Team:Grenoble/Notebook/August

From 2011.igem.org

(Difference between revisions)
Line 13: Line 13:
<div class="noindent">
<div class="noindent">
-
<p>
+
<p>
-
Modifications made on protocols allowed the proper conduct of the experiments even if we didn't get good results.
+
Modifications made on protocols allowed the proper conduct of the experiments even if we didn't get good results.
-
</p>
+
</p>
-
+
-
<ul>
+
-
<li>Sequencing Order :</li>
+
-
<ul>
+
-
<li>RBS-CinI 2</li>
+
-
<li>RBS-CinI 3</li>
+
-
<li>RBS-CinI 5</li>
+
-
<li>MerR-CinR</li>
+
-
<li>MerR-CinR 4</li>
+
-
<li>MerR-CinR 5</li>
+
-
</ul>
+
-
<li>RESULTS :</li>
 
<ul>
<ul>
-
<li>MerR-CinR 5 came back positive from the sequencing</li>
+
<li>Sequencing Order :</li>
-
</ul>
+
-
+
-
<li>Cloning Session (3A Assembly) :</li>
+
-
<ul>
+
-
<li>1st-step constructions :</li>
+
<ul>
<ul>
-
<li>RBS-TetR</li>
+
<li>RBS-CinI 2</li>
-
<li>RBS-LuxR</li>
+
<li>RBS-CinI 3</li>
-
<li>Fha-CinI</li>
+
<li>RBS-CinI 5</li>
-
<li>Fha-CinR</li>
+
<li>MerR-CinR</li>
-
<li>Fha-TetR</li>
+
<li>MerR-CinR 4</li>
-
<li>Fha-LuxI</li>
+
<li>MerR-CinR 5</li>
-
<li>Fha-LuxR</li>
+
-
<p>plasmid vector : pSB1AC3</p>
+
</ul>
</ul>
-
<li>coloration constructions :</li>
+
 +
<li>RESULTS :</li>  
<ul>
<ul>
-
<li>pLux-Lycopene</li>
+
<li>MerR-CinR 5 came back positive from the sequencing</li>
-
<li>pCin-Lycopene</li>
+
-
<p>plasmid vector : pSB1AK3</p>
+
</ul>
</ul>
-
<li>constructions for the tests :</li>
+
 +
<li>Cloning Session (3A Assembly) :</li>
<ul>
<ul>
-
<li>pConst-GFP</li>
+
<li>1st-step constructions :</li>
-
<li>pCin-GFP</li>
+
<ul>
-
<li>pLux-GFP</li>
+
<li>RBS-TetR</li>
-
<li>pTet-GFP</li>
+
<li>RBS-LuxR</li>
-
<li>pLac-GFP</li>
+
<li>Fha-CinI</li>
-
<p>plasmid vector : pSB1AT3</p>
+
<li>Fha-CinR</li>
-
<br/>
+
<li>Fha-TetR</li>
-
<li>pConst-(RBS-CinR)</li>
+
<li>Fha-LuxI</li>
-
<li>pConst-(RBS-LuxR)</li>
+
<li>Fha-LuxR</li>
-
<li>plasmid vector: pSB3C5</li>
+
<p>plasmid vector : pSB1AC3</p>
 +
</ul>
 +
<li>coloration constructions :</li>
 +
<ul>
 +
<li>pLux-Lycopene</li>
 +
<li>pCin-Lycopene</li>
 +
<p>plasmid vector : pSB1AK3</p>
 +
</ul>
 +
<li>constructions for the tests :</li>
 +
<ul>
 +
<li>pConst-GFP</li>
 +
<li>pCin-GFP</li>
 +
<li>pLux-GFP</li>
 +
<li>pTet-GFP</li>
 +
<li>pLac-GFP</li>
 +
<p>plasmid vector : pSB1AT3</p>
 +
<br/>
 +
<li>pConst-(RBS-CinR)</li>
 +
<li>pConst-(RBS-LuxR)</li>
 +
<li>plasmid vector: pSB3C5</li>
 +
</ul>
 +
<li>Digestions :</li>
 +
<p>a gel checking of the restriction results were performed.</p>
 +
<li>Purification :</li>
 +
<p>In order to check and extract the wright insert.<p>
 +
<li>Ligations :</li>
 +
<p> with the ratio : 3X of insert 1X of plasmid</p>
 +
<li>Spreading over Petri dish</li>
 +
<li>Results :</li>
 +
<p>No result on the PCR checking of the colonies.</p>
</ul>
