"
Team:Toulouse/Notebook/project-monitoring
From 2014.igem.org
| (11 intermediate revisions not shown) | |||
| Line 157: | Line 157: | ||
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"> | <div class="centering" style="padding-top: 85px; padding-bottom:40px;"> | ||
| - | <p style="font-size:1.3em;"><a href="#" onClick="ddaccordion. | + | <p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p> |
<div class="technology">Binding module</div> | <div class="technology">Binding module</div> | ||
<div class="thelanguage"> | <div class="thelanguage"> | ||
| - | <p class="title2">1. Amplification of Binding Module (pEX-K4) into E.coli</p> | + | <p class="title2">1. Amplification of Binding Module (pEX-K4) into <i>E. coli</i></p> |
| - | <p class="title3">Transformation of Binding module (pEX-K4) into E. coli</p> | + | <p class="title3">Transformation of Binding module (pEX-K4) into <I>E. coli</i></p> |
<p class="texte">Gene "Binding module" synthesized by Eurofins, resuspended in 20μL Tris 10mM | <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> | <br><b>Result:</b> We obtained distinct colonies on plates LB + Kanamycin (50 µg/mL)</p> | ||
| - | <p class="title3">Liquid culture of | + | <p class="title3">Liquid culture of two clones: 1 et 2 (pEX-K4) transformed into <i>E. coli</i></p> |
<p class="texte"><b>Date:</b> 01/08/2014</p> | <p class="texte"><b>Date:</b> 01/08/2014</p> | ||
| - | <p class="title3">Miniprep with QIAprep Spin Miniprep Kit: | + | <p class="title3">Miniprep with QIAprep Spin Miniprep Kit: two clones of Binding Module (pEX-K4) into <i>E. coli</i></p> |
<p class="texte"><b>Date:</b> 04/08/2014 | <p class="texte"><b>Date:</b> 04/08/2014 | ||
<br><b>Result:</b> Binding Module (pEX-K4) obtained</p> | <br><b>Result:</b> Binding Module (pEX-K4) obtained</p> | ||
| Line 177: | Line 177: | ||
<p class="texte"><b>Date:</b> 04/08/2014 | <p class="texte"><b>Date:</b> 04/08/2014 | ||
<br><b>Result:</b> | <br><b>Result:</b> | ||
| - | <br>- 3 bands : 1500bp (vector with Kanamycin resistance), 1300bp (Module Binding) and | + | <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> | <br>- The two Binding Module clones are ok</p> | ||
| - | <p class="title2">2. Cloning Binding Module on pEX-K4 with pSB1C3 (BBa_K606013 without RFP) into E. coli</p> | + | <p class="title2">2. Cloning Binding Module on pEX-K4 with pSB1C3 (<a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a> without RFP) into <i>E. coli</i></p> |
| - | <p class="title3">Digestion Binding Module on pEX-K4 and BBa_K606013</p> | + | <p class="title3">Digestion Binding Module on pEX-K4 and <a href="http://parts.igem.org/Part:BBa_K606013">BBa_K606013</a></p> |
<p class="texte"><b>Date:</b> 23/08/2014 | <p class="texte"><b>Date:</b> 23/08/2014 | ||
| - | <br><b>Expected bands after digestion | + | <br><b>Expected bands after digestion:</b> |
| - | <br>- BBa_K606013 : 860 bp for RFP and 2100 bp for vector pSB1C3 | + | <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>- 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> | <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> | ||
| Line 195: | Line 195: | ||
<p class="texte"><b>Date:</b> 04/08/2014 | <p class="texte"><b>Date:</b> 04/08/2014 | ||
<br>Transformation with 10 µL of the ligation mix and plate on Chloremphenicol LA plate | <br>Transformation with 10 µL of the ligation mix and plate on Chloremphenicol LA plate | ||
| - | <br>Result : many wrong clones</p> | + | <br><b>Result:</b> many wrong clones</p> |
| - | <p class="title2">3. Cloning Binding Module on pEX-K4 with pVeg on pSB1C3 (BBa_K823003) into E. coli</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 BBa_K823003</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 | <p class="texte"><b>Date:</b> 04/08/2014 | ||
| - | <br>BBa_K823003 digested by XbaI and PstI and Binding Module digested by SpeIand PstI | + | <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>Gel Electrophoresis | ||
<br><b>Result:</b> | <br><b>Result:</b> | ||
