09" height="1px"></td> </tr></table>
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<div class="technology">Binding module</div>
-
<br/>
+
<div class="thelanguage">
-
<div class="Article">
+
 
-
<p>
+
<p class="title2">1. Amplification of Binding Module (pEX-K4) into <i>E. coli</i></p>
-
-   iGEM competition presentation and explanations
+
<p class="title3">Transformation of Binding module (pEX-K4) into <I>E. coli</i></p>
-
<br/>
+
<p class="texte">Gene "Binding module" synthesized by Eurofins, resuspended in 20μL Tris 10mM
-
-   Constitution of the team by interviewing different students from Université Paul Sabatier and INSA
+
<br><b>Result:</b> We obtained distinct colonies on plates LB + Kanamycin (50 µg/mL)</p>
-
<br/> <CENTER><B> The adventure begins for the Toulouse iGEM Team 2014! </B></CENTER>
+
 
-
<br/>
+
<p class="title3">Liquid culture of two clones: 1 et 2 (pEX-K4) transformed into <i>E. coli</i></p>
-
</p>
+
<p class="texte"><b>Date:</b> 01/08/2014</p>
-
<br/>
+
 
-
<div class="Sub_title"> January – June 2014: Projects brainstorming</div>
+
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit: two clones of Binding Module (pEX-K4) into <i>E. coli</i></p>
-
<table width="100%"><tr><td bgColor="#097F09" height="1px"></td> </tr></table>
+
<p class="texte"><b>Date:</b> 04/08/2014
-
<br/>
+
<br><b>Result:</b> Binding Module (pEX-K4) obtained</p>
-
<p>
+
 
-
- Thanks to weekly meetings, our team was able to discuss about new project ideas and publications.
+
<p class="title3">Digestion of Binding Module (pEX-K4) with EcoRI and PstI</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 04/08/2014
-
- Many ideas were approached and debated regarding the feasibility thanks to the literature and supervisors including teachers and researchers
+
<br><b>Result:</b>  
-
<br/>
+
<br>- 3 bands : 1500bp (vector with Kanamycin resistance), 1300bp (Module Binding) and 1000bp (vector with pUC Ori)
-
This is a list of the main projects:  
+
<br>- The two Binding Module clones are ok</p>
-
<br/>
+
 
-
- Let's save our trees with SubtiTree: the purpose is to use Bacillus subtilis as an optimized bacterium to detect, target the fungi and secrete fungicides to destroy the pathogen
+
 
-
<br/>
+
<p class="title2">2. Cloning Binding Module on pEX-K4 with pSB1C3 (<a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a> without RFP) into <i>E. coli</i></p>
-
- E. coli cancer: the concept was to create a bacterium able to colonize specifically the hypoxic regions of the tumor. The oxygen-sensitive bacterium would not be able to develop in the healthy zones
+
<p class="title3">Digestion Binding Module on pEX-K4 and <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a></p>
-
<br/>
+
<p class="texte"><b>Date:</b> 23/08/2014
-
- Reduction of ruminants’methane production: the goal is to reduce the production of methane by ingesting a microorganism in the animal’s rumen. This bacterium would be capable of reducing the quantity of metabolic hydrogen and eliminating the methanogenic Archaes population
+
<br><b>Expected bands after digestion:</b>
-
<br/>
+
<br>- <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a>: 860 bp for RFP and 2100 bp for vector  pSB1C3
-
</p>
+
<br>- Binding Module: 1400 bp for Binding Module and 1500bp + 1000bp for vector pEX_K4
-
<p>
+
<br>We decide to do a ligation between pSB1C3 and Binding Module without gel cutting and purification but after a PCR clean-up (Thermo scientific, GeneJET PCR purifiction kit #K0701) to remove the enzymes.</p>
-
  <br/>
+
 
-
<div class="Sub_title"> June 2014: Choice of SubtiTree project </div>
+
<p class="title3">Ligation Binding Module on pSB1C3</p>
-
<table width="100%"><tr><td bgColor="#097F09" height="1px"></td> </tr></table>
+
<p class="texte"><b>Date:</b> 04/08/2014</p>
-
<br/>
+
 
-
<p>
+
<p class="title3">Transformation of Binding Module on pSB1C3 into E. coli</p>
-
The discussion has been long to estimate the practicabilty of our final project. By the end of June, most of the ideas were eliminated but two of them remained: controlling the cancer or saving the trees. It became easily clear that SubtiTree was successfully completed whether regarding the scientific part or the ethical aspect. Indeed, our knowledge in medicine was not sufficient enough to evaluate the consequences of a bacterium injection in the human body for E. coli cancer.
+
<p class="texte"><b>Date:</b> 04/08/2014
-
Therefore, the whole team agreed on focusing on a local issue as the preservation of the Canal du Midi plane trees. However, the final goal was based on the idea to allow our optimized bacteria to be efficient for all fungi infections in the environment.
+
<br>Transformation with 10 µL of the ligation mix and plate on Chloremphenicol LA plate
-
By this period, we evaluated our budget to approximately <B> 40 k€ </B>.
+
<br><b>Result:</b> many wrong clones</p>
-
</p>  
+
 
-
</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>
-
<div class="Article">
+
 
-
    <p>
+
<div class="technology">Chemotaxis</div>
-
  <br/>
+
<div class="thelanguage">
-
  <FONT SIZE= 2%>
+
 
