Team:Grenoble/Notebook/August

From 2011.igem.org

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<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.
 +
<math>\frac ab</math>
</p>
</p>
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</ul>
</ul>
<li>coloration constructions:</li>
<li>coloration constructions:</li>
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</ul>
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<ul>
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-> <br/>
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<li>pLux-Lycopene</li>
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pLux-Lycopene pCin-Lycopene<br/>
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<li>pCin-Lycopene</li>
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Promoter plasmids are used as vector.<br/><br/>
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<p>Promoter plasmids are used as vector.</p>
-
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</ul>
-
-> constructions for the tests:
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<li>constructions for the tests:</li>
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pConst-GFP pCin-GFP pLux-GFP pTet-GFP pLac-GFP<br/>
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<ul>
-
Promoter plasmids are used as vector.<br/>
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<li>pConst-GFP</li>
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pConst-(RBS-CinR) pConst-(RBS-LuxR)<br/>
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<li>pCin-GFP</li>
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Promoter plasmid is used as vector.<br/></li>
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<li>pLux-GFP</li>
-
+
<li>pTet-GFP</li>
-
<p>Digestions: a gel checking of the restriction results were performed.</p>
+
<li>pLac-GFP</li>
-
<p>Purification: In order to check and extract the wright insert.</p>
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<p>Promoter plasmids are used as vector.</p>
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<p>Ligations</p>
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<li>pConst-(RBS-CinR)</li>
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<p>Spreading over Petri Dish</p>
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<li>pConst-(RBS-LuxR)</li>
-
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<p>Promoter plasmid is used as vector.</p>
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<p>RESULTS : <br/>PCR result from colonies:<br/>
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</ul>
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5 potentially successfull constructions RBS-TetR Fha-CinI Fha-LuxI pLux-Lyco pCin-Lyco<br/>
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<li>Digestions: </li>
-
Double cheking with a restriction gel:<br/>
+
<p>a gel checking of the restriction results were performed.</p>
-
from minpirep of the newly obtained constructions, we performed a digestion. Length of the interest fragment are then given by electroporesis<br/>
+
<p>Purification: In order to check and extract the wright insert.</p>
-
-> same result as previously</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/>
<li>Cloning Session :<br/>

Revision as of 21:24, 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. \frac ab

  • 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

      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.

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