| - | <br>- Binding Module : 1400 bp for Binding Module and 1500bp+1000bp for vector pEX_K47 | + | <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> | <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="title3">Ligation Binding Module on pVeg with pSB1C3</p> | ||
<p class="texte"><b>Date:</b> 04/08/2014 | <p class="texte"><b>Date:</b> 04/08/2014 | ||
| - | <br>BBa_K823003 digested by XbaI and PstI and Binding Module digested by | + | <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="title3">Transformation of Binding Module on pVeg into <i>E. coli</i></p> | ||
| Line 214: | Line 214: | ||
<br><b>Result:</b> Many good clones (check on 06/08/2014)</p> | <br><b>Result:</b> Many good clones (check on 06/08/2014)</p> | ||
| - | <p class="title2">4. Cloning Binding Module with | + | <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 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 | <p class="texte"><b>Date:</b> 07/08/2014 | ||
| - | <br>BBa_K823022 and Binding Module with | + | <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><b>Result:</b> | ||
<br>- Binding Module with Pveg 1600 bp for Binding Module and 2100bp for vector pSB1C3</p> | <br>- Binding Module with Pveg 1600 bp for Binding Module and 2100bp for vector pSB1C3</p> | ||
| Line 224: | Line 224: | ||
<p class="title3">Ligation Binding Module with Pveg on pSBbs4S</p> | <p class="title3">Ligation Binding Module with Pveg on pSBbs4S</p> | ||
<p class="texte"><b>Date:</b> 07/08/2014 | <p class="texte"><b>Date:</b> 07/08/2014 | ||
| - | <br>BBa_K823022 and Binding Module with | + | <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>Ligation | ||
| - | <br><b>Result:</b> Ligation between Binding Module with | + | <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 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 | <p class="texte"><b>Date:</b> 04/08/2014 | ||
| - | <br>Binding Module with | + | <br>Binding Module with P<sub>veg</sub> on pSB<sub>BS</sub>4S |
<br>Plate on Ampicillin LA plate | <br>Plate on Ampicillin LA plate | ||
<br><b>Result:</b> Many good clones (check on 13/08/2014)</p> | <br><b>Result:</b> Many good clones (check on 13/08/2014)</p> | ||
| - | <p class="title3">Transformation of Binding Module with | + | <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 | <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>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>Result : One good clone : PCR (check on 09/09/2014) and threonine test (check on 09/09/2014)</p> | + | <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="title2">5. Binding Test</p> | ||
| Line 262: | Line 262: | ||
<br><b>Result:</b> 4*50µL of chemotaxis (Puc57) obtained</p> | <br><b>Result:</b> 4*50µL of chemotaxis (Puc57) obtained</p> | ||
| - | <p class="title2">2. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested pSB1C3 with EcoRI and PstI into <i>E. coli</i></p> | + | <p class="title2">2. Cloning chemotaxis <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> (chemotaxis_Puc57) with digested pSB1C3 with EcoRI and PstI into <i>E. coli</i></p> |
| - | <p class="title3">Digestion BBa_K1364000 (chemotaxis_Puc57) with EcoRI and PstI - PCR kit Clean up</p> | + | <p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> (chemotaxis_Puc57) with EcoRI and PstI - PCR kit Clean up</p> |
<p class="texte"><b>Date:</b> 07/08/2014 | <p class="texte"><b>Date:</b> 07/08/2014 | ||
| - | <br><b>Result:</b> Expected band after digestion for BBa_K1364000: 2300 bp | + | <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> | <br><b>Problem:</b> We can't distinguish the vector band (2500 bp)</p> | ||
| - | <p class="title3">Gel extraction of BBa_K1364000</p> | + | <p class="title3">Gel extraction of <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a></p> |
<p class="texte"><b>Date:</b> 07/08/2014</p> | <p class="texte"><b>Date:</b> 07/08/2014</p> | ||
| - | <p class="title3">Ligation BBa_K1364000 and digested | + | <p class="title3">Ligation <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> and digested PSB1C3 with EcoRI and PstI</p> |