-
<div class="Sub_title"> Week 1(16-22 June)</div>
+
<p class="title2">1. Transformation of chemotaxis (Puc-57) into <i>E. coli</i></p>
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
<p class="texte">Gene chemotaxis synthesized by Eurofins, resuspended in 20μL Tris 10mM
-
<br/> </FONT>
+
<br><b>Date:</b> 01/08/2014
-
<p>
+
<br><b>Result:</b> We obtained distinct colonies on LA + Ampicillin (100 µg/mL) plates</p>
-
<B> Preparation period for the laboratory work: </B>
+
 
-
<br/>
+
<p class="title3">Culture of 4 clones: A, B, C, D of chemotaxis (Puc57) transformed into <i>E. coli</i></p>
-
<p>
+
<p class="texte"><b>Date:</b> 04/08/2014</p>
-
- Bibliography researches about our subject
+
 
-
<br/>
+
<p class="title3">Miniprep with QIAprep Spin Miniprep Kit Using a Microcentrifuge: 4 clones of chemotaxis (Puc57) into <i>E. coli</i></p>
-
- Cleaning the whole lab to allow good manipulations and avoid any contamination
+
<p class="texte"><b>Date:</b> 05/08/2014
-
<br/>
+
<br><b>Result:</b> 4*50µL of chemotaxis (Puc57) obtained</p>
-
- Inventory of necessary lab equipment and biobricks
+
 
-
<br/>
+
<p class="title2">2. Cloning chemotaxis  <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> (chemotaxis_Puc57) with digested pSB1C3 with EcoRI and PstI into <i>E. coli</i></p>
-
- Summarize the iGEM protocols
+
 
-
<br/>
+
<p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> (chemotaxis_Puc57) with EcoRI and PstI - PCR kit Clean up</p>
-
- Prepare all growing media
+
<p class="texte"><b>Date:</b> 07/08/2014
-
<br/>
+
<br><b>Result:</b> Expected band after digestion for <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>: 2300 bp
-
- Preparing competent cells by optimizing protocols
+
<br><b>Problem:</b> We can't distinguish the vector band (2500 bp)</p>
-
<br/>
+
 
-
- Transformation of RFP plasmid to practice
+
<p class="title3">Gel extraction of <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a></p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/08/2014</p>
-
<p>
+
 
-
<B> Communication and financial support: </B>
+
<p class="title3">Ligation <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> and digested PSB1C3 with EcoRI and PstI</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 08/08/2014</p>
-
<p>
+
 
-
- Increase in our communication strategy to find more sponsors specialized in environment and ecological matters
+
<p class="title3">Transformation <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> in <i>E.coli</i></p>
-
<br/>
+
<p class="texte"><b>Date:</b> 08/08/2014
-
- Support of Adisseo, Toulouse White Biotechnology, LISBP, INSA Toulouse
+
<br><b>Result:</b> We obtained distinct colonies on LA + Cm (15 µg/mL) plates and resuspended 15 colonies in LB+Cm (15 µg/mL)</p>
-
<br/>
+
 
-
- Organization of a weekly meeting each Friday with our supervisors to tackle any problem encountered
+
<p class="title3">Test of sensibility on Ampicillin</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 10/08/2014
-
<p>
+
<br><b>Result:</b> we can determine which colonies are sensible for ampicillin and know which bacterium is carrying the chemotaxis gene.</p>
-
<CENTER> <I> Weekly meeting with the instructors (06/20/2014)</CENTER> </I>
+
 
-
 Distribution of the non-scientific tasks regarding the wiki, communication, sponsorship, ethical problems, safety but also the lab work
+
<p class="title3">PCR</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 11/08/2014</p>
-
 Discussion about the different construction parts of the project
+
 
-
<br/>
+
<p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> on pSB1C3 with EcoRI and PstI</p>
-
 Need to check the absence of stop codon in fungicides sequences
+
<p class="texte"><b>Date:</b> 11/08/2014
-
<br/>
+
<br><b>Result:</b> There is one colony which presents the right construction.</p>
-
</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>
+
<p class="title2">2. Cloning D4E1 in pSB1C3</p>
-
<br/>
+
<p class="title3">Digestion of D4E1 on pEX-A2 and pSB1C3</p>
-
<FONT SIZE= 2%>
+
<p class="texte"><b>Date:</b> 07/23/2014</p>
-
<div class="Sub_title"> Week 2 (23-29 June)</div>
+
<p class="title3">Ligation of D4E1 in pSB1C3</p>
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
<p class="texte"><b>Date:</b> 07/23/2014</p>
-
<br/> </FONT>
+
<p class="title3">Transformation in <i>E.coli</i></p>
-
<p>
+
<p class="texte"><b>Date:</b> 07/23/2014</p>
-
<B> Preparation period for the laboratory work: </B>
+
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of D4E1 (pSB1C3) transformed into <i>E. coli</i></p>
-
<br/>
+
<p class="texte">Processing details: 5mL of LB + chloramphenicol (15µg/mL) , using sterile plastic loop to culture colonies
-
<p>
+
<br><b>Date:</b> 07/24/2014
-
- Ordering the list of necessary products for the laboratory and biobricks
+
<br><b>Result:</b> Culture of 4 clones ok</p>
-
<br/>
+
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
-
- Checking and validation of the genes sequences
+
<p class="texte"><b>Date:</b> 07/25/2014</p>
-
<br/>
+
<p class="title3">Digestion of D4E1 (pSB1C3) A, B, C, D, E, F with EcoRI and PstI - PCR kit Clean up + electrophoresis</p>
-
Communication and financial support
+
<p class="texte"><b>Date:</b> 07/25/2014
-
<br/>
+
<br><b>Result:</b> clones C, D have the expected construction</p>
-
- BioSynSyS conference work: powerpoint, logo in order to present our SubtiTree project to a whole audience of scientific and researchers (http://biosynsys2014.sciencesconf.org/
+
<p class="title3">PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/25/2014
 +
<br><b>Result:</b> clones C, D have the expected construction, and placed in cryopreservation.</p>
-
<p>
+
<p class="title2">3. Cloning Pveg+D4E1 on Pveg plasmid (pSB1C3)</p>
-
<B> Lab work: </B>
+
-
<br/>
+
-
<p>
+
-
- E. coli transformation with BBA_J004450 (pSB1C3)
+
-
<br/>
+
-
<p>
+
-
<CENTER> <I> Weekly meeting with the instructors (06/27/2014) </CENTER> </I>
+
-
 Concentration of antibiotics in media must be checked
+
-
<br/>
+
-
 The transformation rate for pUC19 must be evaluated
+
-
<br/>
+
-
 The transformation efficiency must be calculated regarding the quantity of DNA
+
-
<br/>
+
-
 Possibility to contact the cities halls for the sponsorship
+
-
<br/>
+
-
 Check the mechanism of action of each fungicide to complete the ethical part
+
-
<br/>
+
-
</p>
+
-
<div class="Sub_title"> July 2014 </div>
+
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
-
<br/>
+
-
<p>
+
-
Main activites
+
-
<br/>
+
-
- Beginning of the laboratory work to create our optimized bacterium
+
-
<br/>
+
-
- Research of sponsors and the communication thanks to the press
+
-
<br/>
+
-
</p>  
+
-
<p>
+
<p class="title3">Digestion of D4E1 on pEX-A2 and K823003 (Pveg on pSB1C3)</p>
-
<br/>
+
<p class="texte"><b>Dated:</b></b> 07/24/2014
-
<FONT SIZE= 2%> 
+
<br><b>Result:</b> 20µL digestion of D4E1 on pEX-A2 and of K823003</p>
-
<div class="Sub_title"> Week 3 (30 June -6 July) and Week 4 (7-13 July)</div>
+
 
