Team:Grenoble/Notebook/August

From 2011.igem.org

(Difference between revisions)
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<div class="body">
<div class="body">
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<div class="left">
 
-
  <h1 id="week1">August 4<SUP>th</SUP> to 10<SUP>th</SUP></h1>
+
<div class="left">
-
<div class="blocbackground">
+
-
    <img src="https://static.igem.org/mediawiki/2011/5/56/Marion.png" class="icon"/>
+
<h1 id="week1">August 4<SUP>th</SUP> to 10<SUP>th</SUP></h1>
-
      <h2 >Just Team Beaver<span>Marion</span></h2>
+
-
      <p>Modifications made on protocols allowed the proper conduct of the experiments even if we didn't get good results.</p>
+
-
<ul>
+
<div class="blocbackground">
-
  <li>Sequencing Order</li>
+
<img src="https://static.igem.org/mediawiki/2011/5/56/Marion.png" class="icon"/>
-
    <p>RBS-CinI 2 <br/>
+
<h2>Just Team Beaver<span>Marion</span></h2>
-
      RBS-CinI 3<br/>
+
-
      RBS-CinI 5<br/>
+
<div class="noindent">
-
      MerR-CinR<br/>
+
<p>
-
      MerR-CinR 4<br/>
+
Modifications made on protocols allowed the proper conduct of the experiments even if we didn't get good results.
-
      MerR-CinR 5</p>
+
</p>
-
    <p>RESULTS :<br/> MerR -CinR 5 comeback positiv from the sequencing</p>
+
-
  <li>Cloning Session (3A Assembly):<br/>
+
<ul>
-
    -> 1st-step constructions:<br/>
+
<li>Sequencing Order :</li>
-
    RBS-TetR RBS-LuxR Fha-CinI Fha-CinR Fha-TetR Fha-LuxI Fha-LuxR<br/>
+
<ul>
-
    plasmid vector : pSB1AC3<br/><br/>
+
<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>
 +
<li>MerR-CinR 5 came back positive from the sequencing</li>
 +
</ul>
 +
 +
<li>Cloning Session (3A Assembly) :</li>
 +
<ul>
 +
<li>1st-step constructions :</li>
 +
<ul>
 +
<li>RBS-TetR</li>
 +
<li>RBS-LuxR</li>
 +
<li>Fha-CinI</li>
 +
<li>Fha-CinR</li>
 +
<li>Fha-TetR</li>
 +
<li>Fha-LuxI</li>
 +
<li>Fha-LuxR</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>
 +
</ul>
 +
-
    -> coloration constructions:<br/>
+
-
    pLux-Lycopene pCin-Lycopene<br/>
+
-
    plasmid vector : pSB1AK3<br/><br/>
+
 +
<p>Spreading over Petri dish</p>
 +
<p>RESULTS :<br/>
 +
No result on the PCR checking of the colonies.</p>
 +
<li>Plasmid transfer of Fha: from PCR BlentII to pSB1AT3.</li>
 +
<p>Restriction: between E and P (same for pSB1AT3)</br>
 +
No need of purification because PCR BlentII is Kanamycin resistant hereas the selection of pSB1AT3 is made on Tetracycline.</br>
 +
Ligation<br/>
 +
Spreading over Petri Dish<br/></p>
 +
<p> RESULTS :<br/> PCR result: successful transfer.</p>
 +
 +
<li>Cloning Session (Standard Assembly):<br/>
 +
-> 1st-step constructions:<br/>
 +
RBS-TetR RBS-LuxR Fha-CinI Fha-CinR Fha-TetR Fha-LuxI Fha-LuxR<br/>
 +
Fha plasmid (pSB1AT3) is used as vector.<br/><br/>
 +
 +
-> coloration constructions:<br/>
 +
pLux-Lycopene pCin-Lycopene<br/>
 +
Promoter plasmids are used as vector.<br/><br/>
 +
 +
-> constructions for the tests:
 +
pConst-GFP pCin-GFP pLux-GFP pTet-GFP pLac-GFP<br/>
 +
Promoter plasmids are used as vector.<br/>
 +
pConst-(RBS-CinR) pConst-(RBS-LuxR)<br/>
 +
Promoter plasmid is used as vector.<br/></li>
 +
 +
<p>Digestions: 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>
 +
 +
<p>RESULTS : <br/>PCR result from colonies:<br/>
 +
5 potentially successfull constructions RBS-TetR Fha-CinI Fha-LuxI pLux-Lyco pCin-Lyco<br/>
 +
Double cheking with a restriction gel:<br/>
 +
from minpirep of the newly obtained constructions, we performed a digestion. Length of the interest fragment are then given by electroporesis<br/>
 +
-> same result as previously</p>
 +
 +
 +
<li>Cloning Session :<br/>
 +
-> Constructions for the tests :<br/>
 +
3A Assembly<br/>
 +
pConst-(RBS-CinR) pConst-(RBS-LuxR)<br/>
 +
Plasmid vector: pSB3C5<br/><br/>
 +
 +
Standard Assembly<br/>
 +
pConst-GFP pLac-GFP pTet-GFP pLux-GFP pCin-GFP</li>
 +
 +
 +
 +
<p>Digestions: a gel checking of the restriction results were performed.</p>
 +
<p>Purification: In order to check and extract the right insert.</p>
 +
<p>Ligations : We add a control for the ligations: plasmids without its inserts.</p>
 +
<p>Spreading over Petri dish</p>
 +
<p>RESULTS : Restriction gel checking:<br/>
 +
Only inserts from pConst-GFP and pLac-GFP constructions have the right size</p>
 +
 +
<li>Sequencing Order</li>
 +
<p>pConst-GFP Fha-LuxR Fha-LuxI Fha-CinI pLux-Lycopene pCin-Lycopene pLac-GFP RBS-TetR</p>
 +
<p>RESULTS : Correct sequences for pCin-Lycopene ad RBS-TetR</p>
-
    -> constructions for the tests:<br/>
+
</ul>
-
    pConst-GFP pCin-GFP pLux-GFP pTet-GFP pLac-GFP<br/>
+
-
    plasmid vector : pSB1AT3<br/>
+
</div>
-
    pConst-(RBS-CinR) pConst-(RBS-LuxR)<br/>
+
-
    plasmid vector: pSB3C5<br/><br/>
+
</div>
-
    </li>
+
-
  <p>Digestions:<br/>a gel checking of the restriction results were performed.</p>
+
<h1 id="week2">August 11<SUP>th</SUP> to 17<SUP>th</SUP></h1>
-
  <p>Purification: <br/>In order to check and extract the wright insert.<p>
+
<div class="blocbackground">
-
  <p>Ligations : with the ratio: <br/>
+
<img src="https://static.igem.org/mediawiki/2011/5/56/Marion.png" class="icon"/>
-
    3X of insert 1X of plasmid</p>
+
<h2 >Just Team Beaver<span>Marion</span></h2>
-
  <p>Spreading over Petri dish</p>
+
<p>Few manipulation this week, but we also worked on the tests of our project for the Human Practice.</p>
-
  <p>RESULTS :<br/>
+
<ul>
-
    No result on the PCR checking of the colonies.</p>
+
<li>Cloning Session (Standard Assembly):<br/>
-
  <li>Plasmid transfer of Fha: from PCR BlentII to pSB1AT3.</li>
+
-
    <p>Restriction: between E and P (same for pSB1AT3)</br>
+
-
    No need of purification because PCR BlentII is Kanamycin resistant hereas the selection of pSB1AT3 is made on Tetracycline.</br>
+
-
    Ligation<br/>
+
-
    Spreading over Petri Dish<br/></p>
+
-
    <p> RESULTS :<br/> PCR result: successful transfer.</p>
+
-
   