<p class="texte"><b>Date:</b> 08/08/2014</p> | <p class="texte"><b>Date:</b> 08/08/2014</p> | ||
| - | <p class="title3">Transformation BBa_K1364000 in E.coli</p> | + | <p class="title3">Transformation <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> in <i>E.coli</i></p> |
<p class="texte"><b>Date:</b> 08/08/2014 | <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> | <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> | ||
| Line 286: | Line 286: | ||
<p class="texte"><b>Date:</b> 11/08/2014</p> | <p class="texte"><b>Date:</b> 11/08/2014</p> | ||
| - | <p class="title3">Digestion BBa_K1364000 on pSB1C3 with EcoRI and PstI</p> | + | <p class="title3">Digestion <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a> on pSB1C3 with EcoRI and PstI</p> |
<p class="texte"><b>Date:</b> 11/08/2014 | <p class="texte"><b>Date:</b> 11/08/2014 | ||
<br><b>Result:</b> There is one colony which presents the right construction.</p> | <br><b>Result:</b> There is one colony which presents the right construction.</p> | ||
| Line 292: | Line 292: | ||
| - | <p class="title2">3. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested BBa_823003 ( | + | <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 BBa_K1364000 and digested BBa_823003 on PsB1C3 with SpeI and PstI</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="texte"><b>Date</b>: 08/08/2014</p> | ||
| - | <p class="title3">Transformation BBa_1364004 in E.coli</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="texte"><b>Date:</b> 8/08/2014</p> | ||
<p class="title3">Test of sensibility on Ampicillin</p> | <p class="title3">Test of sensibility on Ampicillin</p> | ||
| Line 301: | Line 301: | ||
<br><b>Result:</b> We can determine which colony is sensible for ampicillin and know which bacterium is carrying the chemotaxis gene. There is one colony which resists on ampicillin.</p> | <br><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 BBa_K1364004 on pSBC3 with EcoRI and PstI</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 | <p class="texte"><b>Date:</b> 12/08/2014 | ||
<br><b>Result:</b> We did not see any colony with chemotaxis insert.</p> | <br><b>Result:</b> We did not see any colony with chemotaxis insert.</p> | ||
| - | <p class="title2">4. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested BBa_823003 ( | + | <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 BBa_K1364000 and digested BBa_823003 on PsB1C3 with SpeI and PstI</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="texte"><b>Date:</b> 11/08/2014</p> | ||
| - | <p class="title3">Transformation BBa_1364004 in E.coli</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="texte"><b>Date:</b> 11/08/2014</p> | ||
<p class="title3">Test of sensibility on Ampicillin</p> | <p class="title3">Test of sensibility on Ampicillin</p> | ||
| Line 314: | Line 314: | ||
<br><b>Result:</b> we obtained 4 colonies sensible at Ampicilline</p> | <br><b>Result:</b> we obtained 4 colonies sensible at Ampicilline</p> | ||
| - | <p class="title3">Digestion BBa_K1364004 on PsB1C3 with EcoRI and PstI</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="texte"><b>Date:</b> 18/08/2014</p> | ||
| - | <p class="title3">Gel extraction of BBa_K1364000</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="texte"><b>Date:</b> 19/08/2014</p> | ||
| - | <p class="title2">5. Cloning chemotaxis BBa_K1364004 with digested | + | <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 BBa_K1364004 digested EcorI and PstI and digested PsB1C3 with EcoRI and PstI</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="texte"><b>Date:</b> 19/08/2014</p> | ||
| - | <p class="title3">Transformation | + | <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 | <p class="texte"><b>Date:</b> 19/08/2014 | ||
<br><b>Result:</b> We obtained one colony and resuspended it in LB+ Amp</p> | <br><b>Result:</b> We obtained one colony and resuspended it in LB+ Amp</p> | ||
| Line 333: | Line 333: | ||