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
<p class="title3">Ligation of D4E1 in K823003 (Pveg on pSB1C3)</p>
-
<br/> </FONT>
+
<p class="texte"><b>Date:</b> 07/24/2014
-
<p>
+
<br><b>Result:</b> 20µL ligation of D4E1 in K823003</p>
-
<B> Communication and financial support: </B>
+
 
-
<br/>
+
<p class="title3">Transformation of ligation products in <i>E.coli</i></p>
-
<p>
+
<p class="texte"><b>Date:</b> 07/24/2014
-
- Participation at the BioSynSys conferences: presentation of SubtiTree
+
<br><b>Result:</b> <i>E.coli</i> transformed by D4E1+K823003</p>
-
<br/>
+
 
-
- We have put in place a newsletter system the first day of each month to keep all our sponsors and people who support us updated about the project, our financial state.
+
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of transformed <i>E. coli</i></p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/26/2014
-
<p>
+
<br><b>Result:</b> Culture of 4 clones ok</p>
-
<B> Lab work: </B>
+
 
-
<br/>
+
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
-
<p>
+
<p class="texte"><b>Date:</b> 07/28/2014</p>
-
- Design of all the constructions needed with cloning and restriction enzymes to save some time and allow a better comprehension of our bacterium system
+
 
-
Problem: A problem was reported in the use of the CYP. Indeed, the biobrick is not expressed properly in Bacillus subtilis strain. Therefore we had to look at the literature to find a new fungicide called GAFP-1.
+
<p class="title3">PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p>
-
<br/>
+
<p class="texte"><b>Dated:</b> 07/28/2014
-
- A bioreactor was conceived to reproduce the composition of the sap in the plane trees. The main goal was to identify the behavior of Bacillus strain in a medium composed of many different carbon sources.
+
<br><b>Result:</b> clones E, F seem to have the expected construction</p>
-
<br/>
+
<p class="title3">Digestion of clones E, F of D4E1+K823003 (Pveg on pSB1C3) with EcoRI and PstI + electrophoresis</p>
-
- Competent cells and transformation practice using GFP and RFP.
+
<p class="texte"><b>Date:</b> 28/07/2014
-
<br/>
+
<br><b>Result:</b> clones C, D have the expected construction and are placed in cryopreservation.</p>
-
<p>
+
-
<CENTER> <I> Weekly meetings with the instructors (07/04/2014) and (07/11/2014) </CENTER> </I>
+
-
 iGEM efficiency kit does not work, we shall not use it anymore
+
-
<br/>
+
-
 Organization of a timetable to check the stored  and sterile equipment everyday
+
-
<br/>
+
-
 Try a transformation in Bacillus subtilis
+
-
<br/>
+
-
</p>  
+
   
   
-
<p>
+
<p class="title2">4. Cloning P<sub>veg</sub> + D4E1 on pSB<sub>BS</sub>4S (K823022) </p>
-
<br/>
+
<p class="texte"><b>Date:</b> 08/13/2014</p>
-
<FONT SIZE= 2%>   
+
   
-
<div class="Sub_title"> Week 5 (14-20 July)</div>
+
<p class="title2">5. Cloning P<sub>veg</sub> + D4E1 on pSB<sub>BS</sub>1C lacZ (23)</p>
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
 
-
<br/> </FONT>
+
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p>
-
<p>
+
</div>
-
<B> Communication and financial support: </B>
+
 
-
<br/>
+
<div class="technology2">GAFP1</div>
-
<p>
+
<div class="thelanguage2">
-
- Our team was interviewed by many local newspapers such as 20minutes, Metronews, La Voix du Midi, La Dépêche but also by the local television channel France3 Midi-Pyrénées to present our draft to the community. Moreover, SubtiTree was approached in some radio programs like Virgin Radio Toulouse and France info.
+
 
