Team:INSA-Lyon/Notebook

From 2014.igem.org

(Difference between revisions)
 
(28 intermediate revisions not shown)
Line 2: Line 2:
<html>
<html>
<head>
<head>
-
<link rel="stylesheet" type="text/css" href="https://2014.igem.org/Team:INSA-Lyon/css_contenu?action=raw&ctype=text/css" />
+
<link rel="stylesheet" type="text/css" href="https://2014.igem.org/Team:INSA-Lyon/css_soustitre?action=raw&ctype=text/css" />
</head>
</head>
-
<div id="contenu" style="background-image: url('https://static.igem.org/mediawiki/2014/d/de/Fond_wetlab.jpg');">
+
<div id="soustitre" style="background-image: url('https://static.igem.org/mediawiki/2014/d/de/Fond_wetlab.jpg');">
-
<div id="igem"><a href="https://2014.igem.org/Main_Page"><img src="https://static.igem.org/mediawiki/2014/2/23/Insa_igem.png" alt="IGEM" /></a>
+
<div id="igem">
 +
<a href="https://2014.igem.org/Main_Page"><img src="https://static.igem.org/mediawiki/2014/2/23/Insa_igem.png" alt="IGEM" /></a>
</div>
</div>
-
 
<h4>NOTEBOOK</h4>
<h4>NOTEBOOK</h4>
-
</div>
+
<div id="icones">
 +
<ul>
 +
<a href="https://2014.igem.org/Team:INSA-Lyon/Biology" class="hu-icon"><li class="iconmulti">WETLAB SUMMARY</li></a>
 +
<a href="https://2014.igem.org/Team:INSA-Lyon/Results" class="hu-icon"><li class="icon">RESULTS</li></a>
 +
<a href="https://2014.igem.org/Team:INSA-Lyon/Notebook" class="hu-icon"><li class="icon">NOTEBOOK</li></a>
 +
<a href="https://2014.igem.org/Team:INSA-Lyon/Protocol" class="hu-icon"><li class="icon">PROTOCOLS</li></a>
 +
<a href="https://2014.igem.org/Team:INSA-Lyon/DataPage" class="hu-icon"><li class="icon">DATA PAGE</li></a>
 +
</ul>
 +
</div>
 +
</div>
<div id="presentation">
<div id="presentation">
 +
<p>
 +
Here you can find a summary of all the experiments we made throughout the project.
 +
</p>
 +
<p> To download a pdf with plasmids' description, resistance and names click <a href="https://static.igem.org/mediawiki/2014/9/98/Plasmids.pdf" target="_blank">here</a>
 +
</p>
 +
<p> To download a pdf with the strains' description, resistance and names click <a href="https://static.igem.org/mediawiki/2014/1/14/Strains.pdf" target="_blank">here</a>
 +
</p>
-
         
+
</br>
-
 
+
-
 
+
<ul style="list-style-type: none !important;">
<ul style="list-style-type: none !important;">
-
     <li><a href="#contenu1" onclick="$('#contenu1').slideToggle('slow')"><h1>February-Mai</h1></a><hr/></li>
+
     <li><a href="#contenu1" onclick="$('#contenu1').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />February-May</h1></a><hr/></li>
           <ul id="contenu1" style="list-style-type: none !important;display:none;">
           <ul id="contenu1" style="list-style-type: none !important;display:none;">
-
               <li><p>Literature searches were performed about Peptide Display, Curli biogenesis and metal trapping.
+
               <li><p>Literature searches were performed about Peptide Display, Curli biogenesis and metal trapping.</p></li>
 +
          </ul>
-
</p></li></ul>
 
-
         
+
     <li><a href="#contenu2" onclick="$('#contenu2').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />June</h1></a><hr/></li>
-
 
+
           <ul id="contenu2" style="list-style-type: none !important;display:none;">
-
          </ul>
+
-
     <li><a href="#contenu3" onclick="$('#contenu3').slideToggle('slow')"><h1>June</h1></a><hr/></li>
+
-
           <ul id="contenu3" style="list-style-type: none !important;display:none;">
+
               <li><p>
               <li><p>
-
 
-
 
<h6>11/06 </h6>
<h6>11/06 </h6>
Line 65: Line 74:
<br/>
<br/>
<p> Extraction of the different plasmids with mini prep ABC protocol.</p>
<p> Extraction of the different plasmids with mini prep ABC protocol.</p>
-
<br/>
+
<br/></p></li>
 +
          </ul>
-
</p></li></ul>
+
     <li><a href="#contenu3" onclick="$('#contenu3').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />July</h1></a><hr/></li>
-
 
+
-
 
+
-
 
+
-
          </ul>
+
-
     <li><a href="#contenu3" onclick="$('#contenu3').slideToggle('slow')"><h1>July</h1></a><hr/></li>
+
           <ul id="contenu3" style="list-style-type: none !important;display:none;">
           <ul id="contenu3" style="list-style-type: none !important;display:none;">
               <li><p>
               <li><p>
-
 