<div class="thelanguage"> | <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="title2">1. Amplification of synthetic gene (D4E1 on pEX-A2)</p> | ||
| Line 386: | Line 387: | ||
<p class="title3">Transformation of ligation products in <i>E.coli</i></p> | <p class="title3">Transformation of ligation products in <i>E.coli</i></p> | ||
<p class="texte"><b>Date:</b> 07/24/2014 | <p class="texte"><b>Date:</b> 07/24/2014 | ||
| - | <br><b>Result:</b> E.coli transformed by D4E1+K823003</p> | + | <br><b>Result:</b> <i>E.coli</i> transformed by D4E1+K823003</p> |
<p class="title3">Culture of 6 clones: A, B, C, D, E, F of transformed <i>E. coli</i></p> | <p class="title3">Culture of 6 clones: A, B, C, D, E, F of transformed <i>E. coli</i></p> | ||
| Line 402: | Line 403: | ||
<br><b>Result:</b> clones C, D have the expected construction and are placed in cryopreservation.</p> | <br><b>Result:</b> clones C, D have the expected construction and are placed in cryopreservation.</p> | ||
| - | <p class="title2">4. Cloning | + | <p class="title2">4. Cloning P<sub>veg</sub> + D4E1 on pSB<sub>BS</sub>4S (K823022) </p> |
<p class="texte"><b>Date:</b> 08/13/2014</p> | <p class="texte"><b>Date:</b> 08/13/2014</p> | ||
| - | <p class="title2">5. Cloning | + | <p class="title2">5. Cloning P<sub>veg</sub> + D4E1 on pSB<sub>BS</sub>1C lacZ (23)</p> |
| + | |||
| + | <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p> | ||
| + | </div> | ||
| + | |||
| + | <div class="technology2">GAFP1</div> | ||
| + | <div class="thelanguage2"> | ||
| - | |||
<p class="title2">1. Amplification of synthetic gene (GAFP1 on pEX-A2)</p> | <p class="title2">1. Amplification of synthetic gene (GAFP1 on pEX-A2)</p> | ||
| - | <p class="title3">Transformation of GAFP1 (pEX-A2) into E.coli</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 | <p class="texte">Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM | ||
<br>Concentration of GAFP1 : 145ng/µL | <br>Concentration of GAFP1 : 145ng/µL | ||
| Line 428: | Line 434: | ||
<p class="title2">2. Cloning GAFP1 gene on PSB1C3 = K1364002 in E. coli</p> | <p class="title2">2. Cloning GAFP1 gene on PSB1C3 = K1364002 in E. coli</p> | ||
| - | <p class="title3">Digestion GAFP1_pEX-A2 and BBa_K606013 (RFP_pSB1C3)</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 | <p class="texte"><b>Date:</b> 07/23/2014 | ||
| - | <br><b>Result:</b> BBa_K606013 : 860 bp | + | <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 BBa_K606013</p> | + | <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 BBa_K606013</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="texte"><b>Date:</b> 07/23/2014</p> | ||
| Line 493: | Line 499: | ||
<br><b>Result:</b> 20µL ligation of GAFP1+ter in K823003</p> | <br><b>Result:</b> 20µL ligation of GAFP1+ter in K823003</p> | ||
| - | <p class="title3">Transformation of ligation products in E.coli</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="texte"><b>Date:</b> 07/29/2014</p> | ||
| Line 512: | Line 518: | ||
| - | <p class="title2">5. Cloning Pveg+GAFP1+Ter B0015 (BBa_K1364008) on pSBBS4S (BBa_K823022)</p> | + | <p class="title2">5. Cloning Pveg+GAFP1+Ter B0015 (<a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>) on pSBBS4S (<a href="http://parts.igem.org/Part:BBa_K823022">BBa_K823022</a>)</p> |
| - | <p class="title3">Digestion of Pveg+GAFP1+ ter B0015 ( | + | <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="texte"><b>Date:</b> 08/01/2014</p> | ||
| Line 520: | Line 526: | ||
<p class="texte"><b>Date:</b> 08/01/2014</p> | <p class="texte"><b>Date:</b> 08/01/2014</p> | ||
| - | <p class="title3">Transformation of ligation products in E.coli</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="texte"><b>Date:</b> 08/01/2014</p> | ||
| Line 533: | Line 539: | ||
<br><b>Result:</b> clones A, E, F have the right construction</p> | <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="title2">1. Transformation of EcAMP in Escherichia coli MC 1061</p> | + | |
<p class="texte"><b>Date:</b> 07/25/2014</p> | <p class="texte"><b>Date:</b> 07/25/2014</p> | ||