</ul>
-
<li>Digestions :</li>
+
-
<p>a gel checking of the restriction results were performed.</p>
+
<li>Plasmid transfer of Fha: from PCR BlentII to pSB1AT3.</li>
-
<li>Purification :</li>
+
-
<p>In order to check and extract the wright insert.<p>
+
-
<li>Ligations :</li>
+
-
<p> with the ratio : 3X of insert 1X of plasmid</p>
+
-
<li>Spreading over Petri dish</li>
+
-
<li>Results :</li>
+
-
<p>No result on the PCR checking of the colonies.</p>
+
-
</ul>
+
-
+
-
<li>Plasmid transfer of Fha: from PCR BlentII to pSB1AT3.</li>
+
-
<ul>
+
-
<li>Restriction :</li>
+
-
<p>
+
-
Between E and P (same for pSB1AT3). No need of purification because PCR BlentII is Kanamycin resistant whereas the selection of pSB1AT3 is made on Tetracycline.
+
-
</p>
+
-
<li>Ligation</li>
+
-
<li>Spreading over Petri Dish</li>
+
-
<li>Results :</li>
+
-
<p>PCR result: successful transfer.</p>
+
-
</ul>
+
-
+
-
<li>Cloning Session (Standard Assembly):<br/>
+
-
<ul>
+
-
<li>1<sup>st</sup>-step constructions:</li>
+
<ul>
<ul>
-
<li>RBS-TetR</li>
+
<li>Restriction :</li>
-
<li>RBS-LuxR</li>
+
<p>
-
<li>Fha-CinI</li>
+
Between E and P (same for pSB1AT3). No need of purification because PCR BlentII is Kanamycin resistant whereas the selection of pSB1AT3 is made on Tetracycline.
-
<li>Fha-CinR</li>
+
</p>
-
<li>Fha-TetR</li>
+
<li>Ligation</li>
-
<li>Fha-LuxI</li>
+
<li>Spreading over Petri Dish</li>
-
<li>Fha-LuxR</li>
+
<li>Results :</li>
-
<p>Fha plasmid (pSB1AT3) is used as vector.</p>
+
<p>PCR result: successful transfer.</p>
</ul>
</ul>
-
<li>coloration constructions:</li>
+
 +
<li>Cloning Session (Standard Assembly):<br/>
<ul>
<ul>
-
<li>pLux-Lycopene</li>
+
<li>1<sup>st</sup>-step constructions:</li>
-
<li>pCin-Lycopene</li>
+
-
<p>Promoter plasmids are used as vector.</p>
+
-
</ul>
+
-
<li>constructions for the tests:</li>
+
-
<ul>
+
-
<li>pConst-GFP</li>
+
-
<li>pCin-GFP</li>
+
-
<li>pLux-GFP</li>
+
-
<li>pTet-GFP</li>
+
-
<li>pLac-GFP</li>
+
-
<p>Promoter plasmids are used as vector.</p>
+
-
<li>pConst-(RBS-CinR)</li>
+
-
<li>pConst-(RBS-LuxR)</li>
+
-
<p>Promoter plasmid is used as vector.</p>
+
-
</ul>
+
-
<li>Digestions: </li>
+
-
<p>a gel checking of the restriction results were performed.</p>
+
-
<p>Purification: In order to check and extract the wright insert.</p>
+
-
<p>Ligations</p>
+
-
<p>Spreading over Petri Dish</p>
+
-
<ul>
+
-
<li>PCR result from colonies :</li>
+
-
<p>5 potentially successfull constructions:</p>
+
<ul>
<ul>
<li>RBS-TetR</li>
<li>RBS-TetR</li>
 +
<li>RBS-LuxR</li>
<li>Fha-CinI</li>
<li>Fha-CinI</li>
 +
<li>Fha-CinR</li>
 +
<li>Fha-TetR</li>
<li>Fha-LuxI</li>
<li>Fha-LuxI</li>
-
<li>pLux-Lyco</li>
+
<li>Fha-LuxR</li>
-
<li>pCin-Lyco</li>
+
<p>Fha plasmid (pSB1AT3) is used as vector.</p>
</ul>
</ul>
-
</ul>
+
<li>coloration constructions:</li>
-
 