-
<br/>
+
<p class="title2">1. Amplification of synthetic gene (GAFP1 on pEX-A2)</p>
-
- Support of ThermoFisher, New Englands BioLabs, Qiagen, Eurofins but also GenoToul, Labex Tulip, L’Université Paul Sabatier, CROUS.
+
 
-
<br/>
+
<p class="title3">Transformation of GAFP1 (pEX-A2) into <i>E.coli</i></p>
-
<p>
+
<p class="texte">Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
-
<B> Lab work: </B>
+
<br>Concentration of GAFP1 : 145ng/µL
-
<br/>
+
<br><b>Date:</b> 07/21/2014
-
<p>
+
<br><b>Result:</b> We obtained distinct colonies on plates LB + Ampicillin (100 µg/mL)</p>
-
- GFP and RFP digestion amplified by miniprep and gel electrophoresis
+
 
-
<br/>Problem: the digested GFP doesn’t appear on the gel contrary to RFP plasmid. We assumed a problem with the GFP miniprep. We tried the miniprep from the INSA Toulouse team from 2013 and the GFP plasmid was fully digested.
+
<p class="title3">Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/22/2014
-
- Transformation and cryopreservation of biobricks BBa_K823002 (PlepA), BBa_K823003 (Pveg), BBa_K606061 (RBS SpoVG) and BBa_B0015 (double terminator), BBa_K823023 (pSBBS1C), BBa_K733013 (Pveg+ RBS) in E. coli
+
<br><b>Result:</b> Culture of 4 clones ok</p>
-
<br/>
+
 
-
- Transformation of the Munich B. subtilis backbones
+
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/23/2014</p>
-
- Cloning of BBa_K606061 (RBS SpoVG) with BBa_K823003 (PvEG) and BBa_K823002 (PlepA)
+
 
-
<br/>
+
<p class="title3">Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel Electrophoresis</p>
-
<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>
-
<CENTER> <I> Weekly meeting with the instructors (07/18/2014) </CENTER> </I>
+
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge: 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) into <i>E. coli</i></p>
-
 Transformation of the Eurofins genes
+
<p class="texte"><b>Date:</b> 07/25/2014</p>
-
<br/>
+
-
 Start assembling the biobricks for the fungicides
+
-
<br/>
+
-
 Check all the cloning
+
-
<p>
+
<p class="title3">PCR on 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) + electrophoresis</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/25/2014
-
<FONT SIZE= 2%> 
+
<br><b>Result:</b> clones A, C, D, F seem to have the right construction</p>
-
<div class="Sub_title"> Week 6 (21 July-27 July) </div>
+
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
-
<br/> </FONT>
+
-
<p>
+
-
<B> Communication and financial support: </B>
+
-
<br/>
+
-
<p>
+
-
- Description of SubtiTree project and determination of the goodies for a crowdfunding campaign on Ulule website
+
-
<br/>
+
-
<p>
+
-
<B> Lab work: </B>
+
-
<br/>
+
-
<p>
+
-
- Transformation of BBA_1364002 (GAFP1)  and BBA_1364003 (D4E1) fungicides genes
+
-
<br/>
+
-
- Subculture of the clones for each gene
+
-
<br/>
+
-
- PCR and migration on electrophoresis gel
+
-
<br/>
+
-
- Transformation BBA_1364003 (D4E1)  on pSB1C3 and BBA_1364002 (GAFP1)  on pSB1C3
+
-
<br/>
+
-
- Cloning BBA_1364002 (GAFP1) with BBa_B0015 (double terminator) and BBA_1364003 (D4E1)  with BBa_K823003 (Pveg)
+
-
<br/>
+
-
<p>
+
-
<B> Others: </B>
+
-
<br/>
+
-
<p>
+
-
- Research for plane tickets and hotels in Boston for the Giant Jamboree
+
-
<br/>
+
-
- New idea: analyze the plane tree sap to determine the composition
+
-
<br/>
+
-
<div class="Sub_title"> August 2014 </div>
+
-
<table width="100%"><tr><td bgColor="#097F09" height="1px"></td> </tr></table>
+
-
<br/>
+
-
<p>
+
-
Main activites
+
-
<br/>
+
-
- Finishing the cloning for the different parts of the bacterium
+
-
<br/>
+
-
- Putting in place the fungicides, binding and chemotaxis tests
+
-
<br/>
+
-
</p>  
+
-
<p>
+
<p class="title3">Digestion of 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) by EcoR1 and Pst1 + electrophoresis</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/25/2014</p>
-
  <FONT SIZE= 2%> 
+
-
<div class="Sub_title"> Week 7 (28 July-3 August)</div>
+
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
-
<br/> </FONT>
+
-
<p>
+
-
<B> Communication and financial support: </B>
+
-
<br/>
+
-
<p>
+
-
- Financial supports of the Ministery (Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt) and Voies Navigables de France
+
-
<br/>
+
-
- Interview with Johan Langot from Sciences Animation for a possible presentation at the Toulouse Festival of Science
+
-
<br/>
+
-
- Launch of the crowdfunding campaign on Ulule
+
-
<br/>
+
-
<p>
+
-
<B> Lab work: </B>
+
-
<br/>
+
-
<p>
+
-
- Cloning BBa_K823003 (Pveg) + RFP and BBa_K823002 (PlepA) + RFP in pSBBs1C
+
-
<br/>
+
-
- Test of every pSBBs vector : BBa_K823021 (pSBBS1C-lacZ), BBa_K823022 (pSBBS4S), BBa_K823023 (pSBBS1C), minipreps and cryopreservation of the best clones
+
-
<br/>
+
-
- Checking of BBA_1364002 (GAFP1)  + BBa_B0015 (double terminator) transformants (biobrick BBa_K1364007) and BBA_1364003 (D4E1)  + BBa_K823003 (Pveg) biobrick BBa_K1364009
+
-
<br/>
+
-
- Cloning of BBA_1364003 (D4E1)  + pSBBS4S (BBa_K823022) and subculture of the colonies
+
-
<br/>
+
-
- Cloning of BBa_K823003 (Pveg) + BBA_1364003 (D4E1)  with BBa_B0015 (double terminator)
+
-
<br/>
+
-
- Cloning of BBa_K823003 (Pveg) + RFP (BBa_K1364017)  in pSBBS4S (BBa_K823022), subculture and minipreps
+
-
<br/>
+
-
- Cloning of BBa_K823002 (PlepA) + RFP (BBa_K1364016)  in pSBBs4S (BBa_K823022), subculture and minipreps
+
-
<br/>
+
-
- Cloning of BBa_1364018 (Strong Promotor + RFP)
+
-
<br/>
+
-
- Cloning of BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) + Promotor BBa_K823003 (Pveg) biobirck BBa_K1364012 with gel extraction for BBA_1364003 (D4E1) + BBA_1364002 (GAFP1)  and a PCR cleanup for BBa_K823003 (Pveg), subculture
+
-
<br/>
+
-
- Cloning BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBS4S in E. coli
+
-
<br/>
+
-
- Transformation of binding gene with E. coli competent cells
+
-
<br/>
+
-
- Transformation of BBa_K1364000, the chemotaxis gene in E. coli
+
-
<br/>
+
-
Problem: the transformation worked but a cell layer appeared. A new subculture of the colonies was made as well as a new spreading on petri dishes.
+
-
<br/>
+
-
- Spreading of BBa_K1162001 (EcAMP), miniprep and digestion
+
-
<br/>
+
-
Problem: the digestion did not work  Issue with the quantity of DNA in the miniprep is assumed
+
-
<br/>
+
-
- First experience of chemotaxis with a glucose chemoattractant
+
-
<br/>
+
-
<p>
+
-
<CENTER> <I> Weekly meeting with the instructors(08/01/2014)</CENTER> </I>
+
-
 Possible problem with the restriction enzymes regarding the digestion: new recommendations
+
-
<br/>
+
-
 Discussion about EcAMP cloning which presents some issues
+
-
<br/>
+
-
 Discussion about the transfer of pSB1C3 from E. coli to Bacillus strain
+
-
<br/>
+
-
</p>  
+
   