-
 
 
-
 
<h6>1/07 </h6>
<h6>1/07 </h6>
<br/>
<br/>
Line 221: Line 223:
<br/>
<br/>
-
<h6>23/05 </h6>
+
<h6>23/07 </h6>
<br/>
<br/>
<p>DNA extraction with ABC mini prep of 24 c transformed clones with pHC25 ligations: few amounts of DNA were extracted because cultures were incubated for 7 hours and only 1,5mL of them were used for mini prep. </p>
<p>DNA extraction with ABC mini prep of 24 c transformed clones with pHC25 ligations: few amounts of DNA were extracted because cultures were incubated for 7 hours and only 1,5mL of them were used for mini prep. </p>
Line 290: Line 292:
<br/>
<br/>
<br/>
<br/>
 +
</p></li>
 +
          </ul>
-
 
+
     <li><a href="#contenu4" onclick="$('#contenu4').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />August</h1></a><hr/></li>
-
</p></li></ul>
+
           <ul id="contenu4" style="list-style-type: none !important;display:none;">
-
 
+
-
 
+
-
 
+
-
          </ul>
+
-
     <li><a href="#contenu3" onclick="$('#contenu3').slideToggle('slow')"><h1>August</h1></a><hr/></li>
+
-
           <ul id="contenu3" style="list-style-type: none !important;display:none;">
+
               <li><p>
               <li><p>
-
 
-
 
-
 
<h6>1/08  </h6>
<h6>1/08  </h6>
<br/>
<br/>
Line 515: Line 510:
<p> New tests with UV light to kill bacteria : 5 LB plates were spotted after exposure of 1,3,5 and 7 min to UV and genomic DNA was extracted from 50µL of each culture by heating 5min at 100°C and centrifuging 5 min at 14000 rpm.</p>
<p> New tests with UV light to kill bacteria : 5 LB plates were spotted after exposure of 1,3,5 and 7 min to UV and genomic DNA was extracted from 50µL of each culture by heating 5min at 100°C and centrifuging 5 min at 14000 rpm.</p>
<p> Electrophoresis gel revealed no bands, more DNA had to be dropped off on gel.</p>
<p> Electrophoresis gel revealed no bands, more DNA had to be dropped off on gel.</p>
-
<p> Characterization of Curli promoter : Miniprep of pIG57 for future digestions and ligations.</p>
+
Characterization of Curli promoter : Miniprep of pIG57 for future digestions and ligations.</p>
<br/>
<br/>
Line 527: Line 522:
<p> Characterization of Curli promoter : digested pIG57 (E + X), ligated to PC750 (E + S) or PC70 (E + S) and then transformed in Kanamycin plates.</p>
<p> Characterization of Curli promoter : digested pIG57 (E + X), ligated to PC750 (E + S) or PC70 (E + S) and then transformed in Kanamycin plates.</p>
<p> Culture of s225, s226+His1, s226+His2, s228, s229 for epifluorescence observation and immunocytochemistry. Culture of the same strains in 96 wells plates for Immunocytochemistry and for ThioflavineS staining.</p>
<p> Culture of s225, s226+His1, s226+His2, s228, s229 for epifluorescence observation and immunocytochemistry. Culture of the same strains in 96 wells plates for Immunocytochemistry and for ThioflavineS staining.</p>
-
 
+
<p>Ni-DMG test at normal pH</p>
<br/>
<br/>
Line 545: Line 540:
<p> Mesure of the fluorescence and OD of the 96 wells plate with ThS with the Tecan with or without biofilm resuspension.</p>
<p> Mesure of the fluorescence and OD of the 96 wells plate with ThS with the Tecan with or without biofilm resuspension.</p>
<p> Culture of s225 and s226His2.</p>
<p> Culture of s225 and s226His2.</p>
 +
<p> Culture of each strain for icp-ms nickel test pH</p>
<br/>
<br/>
Line 559: Line 555:
<p> Culture of s204, s227, His1, His2, WT and CsgB- pkkCsgD for MET observation.</p>
<p> Culture of s204, s227, His1, His2, WT and CsgB- pkkCsgD for MET observation.</p>
<p>Culture of s225, s226+His2 in 96 wells plate. </p>
<p>Culture of s225, s226+His2 in 96 wells plate. </p>
 +
<p> ICP-MS nickel test at 3 concentrations</p>
<br/>
<br/>
Line 567: Line 564:
<p> Immuno staining with the primary antibody (without blocking) overweekend (anti His tag and anti Curli).</p>
<p> Immuno staining with the primary antibody (without blocking) overweekend (anti His tag and anti Curli).</p>
<p> Characterization of Curli promoter : Digestion (EcoRI + PstI) of the following miniprep samples. Letters have a double digestion, numbers have a single digestion and T are the controls for non-digestion:</p>
<p> Characterization of Curli promoter : Digestion (EcoRI + PstI) of the following miniprep samples. Letters have a double digestion, numbers have a single digestion and T are the controls for non-digestion:</p>
-
<br/>
 
<br/>
<br/>
 +
</p></li>
 +
          </ul>
-
</p></li></ul>
+
     <li><a href="#contenu5" onclick="$('#contenu5').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />September</h1></a><hr/></li>
-
 