+
<ul>
-
Double cheking with a restriction gel:<br/>
+
<li>pLux-Lycopene</li>
-
from minpirep of the newly obtained constructions, we performed a digestion. Length of the interest fragment are then given by electroporesis<br/>
+
<li>pCin-Lycopene</li>
-
-> same result as previously</p>
+
<p>Promoter plasmids are used as vector.</p>
 +
</ul>
 +
<li>constructions for the tests:</li>
 +
<ul>
 +
<li>pConst-GFP</li>
 +
<li>pCin-GFP</li>
 +
<li>pLux-GFP</li>
 +
<li>pTet-GFP</li>
 +
<li>pLac-GFP</li>
 +
<p>Promoter plasmids are used as vector.</p>
 +
<li>pConst-(RBS-CinR)</li>
 +
<li>pConst-(RBS-LuxR)</li>
 +
<p>Promoter plasmid is used as vector.</p>
 +
</ul>
 +
<li>Digestions: </li>
 +
<p>a gel checking of the restriction results were performed.</p>
 +
<p>Purification: In order to check and extract the wright insert.</p>
 +
<p>Ligations</p>
 +
<p>Spreading over Petri Dish</p>
 +
<ul>
 +
<li>PCR result from colonies :</li>
 +
<p>5 potentially successfull constructions:</p>
 +
<ul>
 +
<li>RBS-TetR</li>
 +
<li>Fha-CinI</li>
 +
<li>Fha-LuxI</li>
 +
<li>pLux-Lyco</li>
 +
<li>pCin-Lyco</li>
 +
</ul>
 +
</ul>
 +
<li>Double cheking with a restriction gel :</li>
 +
<p>
 +
From minpirep of the newly obtained constructions, we performed a digestion. Length of the interest fragment are then given by electroporesis.
 +
</p>
 +
<ul>
 +
<li>same result as previously</li>
 +
</ul>
 +
</ul>
-
+
<li>Cloning Session :</li>
-
<li>Cloning Session :<br/>
+
<ul>
-
-> Constructions for the tests :<br/>
+
<li>Constructions for the tests (3A Assembly):</li>
-
3A Assembly<br/>
+
<ul>
-
pConst-(RBS-CinR) pConst-(RBS-LuxR)<br/>
+
<li>pConst-(RBS-CinR)</li>
-
Plasmid vector: pSB3C5<br/><br/>
+
<li>pConst-(RBS-LuxR)</li>
-
+
<p>Plasmid vector: pSB3C5</p>
-
Standard Assembly<br/>
+
</ul>
-
pConst-GFP pLac-GFP pTet-GFP pLux-GFP pCin-GFP</li>
+
<li>Standard Assembly</li>
-
+
<ul>
-
+
<li>pConst-GFP</li>
-
+
<li>pLac-GFP</li>
-
<p>Digestions: a gel checking of the restriction results were performed.</p>
+
<li>pTet-GFP</li>
-
<p>Purification: In order to check and extract the right insert.</p>
+
<li>pLux-GFP</li>
-
<p>Ligations : We add a control for the ligations: plasmids without its inserts.</p>
+
<li>pCin-GFP</li>
-
<p>Spreading over Petri dish</p>
+
</ul>
-
<p>RESULTS : Restriction gel checking:<br/>
+
<li>Digestions : a gel checking of the restriction results were performed.</li>
-
Only inserts from pConst-GFP and pLac-GFP constructions have the right size</p>
+
<li>Purification : In order to check and extract the right insert.</li>
-
+
<li>Ligations : We add a control for the ligations: plasmids without its inserts.</li>
-
<li>Sequencing Order</li>
+
<li>Spreading over Petri dish</li>
-
<p>pConst-GFP Fha-LuxR Fha-LuxI Fha-CinI pLux-Lycopene pCin-Lycopene pLac-GFP RBS-TetR</p>
+
<li>Results :</li>
-
<p>RESULTS : Correct sequences for pCin-Lycopene ad RBS-TetR</p>
+
<p>Restriction gel checking : Only inserts from pConst-GFP and pLac-GFP constructions have the right size</p>
-
 