   
-
<p>
 
-
<br/>
 
-
<FONT SIZE= 2%> 
 
-
<div class="Sub_title"> Week 8 (04-10 August)</div>
 
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
 
-
<br/> </FONT>
 
-
<p>
 
-
<B> Communication and financial support: </B>
 
-
<br/>
 
-
<p>
 
-
- Participation at the “Passion Jeune” competition organized by a French bank foundation named  Fondation Crédit Agricole Toulouse
 
-
<br/>
 
-
<p>
 
-
<B> Lab work: </B>
 
-
<br/>
 
-
<p>
 
-
- Check the cloning of BBa_K823003 (Pveg)+BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBs4S and cryopreservation
 
-
<br/>
 
-
- Transformation of BBa_K823003 (Pveg) +BBA_1364002 (GAFP1) + BBa_B0015 (double terminator)  (biobrick BBa_ K1364008) in BBa_K823022 (pSBBS4S)
 
-
<br/>
 
-
- Cloning of BBa_K1364001, the binding gene + BBa_K823003 (Pveg) on pSB1C3 and pSBBS 4S
 
-
<br/>
 
-
Problem: the band is at 1500 bp instead of 1300 bp on the gel
 
-
<br/>
 
-
- Cloning of BBa_K1162001 (EcAMP) + BBa_K823003 (Pveg) + BBa_K606061 (RBS SpoVG), miniprep, PCR and digestion
 
-
<br/>
 
-
Problem: the fragment of DNA is too small (149 bp) to be seen on the gel and was probably lost during the PCR cleanup. Instead of that, we used the heat enzymes inactivation at 95°C for 15 minutes.
 
-
<br/>
 
-
- Cloning of Promotor + BBA_1364003 (D4E1)  + BBA_1364002 (GAFP1) in pSB1C3 and pSBBS4S and cryopreservation
 
-
<br/>
 
-
- Elaboration of an efficient fungicide test protocol
 
-
<br/>
 
-
- Test of the fungus growth on PDA medium + test of growth for Bacillus subtilis liquid culture on PDA
 
-
<br/>
 
-
<p>
 
-
<CENTER> <I> Weekly meeting with the instructors(08/08/2014)</CENTER> </I>
 
-
 Ask iGEM headquarters if all the biobricks must be on pSB1C3 plasmid
 
-
<br/>
 
-
 Use another strain of E. coli (DH5-1 instead of DH5 alpha) to have a faster growth
 
-
<br/>
 
-
 Check which quantity of Bacillus subtilis is necessary to naturally destroy the fungus
 
-
<br/>
 
-
<p>
 
-
<br/>
 
-
<FONT SIZE= 2%> 
 
-
<div class="Sub_title"> Week 9 (11-17 August)</div>
 
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
 
-
<br/> </FONT>
 
-
<p>
 
-
<B> Communication and financial support: </B>
 
-
<br/>
 
-
<p>
 
-
- Publication of more articles about our project in Labiotech, Développement durable (Networkvisio), Midi Libre, l’Indépendant, DigiSchool, La voix du Midi Lauragais, LURIO Addl.
 