| - | <p class="title2">2. Spreading of coli cells transformed with pUC | + | <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="texte"><b>Date:</b> 07/28/2014</p> | ||
| - | <p class="title2">3. Liquid culture | + | <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="texte"><b>Date:</b> 07/30/2014</p> | ||
| - | <p class="title2">4. Cloning 1: EcAMP + | + | <p class="title2">4. Cloning 1: EcAMP + P<sub>veg</sub> + RBS</p> |
| - | <p class="title3">Digestion of EcAMP ( | + | <p class="title3">Digestion of EcAMP (insert) by XbaI and PstI</p> |
<p class="texte"><b>Date:</b> 07/31/2014</p> | <p class="texte"><b>Date:</b> 07/31/2014</p> | ||
| - | <p class="title3">Digestion of | + | <p class="title3">Digestion of P<sup>veg</sup> + RBS (VECTOR)</p> |
<p class="texte"><b>Date:</b> 07/31/2014</p> | <p class="texte"><b>Date:</b> 07/31/2014</p> | ||
| Line 568: | Line 575: | ||
<p class="texte"><b>Date:</b> 08/05/2014 | <p class="texte"><b>Date:</b> 08/05/2014 | ||
| - | <br>The first cloning show a lack of DNA in the Miniprep EcAMP. The bands are hardly visible on the gel for the linearization of EcAMP + Pveg + RBS. The problem came from the PCR cleanup step: the fragment size of EcAMP is lower than 200bp.</p> | + | <br><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="title2">5. Cloning 2: EcAMP + Pveg + RBS </p> | ||
| - | <p class="texte"><b>Date:</b> | + | <p class="texte"><b>Date:</b> 08/06/2014</p> |
| - | <p class="title3">Digestion of EcAMP ( | + | <p class="title3">Digestion of EcAMP (insert) by XbaI and PstI</p> |
| - | <p class="title3">Digestion of | + | <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="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="title3">Ligation and transformation</p> | ||
| + | <p class="texte"><b>Date:</b> 08/06/2014</p> | ||
<p class="title3">PCR test</p> | <p class="title3">PCR test</p> | ||
| - | <p class="texte"><b>Date:</b> | + | <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="title3">Striation on a petri dish to purify the clone</p> | ||
| - | <p class="texte">Purpose: to isolate a clone with vector+insert | + | <p class="texte"><b>Purpose:</b> to isolate a clone with vector+insert |
<br><b>Date:</b> 08/072014</p> | <br><b>Date:</b> 08/072014</p> | ||
| - | <p class="title3">Miniprep of | + | <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="texte"><b>Date:</b> 08/08/2014</p> | ||
| - | <p class="title3">Ligation of Pveg SpoVG EcAMP with double terminateur B0015 + | + | <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="texte"><b>Date:</b> 08/11/2014</p> | ||
| - | <p class="title3">Miniprep of | + | <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="texte"><b>Date:</b> 08/13/2014</p> | ||
| - | <p class="title3">Cloning K1364011 (EcAMP+ | + | <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="texte"><b>Date:</b> 08/19/2014</p> | ||
| - | <p class="title3">Cloning K1364011 + K823023 ( | + | <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="texte"><b>Date:</b> 08/18/2014</p> | ||
| - | <p class="title3">Verification of the insertion of K1364011 + K823022 ( | + | <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 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="title2">1. Cloning GAFP1+D4E1 on pSB1C3: BBa_K1364012 </p> | + | |
<p class="title3">Digestion of D4E1 on pEX-A2 and GAFP1 on pSB1C3</p> | <p class="title3">Digestion of D4E1 on pEX-A2 and GAFP1 on pSB1C3</p> | ||
| Line 622: | Line 633: | ||
<p class="texte"><b>Date:</b> 07/28/2014</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="title3">PCR of GAFP1 + D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis</p> |
<p class="texte"><b>Date:</b> 07/29/2014 | <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> | <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="title3">Digestion of GAFP1 + D4E1 (pSB1C3) A, C, F, G + electrophoresis</p> |
<p class="texte"><b>Date:</b> 07/30/2014 | <p class="texte"><b>Date:</b> 07/30/2014 | ||