+
</ul>
-
</ul>
+
 +
<li>Sequencing Order</li>
 +
<ul>
 +
<li>pConst-GFP</li>
 +
<li>Fha-LuxR</li>
 +
<li>Fha-LuxI</li>
 +
<li>Fha-CinI</li>
 +
<li>pLux-Lycopene</li>
 +
<li>pCin-Lycopene</li>
 +
<li>pLac-GFP</li>
 +
<li>RBS-TetR</li>
 +
<p>RESULTS : Correct sequences for pCin-Lycopene ad RBS-TetR</p>
 +
</ul>
 +
 +
</ul>
</div>
</div>
Line 170: Line 188:
<h1 id="week2">August 11<SUP>th</SUP> to 17<SUP>th</SUP></h1>
<h1 id="week2">August 11<SUP>th</SUP> to 17<SUP>th</SUP></h1>
 +
<div class="blocbackground">
<div class="blocbackground">
-
<img src="https://static.igem.org/mediawiki/2011/5/56/Marion.png" class="icon"/>
 
-
<h2 >Just Team Beaver<span>Marion</span></h2>
 
-
<p>Few manipulation this week, but we also worked on the tests of our project for the Human Practice.</p>
 
-
<ul>
 
-
<li>Cloning Session (Standard Assembly):<br/>
 
-
-> 1st-step constructions:<br/>
 
-
RBS-CinI RBS-LuxI RBS-LacI Fha-CinR Fha-LacI<br/><br/>
 
-
-> Constructions for the tests:<br/>
+
<img src="https://static.igem.org/mediawiki/2011/5/56/Marion.png" class="icon"/>
-
pCin-GFP pLux-GFP pLac-GFP pConst-GFP pConst-(RBS-CinR) pConst-MerR-CinR<br/><br/>
+
<h2 >Just Team Beaver<span>Marion</span></h2>
-
+
-
-> 3rd-step Construction:<br/>
+
<div class="noindent">
-
pLac-MerR-CinR<br/><br/></li>
+
<p>
-
+
Few manipulation this week, but we also worked on the tests of our project for the Human Practice.
-
<p>Digestions: The DNA quantity added into the preparation was increased: 7µl instead of 2µl.
+
</p>
-
a gel checking of the restriction results were performed.</p>
+
-
<p>Purification:  In order to check and extract the wright insert.</p>
+
<ul>
-
<p>Ligations : We add a control for the ligations: plasmids without its inserts.</p>
+
<li>Cloning Session (Standard Assembly):</li>
-
<p>Spreading over Petri dish</p>
+
<ul>
-
<p>RESULTS : Owing to an antibiotic issue, bacterial mats were obtained on every Petri Dishes.
+
<li>1<sup>st</sup>-step constructions:</li>
-
So, remaining preculture were spread again.
+
<ul>
-
And no bacteria growd this time.<br/><br/>
+
<li>RBS-CinI</li>
-
+
<li>RBS-LuxI</li>
-
Ligations were started over using purified DNA from the previous digestions:
+
<li>RBS-LacI</li>
-
PCR checking were performed on colonies, just few inserts had the right length <br/>
+
<li>Fha-CinR</li>
-
pConst-GFP pTet-GFP pConst-(RBS-CinR)</p>
+
<li>Fha-LacI</li>
-
+
</ul>
-
<li>Sequencing Order</li>
+
<li>Constructions for the tests:</li>
-
<p>pLac-GFP pConst-GFP pTet-GFP pConst-(RBS-CinR)<br/>
+
<ul>
-
Fha in pSB1AT3</p>
+
<li>pCin-GFP</li>
-
<p>RESULTS : Sequences matched perfectly with theory for both inserts : pConst-GFP and Fha.</p>
+
<li>pLux-GFP</li>
-
</ul>
+
<li>pLac-GFP</li>
-
</div>
+
<li>pConst-GFP</li>
 +
<li>pConst-(RBS-CinR)</li>
 +
<li>pConst-MerR-CinR</li>
 +
</ul>
 +
<li>3<sup>rd</sup>-step Construction:</li>
 +
<ul>
 +
<li>pLac-MerR-CinR</li>
 +
</ul>
 +
<li>Digestions :</li>
 +
<p>
 +
The DNA quantity added into the preparation was increased: 7µl instead of 2µl. A gel checking of the restriction results were performed.
 +
</p>
 +
<li>Purification :  In order to check and extract the wright insert.</li>
 +
<li>Ligations : We add a control for the ligations: plasmids without its inserts.</li>
 +
<li>Spreading over Petri dish</li>
 +
<p>
 +
Results : Owing to an antibiotic issue, bacterial mats were obtained on every Petri Dishes. So, remaining preculture were spread again. And no bacteria growd this time. Ligations were started over using purified DNA from the previous digestions : PCR checking were performed on colonies, just few inserts had the right length :
 +
</p>
 +
<ul>
 +
<li>pConst-GFP</li>
 +
<li>pTet-GFP</li>
 +
<li>pConst-(RBS-CinR)</li>
 +
</ul>
 +
</ul>
 +
 