-
<br/>
 
-
<p>
 
-
<B> Lab work: </B>
 
-
<br/>
 
-
<p>
 
-
- Evaluation of the bacterial decrease rate from 10°C to 4°C with a sporuling strain of Bacillus subtilis.
 
-
<br/>
 
-
- Chemotaxis test with different concentration of glucose on a 0.3% agar.
 
-
<br/>
 
-
- Miniprep of pSBbs4S with binding gene and transformation in Bacillus subtilis.
 
-
<br/>
 
-
Problem: no digestion was visible on the gel  the cloning failed
 
-
<br/>
 
-
- Transformation of BBa_K1374010 (Pveg + RBS SpoVG + EcAMP) + BBa_K1364012 (RBS + Gafp1 + D4E1 + double terminator), subculture
 
-
<br/>
 
-
- Subculture of Bacillus subtilis clones transformed by BBa_K823003 (Pveg) + BBA_1364002 (GAFP1)  + BBa_B0015 (double terminator) in pSBBs4S, cryopreservation
 
-
<br/>
 
-
 Fungicide test
 
-
<p>
+
<p class="title2">3.  Cloning GAFP1+terminator(B0015) = K1364007 (pSB1C3)</p>
-
<br/>
+
-
<FONT SIZE= 2%> 
+
-
<div class="Sub_title"> Week 10 (18-24 August)</div>
+
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
-
<br/> </FONT>
+
-
<p>
+
-
<B> Communication and financial support: </B>
+
-
<br/>
+
-
<p>
+
-
- Ethics: Interview with a specialist in ethical sciences, Vincent Grégoire Delory to complete our reflexion about the ethical issues in SubtiTree project.
+
-
<br/>
+
-
- Wiki: hiring a web designer, Adrien Nicod, to improve the aesthetic effect and the logo.
+
-
<br/>
+
-
<p>
+
-
<B> Lab work: </B>
+
-
<br/>
+
-
<p>
+
-
- Subculture and transformation in Bacillus subtilis of BBa_K1364011 in pSBBS4S (BBa_K823022) and cloning of BBa_K1364011 on pSBBS1C (BBa_K823023).
+
-
<br/>
+
-
Problem: no clone had the insert on pSBBS1C  the cloning had to be done again
+
-
- Miniprep of BBa_K1364011 in BBa_K823023 (pSBBS1C) + Transformation in B. subtilis.
+
-
<br/>
+
-
- Cloning of BBA_1364003 (D4E1) , BBA_1364002 (GAFP1) , BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) on BBa_K823023 (pSBBS1C)
+
-
<br/>
+
-
- Transformation BBa_1364004 (in pSBBS4S in E.coli
+
-
<br/>
+
-
- Cloning of BBa_K1364014 (Pveg + RBS SpoVG + EcAMP + GAFP1 +D4E1 + double terminator) in K823022 (pSBBS4S) and in BBa_K823023 (pSBBS1C), miniprep and digestion by restriction enzymes to see the size of the insert on a 1% gel
+
-
<br/>
+
-
Problem: no clone had the insert on pSBBS1C  the cloning had to be done again
+
-
<br/>
+
-
<p>
+
<p class="title3">Digestion of GAFP1 on pEX-A2 and B0015 (terminator on pSB1C3)
-
<br/>
+
<p class="texte"><b>Date:</b> 07/24/2014
-
<FONT SIZE= 2%> 
+
-
<div class="Sub_title"> Week 11 (25- 31 August)</div>
+
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
-
<br/> </FONT>
+
-
<p>
+
-
<B> Communication and financial support: </B>
+
-
<br/>
+
-
<p>
+
-
- Beginning of the creation and redaction of the different parts of the wiki by the Toulouse iGEM Toulouse
+
-
<br/>
+
-
<p>
+
<p class="title3">Ligation of GAFP1 in B0015 (terminator on pSB1C3)</p>
-
<B> Lab work: </B>
+
<p class="texte"><b>Date:</b> 07/24/2014</p>
-
<br/>
+
 
-
<p>
+
<p class="title3">Transformation of ligation products in <i>E.coli</i></p>
-
- Cloning of BBa_K1364014 in BBa_K823022 and in BBa_K823023
+
<p class="texte"><b>Date:</b> 07/24/2014</p>
-
<br/>
+
 
-
- Cloning GAFP1 + BBa_K823023 (pSBBS1C) in Bacillus subtilis with a change in the protocol: pre-induction of the culture with 0.125 µg/ml of chloramphenicol one hour after the addition of DNA, and let grow for another hour.
+
<p class="title3">Culture of 6 clones: A, B, C, D, E, F transformed in <i>E. coli</i></p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/26/2014</p>
-
Problem: no colony
+
 
-
<br/>
+
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
-
- Liquid culture of Bacillus + BBA_1364002 (GAFP1)  ;  Bacillus + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1)  ;  Bacillus + BBA_1364003 (D4E1)  for future fungicide tests
+
<p class="texte"><b>Date:</b> 07/28/2014</p>
-
<br/>
+
 
-
- Miniprep of BBa_ K1364011 in BBa_ K823023 (pSBBs1C) and transformation in Bacillus subtilis strain.
+
<p class="title3">PCR of GAFP1+B0015 = K1364007 (pSB1C3) A, B, C, D, E, F + electrophoresis</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/28/2014
-
- Fungicide test of BBa_K1364011 in pSBBs4S,  Promotor  + BBA_1364002 (GAFP1)  + Terminator (biobrick BBa_K1364008) in pSBBS4S, Promotor  + BBA_1364003 (D4E1)  + Terminator  (biobrick BBa_K1364009) in pSBBS4S, Promotor + BBA_1364002 (GAFP1) + BA_1364003 (D4E1)  + Terminator ( biobrick BBa_K1364013) in pSBBS4S
+
<br><b>Result:</b> clones B, C, D, E, F seem to have the expected construction.</p>
-
<br/>
+
 