+
-
    <li>Cloning Session (Standard Assembly):<br/>
+
-
      -> 1st-step constructions:<br/>
+
-
      RBS-TetR RBS-LuxR Fha-CinI Fha-CinR Fha-TetR Fha-LuxI Fha-LuxR<br/>
+
-
      Fha plasmid (pSB1AT3) is used as vector.<br/><br/>
+
-
 
+
-
      -> coloration constructions:<br/>
+
-
      pLux-Lycopene pCin-Lycopene<br/>
+
-
      Promoter plasmids are used as vector.<br/><br/>
+
-
 
+
-
      -> constructions for the tests:
+
-
      pConst-GFP pCin-GFP pLux-GFP pTet-GFP pLac-GFP<br/>
+
-
      Promoter plasmids are used as vector.<br/>
+
-
      pConst-(RBS-CinR) pConst-(RBS-LuxR)<br/>
+
-
      Promoter plasmid is used as vector.<br/></li>
+
-
 
+
-
      <p>Digestions: 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>
+
-
 
+
-
      <p>RESULTS : <br/>PCR result from colonies:<br/>
+
-
      5 potentially successfull constructions RBS-TetR Fha-CinI Fha-LuxI pLux-Lyco pCin-Lyco<br/>
+
-
      Double cheking with a restriction gel:<br/>
+
-
      from minpirep of the newly obtained constructions, we performed a digestion. Length of the interest fragment are then given by electroporesis<br/>
+
-
      -> same result as previously</p>
+
-
 
+
-
 
+
-
    <li>Cloning Session :<br/>
+
-
    -> Constructions for the tests :<br/>
+
-
                  3A Assembly<br/>
+
-
    pConst-(RBS-CinR) pConst-(RBS-LuxR)<br/>
+
-
    Plasmid vector: pSB3C5<br/><br/>
+
-
 