 +
<li>Sequencing Order :</li>
 +
<ul>
 +
<li>pLac-GFP</li>
 +
<li>pConst-GFP</li>
 +
<li>pTet-GFP</li>
 +
<li>pConst-(RBS-CinR)</li>
 +
<li>Fha in pSB1AT3</li>
 +
<p>Results : Sequences matched perfectly with theory for both inserts : pConst-GFP and Fha.</p>
 +
</ul>
 +
 +
</ul>
 +
 +
</div>
 +
 +
</div>
</div>
</div>

Revision as of 00:35, 21 September 2011


Grenoble 2011, Mercuro-Coli iGEM

August 4th to 10th

Just Team BeaverMarion

Modifications made on protocols allowed the proper conduct of the experiments even if we didn't get good results.

  • Sequencing Order :
    • RBS-CinI 2
    • RBS-CinI 3
    • RBS-CinI 5
    • MerR-CinR
    • MerR-CinR 4
    • MerR-CinR 5
  • RESULTS :
    • MerR-CinR 5 came back positive from the sequencing
  • Cloning Session (3A Assembly) :
    • 1st-step constructions :
      • RBS-TetR
      • RBS-LuxR
      • Fha-CinI
      • Fha-CinR
      • Fha-TetR
      • Fha-LuxI
      • Fha-LuxR

        • plasmid vector : pSB1AC3

    • coloration constructions :
      • pLux-Lycopene
      • pCin-Lycopene

        • plasmid vector : pSB1AK3

    • constructions for the tests :
      • pConst-GFP
      • pCin-GFP
      • pLux-GFP
      • pTet-GFP
      • pLac-GFP

        • plasmid vector : pSB1AT3


      • pConst-(RBS-CinR)
      • pConst-(RBS-LuxR)
      • plasmid vector: pSB3C5
    • Digestions :

      • a gel checking of the restriction results were performed.

    • Purification :

      • In order to check and extract the wright insert.

    • Ligations :

      • with the ratio : 3X of insert 1X of plasmid

    • Spreading over Petri dish
    • Results :

      • No result on the PCR checking of the colonies.

  • Plasmid transfer of Fha: from PCR BlentII to pSB1AT3.
    • Restriction :

      • Between E and P (same for pSB1AT3). No need of purification because PCR BlentII is Kanamycin resistant whereas the selection of pSB1AT3 is made on Tetracycline.