-
- Transformation of a new vector PKL 190 (threonine integrative) in Bacillus strain
+
<p class="title3">Digestion of clones E, F of GAFP1+Ter B0015 = K1364007 with EcoRI and PstI + electrophoresis</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/28/2014
-
<p>
+
<br><b>Result:</b> clones B, C, D, E, F have the expected construction.</p>
-
<CENTER> <I> Weekly meeting with the instructors(08/29/2014)</I> </CENTER>  
+
-
 Boston accommodation must be booked
+
-
<br/>
+
-
 Discussion about the contamination seen on the fungicide tests : the issue seems to come from the sterile water which contained contaminants
+
-
<br/>
+
-
 Objectives : chemotaxis, binding and fungicides tests have to be performed again
+
-
<br/>
+
-
</p>  
+
   
   
-
<div class="Sub_title"> September 2014 </div>
 
-
<table width="100%"><tr><td bgColor="#097F09" height="1px"></td> </tr></table>
 
-
<br/>
 
-
<p>
 
-
Main activites
 
-
<br/>
 
-
- Finish the tests of the different modules
 
-
<br/>
 
-
- Establish the final editorial parts for the wiki
 
-
<br/>
 
-
- Design of the wiki
 
-
<br/>
 
-
- Sequencing the different assembled parts of our bacterium
 
-
<br/>
 
-
</p>
 
-
<p>
 
-
<br/>
 
-
<FONT SIZE= 2%> 
 
-
<div class="Sub_title"> Week 12 (1-7 September)</div>
 
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
 
-
<br/> </FONT>
 
-
<p>
 
-
<B> Communication and financial support: </B>
 
-
<br/>
 
-
<p>
 
-
- Different parts of the wiki by the Toulouse iGEM Toulouse
 
-
<br/>
 
-
-  Reservation of Boston accommodation
 
-
<br/>
 
-
<p>
 
-
<B> Lab work: </B>
 
-
<br/>
 
-
<p>
 
-
- Chemotaxis test and modelling
 
-
<br/>
 
-
- Fungicides tests on EcAMP-A,  EcAMP-B, EcAMP-C, EcAMP-D, MaxiOpA, MaxiOpB, MaxiOpC, MaxiOpD from 2ml of overnight cultures. Only with 25000 conidia with both fungi.
 
-
<br/>
 
-
- PCR on pSBBS4S + BBa_K1162001 (EcAMP)
 
-
<br/>
 
-
Problem: failed on both diluted plasmid and colony
 
-
<br/>
 
-
- Spreading of BBa_K1364014 + BBa_ K823022 in threonine medium and in medium without threonine
 
-
<br/>
 
-
<CENTER> <I> Weekly meeting with the instructors(09/05/2014)</I> </CENTER>
 
-
<br/>
 
-
<br/> The first sequencing was not successful  new sequencing with one primer/tube
 
-
<br/> Chemotaxis test: increase the concentration in bacterium, try a new protocol by capillarity
 
-
<br/> End of the binding test: IT WORKS PERFECTLY! SubtiTree has the good phenotype and presents a better binding property than the wild type Bacillus subtilis strain.
 
-
<br/> Fungicides test: contaminants with the fungus. Need to ask for another culture of our fungi.
 