+
           <ul id="contenu5" style="list-style-type: none !important;display:none;">
-
 
+
-
 
+
-
          </ul>
+
-
     <li><a href="#contenu3" onclick="$('#contenu3').slideToggle('slow')"><h1>September</h1></a><hr/></li>
+
-
           <ul id="contenu3" style="list-style-type: none !important;display:none;">
+
               <li><p>
               <li><p>
-
 
-
 
-
 
-
<h5>September </h5>
 
<br/>
<br/>
<p> <u>Dry lab</u>: Worked on a paper where a model for CsgB in vitro polymerisation was proposed : " A Kinetic Study of Amyloid Formation : Fibril Growth and Length Distributions" by John S. Schreck and Jian-Min Yuan.</p>
<p> <u>Dry lab</u>: Worked on a paper where a model for CsgB in vitro polymerisation was proposed : " A Kinetic Study of Amyloid Formation : Fibril Growth and Length Distributions" by John S. Schreck and Jian-Min Yuan.</p>
Line 635: Line 624:
<p> Immunolabeling with 2 antibodies and observation with the epifluorescence microscope.</p>
<p> Immunolabeling with 2 antibodies and observation with the epifluorescence microscope.</p>
<p> Red Congo test on DH5α and 326 in batch.</p>
<p> Red Congo test on DH5α and 326 in batch.</p>
 +
<p> <p>ICP-MS nickel results</p></p>
<br/>
<br/>
Line 742: Line 732:
<p> => All transformation were successful except for 1171 and 206 where negative control was positive. Maybe due to a problem in the strain stored.</p>
<p> => All transformation were successful except for 1171 and 206 where negative control was positive. Maybe due to a problem in the strain stored.</p>
<br/>
<br/>
-
</p></li></ul>
 
 +
</p></li>
 +
          </ul>
-
          </ul>
+
     <li><a href="#contenu6" onclick="$('#contenu6').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />October</h1></a><hr/></li>
-
     <li><a href="#contenu3" onclick="$('#contenu3').slideToggle('slow')"><h1>October</h1></a><hr/></li>
+
           <ul id="contenu6" style="list-style-type: none !important;display:none;">
-
           <ul id="contenu3" style="list-style-type: none !important;display:none;">
+
               <li><p>
               <li><p>
 +
<h6>2/10 </h6>
 +
<p>
 +
Pre-cultures of the negative control, positive control, PHC46 (P70-GFP) and PHC47(P750-GFP).
 +
</p>
 +
<h6>3/10 </h6>
 +
<p>
 +
We distributed the blank, growth control, positive control for GFP expression, PHC46 (P70-GFP) and PHC47 (P750-GFP) in a 96 well plate and explored the GFP expression at 37 degrees.
 +
</p>
 +
<h6>3/10 </h6>
 +
<p>
 +
We gathered the results at 37°C and interpreted the results of GFP expression from the P70 or P750 long promoter upstream.
 +
</p>
Line 774: Line 776:
<p> Petri dish nickel test on the Petri dishes containing the best clones for each one of the newly transformed strains 224, 266, 267.</p>
<p> Petri dish nickel test on the Petri dishes containing the best clones for each one of the newly transformed strains 224, 266, 267.</p>
<br/>
<br/>
 +
 +
<p>
 +
Pre-cultures of the negative control, positive control, PHC46 (P70-GFP) and PHC47(P750-GFP).
 +
</p>
<h6>8/10  </h6>
<h6>8/10  </h6>
<br/>
<br/>
-
<p>Cultured the best clones for each one of the newly transformed strains 224, 266, 267, 325 and 334 in a 24 wells plate. </p>
+
<p>
 +
Cultured the best clones for each one of the newly transformed strains 224, 266, 267, 325 and 334 in a 24 wells plate.
 +
</p>
 +
 
 +
<p>
 +
We distributed the blank, growth control, positive control for GFP expression, PHC46 (P70-GFP) and PHC47 (P750-GFP) in a 96 well plate and explored the GFP expression at 30 degrees.
 +
</p>
 +
 
<br/>
<br/>
Line 783: Line 796:
<br/>
<br/>
<p> Red congo and adherence tests on the newly transformed strains 224, 266, 267, 325 and 334 cultured in the 24 wells plate.</p>
<p> Red congo and adherence tests on the newly transformed strains 224, 266, 267, 325 and 334 cultured in the 24 wells plate.</p>
 +
 +
<p>
 +
We interpreted and compared the results of GFP expression from the P70 or P750 long promoter upstream.
 +
</p>
 +
 +
<br/>
<br/>
 +
 +
</p></li>
 +
          </ul>
-
</p></li></ul>
 
</ul>
</ul>
 +
 +
 +
 +
 +
 +
 +
</div>
</div>

Latest revision as of 00:55, 18 October 2014

Curly'on - IGEM 2014 INSA-LYON

Here you can find a summary of all the experiments we made throughout the project.

To download a pdf with plasmids' description, resistance and names click here

To download a pdf with the strains' description, resistance and names click here


  • February-May


  • June


  • July


  • August


  • September


  • October