    • Ligation
    • Spreading over Petri Dish
    • Results :

      • PCR result: successful transfer.

  • Cloning Session (Standard Assembly):
    • 1st-step constructions:
      • RBS-TetR
      • RBS-LuxR
      • Fha-CinI
      • Fha-CinR
      • Fha-TetR
      • Fha-LuxI
      • Fha-LuxR

        • Fha plasmid (pSB1AT3) is used as vector.

    • coloration constructions:
      • pLux-Lycopene
      • pCin-Lycopene

        • Promoter plasmids are used as vector.

    • constructions for the tests:
      • pConst-GFP
      • pCin-GFP
      • pLux-GFP
      • pTet-GFP
      • pLac-GFP

        • Promoter plasmids are used as vector.

      • pConst-(RBS-CinR)
      • pConst-(RBS-LuxR)

        • Promoter plasmid is used as vector.

    • Digestions:

      • a gel checking of the restriction results were performed.

      Purification: In order to check and extract the wright insert.

      Ligations

      Spreading over Petri Dish

      • PCR result from colonies :

        • 5 potentially successfull constructions:

        • RBS-TetR
        • Fha-CinI
        • Fha-LuxI
        • pLux-Lyco
        • pCin-Lyco
    • Double cheking with a restriction gel :

      • From minpirep of the newly obtained constructions, we performed a digestion. Length of the interest fragment are then given by electroporesis.

      • same result as previously
  • Cloning Session :
    • Constructions for the tests (3A Assembly):
      • pConst-(RBS-CinR)
      • pConst-(RBS-LuxR)

        • Plasmid vector: pSB3C5

    • Standard Assembly
      • pConst-GFP
      • pLac-GFP
      • pTet-GFP
      • pLux-GFP
      • pCin-GFP
    • Digestions : a gel checking of the restriction results were performed.
    • Purification : In order to check and extract the right insert.
    • Ligations : We add a control for the ligations: plasmids without its inserts.
    • Spreading over Petri dish
    • Results :

      • Restriction gel checking : Only inserts from pConst-GFP and pLac-GFP constructions have the right size

  • Sequencing Order
    • pConst-GFP
    • Fha-LuxR
    • Fha-LuxI
    • Fha-CinI
    • pLux-Lycopene
    • pCin-Lycopene
    • pLac-GFP
    • RBS-TetR

      • RESULTS : Correct sequences for pCin-Lycopene ad RBS-TetR

August 11th to 17th

Just Team BeaverMarion

Few manipulation this week, but we also worked on the tests of our project for the Human Practice.

  • Cloning Session (Standard Assembly):
    • 1st-step constructions:
      • RBS-CinI
      • RBS-LuxI
      • RBS-LacI
      • Fha-CinR
      • Fha-LacI
    • Constructions for the tests:
      • pCin-GFP
      • pLux-GFP
      • pLac-GFP
      • pConst-GFP
      • pConst-(RBS-CinR)
      • pConst-MerR-CinR
    • 3rd-step Construction:
      • pLac-MerR-CinR
    • Digestions :

      • The DNA quantity added into the preparation was increased: 7µl instead of 2µl. A gel checking of the restriction results were performed.

    • Purification : In order to check and extract the wright insert.
    • Ligations : We add a control for the ligations: plasmids without its inserts.
    • Spreading over Petri dish

      • Results : Owing to an antibiotic issue, bacterial mats were obtained on every Petri Dishes. So, remaining preculture were spread again. And no bacteria growd this time. Ligations were started over using purified DNA from the previous digestions : PCR checking were performed on colonies, just few inserts had the right length :

      • pConst-GFP
      • pTet-GFP
      • pConst-(RBS-CinR)
  • Sequencing Order :
    • pLac-GFP
    • pConst-GFP
    • pTet-GFP
    • pConst-(RBS-CinR)
    • Fha in pSB1AT3

      • Results : Sequences matched perfectly with theory for both inserts : pConst-GFP and Fha.

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