+
-
            Standard Assembly<br/>
+
-
      pConst-GFP pLac-GFP pTet-GFP pLux-GFP pCin-GFP</li>
+
-
 
+
-
 
+
-
   
+
-
    <p>Digestions: a gel checking of the restriction results were performed.</p>
+
-
    <p>Purification: In order to check and extract the right insert.</p>
+
-
    <p>Ligations : We add a control for the ligations: plasmids without its inserts.</p>
+
-
    <p>Spreading over Petri dish</p>
+
-
    <p>RESULTS : Restriction gel checking:<br/>
+
-
    Only inserts from pConst-GFP and pLac-GFP constructions have the right size</p>
+
-
   
+
-
    <li>Sequencing Order</li>
+
-
    <p>pConst-GFP Fha-LuxR Fha-LuxI Fha-CinI pLux-Lycopene pCin-Lycopene pLac-GFP RBS-TetR</p>
+
-
    <p>RESULTS : Correct sequences for pCin-Lycopene ad RBS-TetR</p>
+
-
     
+
-
     
+
-
 
+
-
 
+
-
</ul>
+
-
  </div>
+
-
 
+
-
  <h1 id="week2">August 11<SUP>th</SUP> to 17<SUP>th</SUP></h1>
+
-
  <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/>
-> 1st-step constructions:<br/>
RBS-CinI RBS-LuxI RBS-LacI Fha-CinR Fha-LacI<br/><br/>
RBS-CinI RBS-LuxI RBS-LacI Fha-CinR Fha-LacI<br/><br/>
-
 
+
-> Constructions for the tests:<br/>
-> Constructions for the tests:<br/>
pCin-GFP pLux-GFP pLac-GFP pConst-GFP pConst-(RBS-CinR) pConst-MerR-CinR<br/><br/>
pCin-GFP pLux-GFP pLac-GFP pConst-GFP pConst-(RBS-CinR) pConst-MerR-CinR<br/><br/>
-
 
+
-> 3rd-step Construction:<br/>
-> 3rd-step Construction:<br/>
pLac-MerR-CinR<br/><br/></li>
pLac-MerR-CinR<br/><br/></li>
-
 
+
<p>Digestions: The DNA quantity added into the preparation was increased: 7µl instead of 2µl.
<p>Digestions: The DNA quantity added into the preparation was increased: 7µl instead of 2µl.
-
  a gel checking of the restriction results were performed.</p>
+
a gel checking of the restriction results were performed.</p>
<p>Purification:  In order to check and extract the wright insert.</p>
<p>Purification:  In order to check and extract the wright insert.</p>
<p>Ligations : We add a control for the ligations: plasmids without its inserts.</p>
<p>Ligations : We add a control for the ligations: plasmids without its inserts.</p>
<p>Spreading over Petri dish</p>
<p>Spreading over Petri dish</p>
<p>RESULTS : Owing to an antibiotic issue, bacterial mats were obtained on every Petri Dishes.
<p>RESULTS : Owing to an antibiotic issue, bacterial mats were obtained on every Petri Dishes.
-
    So, remaining preculture were spread again.
+
So, remaining preculture were spread again.
-
    And no bacteria growd this time.<br/><br/>
+
And no bacteria growd this time.<br/><br/>
-
 
+
-
    Ligations were started over using purified DNA from the previous digestions:
+
Ligations were started over using purified DNA from the previous digestions:
-
    PCR checking were performed on colonies, just few inserts had the right length <br/>
+
PCR checking were performed on colonies, just few inserts had the right length <br/>
-
    pConst-GFP pTet-GFP pConst-(RBS-CinR)</p>
+
pConst-GFP pTet-GFP pConst-(RBS-CinR)</p>
-
 
+
-
<li>Sequencing Order</li>
+
<li>Sequencing Order</li>
-
<p>pLac-GFP pConst-GFP pTet-GFP pConst-(RBS-CinR)<br/>
+
<p>pLac-GFP pConst-GFP pTet-GFP pConst-(RBS-CinR)<br/>
-
Fha in pSB1AT3</p>
+
Fha in pSB1AT3</p>
-
<p>RESULTS : Sequences matched perfectly with theory for both inserts : pConst-GFP and Fha.</p>
+
<p>RESULTS : Sequences matched perfectly with theory for both inserts : pConst-GFP and Fha.</p>
-
    </ul>
+
</ul>
-
    </div>
+
</div>
-
 
+
 +
</div>
-
</div>
 
</div>
</div>

Revision as of 20:31, 20 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 hereas 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

    RESULTS :
    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.

Retrieved from "http://2011.igem.org/Team:Grenoble/Notebook/August"