-
<p>
+
<p class="title2">4. Cloning GAFP1+terminator with Pveg on pSB1C3 (K1364008) in <i>E. coli</i></p>
-
<br/>
+
-
<FONT SIZE= 2%> 
+
-
<div class="Sub_title"> Week 13 (8- 14 September)</div>
+
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
-
<br/> </FONT>
+
-
<p>
+
-
<B> Communication and financial support: </B>
+
-
<br/>
+
-
<p>
+
-
- Telephone interview for an article in a French national review named Fluvial which will be published at the end of September.
+
-
<br/>
+
-
<p>
+
-
<B> Lab work: </B>
+
-
<br/>
+
-
<br/>- PCR on D4E1 colony with different primers
+
-
<br/>- Culture and miniprep of EcAMP to get more DNA + analytic digestion
+
-
<br/>- Transformation of K1364014+K1364006 in E. coli  
+
-
<br/>- Transformation of GAFP1 + D4E1, GAFP1 and  EcAMP+GAFP1+D4E1 on Bacillus subtilis + colony PCR + threonine test
+
-
<br/>NB : good results for the maxi operon and GAFP1 but not for GAFP1 + D4E1 CA
+
-
<br/>- Cloning of K606013+pEX_K4, 823022+PlepA_RFP, 823022+ Pveg_RFP
+
-
<br/>- Chemotaxis tests: different times of LB + agar addition in the LB medium (after 3 minutes, 5 minutes, 8 minutes, 10 minutes…) are performed  No difference of the results after spreading on the plates.
+
-
<br/>- New test of chemotaxis: capillary between two Eppendorf tubes
+
-
<br/>- Spreading of the fungi on a medium which mimics the sap
+
-
<CENTER> <I> Weekly meeting with the instructors(09/11/2014)</I> </CENTER>  
+
<p class="title3">Digestion of GAFP1+ter = K1364007 on pSB1C3 and K823003 (terminator on pSB1C3) + electrophoresis</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/29/2014</p>
-
<br/> Try to use the GFP or RFP to follow the chemotaxis test.
+
-
<br/> Make a new glass process for the chemotaxis capillary test.
+
-
<br/> Possibility of contacting an INRA laboratory to get pictures of the binding test with confocal microscope.
+
-
<br/> Sequencing the binding construction.
+
-
<br/> Try different temperatures for the fungicide tests: 20°C, 25°C, and 37°C to reduce the fungus growth.
+
-
<p>
+
<p class="title3">Gel extraction of K1364007 (extraction of GAFP1+ter gene)</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/29/2014</p>
-
<FONT SIZE= 2%> 
+
-
<div class="Sub_title"> Week 14 (15 - 21 September)</div>
+
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
-
<br/> </FONT>
+
-
<p>
+
-
<B> Communication and financial support: </B>
+
-
<br/>
+
-
<p>
+
-
<br/>-      Support of two French city halls: Mairie de Montréal, Mairie de Ventenac en Minervois.
+
-
<br/>- First interview by the French national radio France Inter which will be aired on October 12th.
+
-
<br/>- Beginning of the organization of the iGEM Toulouse 2014 acknowledgement day: the purpose is to present the project to the students and give a feedback on the competition to the sponsors.
+
-
<br/>- Final design of the wiki
+
-
<br/>
+
-
<p>
+
-
<B> Lab work: </B>
+
-
<br/>
+
-
<br/>- Threonine tests for the BBa_K1364014 maxi operon (EcAMP1 – GAFP1 – D4E1) and mini operon BBa_K1364013 (GAFP1-D4E1)
+
-
<br/>- Colony PCR on the mini operon BBa_K1364013 (GAFP1 - D4E1)
+
-
<br/>- Colony PCR on BBa_K1364011 on BBa_K823022 (pSBBS4S)
+
-
<br/>- Chemotaxis test with wild type Bacillus subtilis strain using Imperial College protocol
+
-
<br/>- Tansformation of BBa_K1364011 + BBa_K823022 (pSBBS4S) in Bacillus subtilis
+
-
<br/>- Culture of fungi with fungicides
+
-
<br/>- Sequencing of plasmids containing the fungicides
+
 +
<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>
-
<CENTER> <I> Weekly meeting with the instructors(09/18/2014)</I> </CENTER>  
+
<p class="title3">Transformation of ligation products in <i>E.coli</i></p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/29/2014</p>
-
<br/> Sequencing worked for D4E1 and GAFP1 and the mini operon BBa_K1364013 (GAFP1-D4E1).
+
-
<br/> The next step is to sequence the maxi operon (EcAMP1 – GAFP1 – D4E1).
+
-
<br/> Try different approaches for the chemotaxis:
+
-
<br/>- New protocol with the capillary assay with a new system made by a glassblower.
+
-
<br/>- Make the Imperial College test again by testing 3 different solutions: chitine, N-acetylglucosamine and another carbon source.
+
-
<br/> Decrease the number of conidia and temperature for the fungicide test.
+
-
<p>
+
<p class="title3">Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 07/30/2014</p>
-
<FONT SIZE= 2%> 
+
-
<div class="Sub_title"> Week 15 (22 - 28 September)</div>
+
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
-
<br/> </FONT>
+
-
<p>
+
-
<B> Communication and financial support: </B>
+
-
<br/>
+
-
<p>
+
-
<br/>- New support of Sallèles d’Aude city hall.
+
-
<br/>- Payment of the plane tickets.
+
-
<br/>- Acknowledgement day (4th December): contact with catering service, reservation of the amphitheater, list of guests…
+
-
<br/>- Order of the T shirt for the Jamboree! We look awesome with them 
+
-
+
-
<br/>
+
-
<p>
+
-
<B> Lab work: </B>
+
-
<br/>
+
-
<br/>- Last experiments regarding chemotaxis tests.
+
 +
<p class="title3">QIAprep Spin Miniprep Kit Using a Microcentrifuge</p>
 +
<p class="texte"><b>Date:</b> 07/31/2014</p>
-
<CENTER> <I> Weekly meeting with the instructors(09/26/2014)</I> </CENTER>  
+
<p class="title3">PCR of GAFP1+B0015 + K823003 = K1364008 (pSB1C3) A, B, C, D, E, F, G, H + electrophoresis</p>
-
<br/>
+
<p class="texte"><b>Date:</b> 31/07/2014
-
<br/> Focus on the wiki design and writing.
+
<br><b>Result:</b> clones A, B, C, D, E, G, H seem to have the expected construction</p>
-
<br/> Start making the power point presentation for Boston and the poster.
+
-
<br/> Division of responsibilities for all the non-scientific work.
+
-
<br>
+
<p class="title3">Digestion of clones A, B, C, D, E, G, H of Pveg+K1364007 = K1364008 with EcoRI and PstI + electrophoresis</p>
-
<p>
+
<p class="texte"><b>Date:</b> 31/07/2014
-
<div class="Sub_title"> October 2014 </div>
+
<br><b>Result:</b> clones A, B, C, D, E, G, H have the expected construction</p>
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
-
<br/>
+
-
<p>
+
-
Main activites
+
-
<br/> - Preparation of the wiki every day… all day long…. so hard to work on this even at night!
+
-
<br/>- Preparation of the poster and the presentation for the Jamboree with our instructors
+
-
<br/>
+
-
- Daily presentations in front of the laboratory teachers to repeat again and again and again… let’s get ready for the Jamboree!
+
-
<br/>
+
-
</p>
+
-
      </div>
+
 
 +
<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>
 +
 
 +
 
 +
 
 +
<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>
 +
</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>
 +
 
 +
<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="clear"></div>
 +
</div>
     </div>
     </div>
   </div>
   </div>

Latest revision as of 01:22, 18 October 2014

           
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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"