| - | <br><b>Result:</b> clone F (BBa_K1364012) has the expected construction and is placed on cryopreservation.</p> | + | <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 (BBa_K1364012) on | + | <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=" | + | |
| + | <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: BBa_K1364014</p> | + | <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 BBa_K1364014 in | + | <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 | + | <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 | + | <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>Date:</b> 08/11/2014 | ||
| - | <br><b>Result:</b> We obtained digested fragments of | + | <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 | + | <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 | <p class="texte"><b>Date:</b> 08/11/2014 | ||
| - | <br><b>Result:</b> We obtained ligation of K1364012 and ~500 bp fragment of | + | <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 | + | <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="texte"><b>Date:</b> 08/12/2014</p> | ||
| - | <p class="title3">Testing 8 E.coli+ | + | <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="texte"><b>Date:</b> 08/14/2014 </p> | ||
| - | <p class="title3">Testing 15 E.coli+ | + | <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 | <p class="texte"><b>Date:</b> 08/18/2014 | ||
| - | <br><b>Result:</b> clones H, J, O, Q, U, V might have the right | + | <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. BBa_K1364014 in | + | <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 | + | <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 | <p class="texte"><b>Date:</b> 08/22/2014 | ||
| - | <br><b>Result</b>: | + | <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 | + | <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 | <p class="texte"><b>Date:</b> 08/22/2014 | ||
| - | <br><b>Result:</b> We obtained 20µL ligation of K1364014 with | + | <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 E.coli</p> | + | <p class="title3">Transformation of ligations in <i>E. coli</i></p> |
| - | <p class="texte">E. coli transformed by | + | <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">E. coli transformed by | + | <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> | <br><b>Date:</b> 08/25/2014</p> | ||
| - | <p class="title3">Testing E.coli+ | + | <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="texte"><b>Date:</b> 08/27/2014</p> | ||
| - | <p class="title2">3. Transformation of | + | <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 | + | <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 | + | <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> | <br><b>Date:</b> 08/28/2014</p> | ||
| - | <p class=" | + | <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="title2">1. D4E1-GAFP1</p> | ||
| - | <p class="title3">Transformation | + | <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="texte"><b>Date:</b> 08/12/2014</p> | ||
<p class="title3">Integration threonine test + fungicide test </p> | <p class="title3">Integration threonine test + fungicide test </p> | ||
| Line 696: | Line 718: | ||
<p class="title2"2. D4E1</p> | <p class="title2"2. D4E1</p> | ||
<p class="title3"> | <p class="title3"> | ||
| - | Cloning D4E1 into | + | Cloning D4E1 into pSB<sub>BS</sub>1C + fungicide test</p> |
<p class="texte"><b>Date:</b>08/15/2014</p> | <p class="texte"><b>Date:</b>08/15/2014</p> | ||
<p class="title3"> | <p class="title3"> | ||
| Line 707: | Line 729: | ||
<div class="clear"></div> | <div class="clear"></div> | ||
| - | + | </div> | |
</div> | </div> | ||
</div> | </div> | ||
Latest revision as of 01:22, 18 October 2014
Notebook > Project monitoring
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
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
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)
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
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
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
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
Socialize
