Team:Grenoble/Notebook/August

From 2011.igem.org

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<h2>Biology<span>Félix</span></h2>
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Screening by enzymatic restriction (E and P) of constructs 19 and 21. Clone 19A,19B, 19E, 21A and 21B were sent to sequencing. Sequencing results shows expected constructs for all clones. Consequently they  were stored at -80°C in 40% glycerol.
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Screening of construction 22, 23 were performed first by colony PCR. Four clones for constructs 22 and 23 were retaineds for a restriction digest screening. Later experiments confirm colony PCR results for every clone tested.
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<p>They were all sent for sequencing.</p>
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Construction 24 was abandoned because two EcoRI restriction sites exist in pTOPO around 5’ and 3’ of blunt restriction sites (where standard rpoSLS was ligated). Construction with fhaLS-pTOPO (SpeII and Pst I) and I13401(XbaI and PstI) in standard assembly. fhaSL-I13401-pTOPO (construction 25) Construction 25 gave colonie but all contained empty plasmids as tested by colony PCR.
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<h1 id="week3">August 18<SUP>th</SUP> to 25<SUP>th</SUP></h1>
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<h2>Biology<span>Eric</span></h2>
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Construction of construct 26 using fhaLS-pSB1AT3 constructed by (SpeI and Pst I) and I13401 (XhoI and PstI)
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<li>fhaSL-I13401-pSB1AT2 (construct 26).</li>
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<p>No colonies were obtained. </p>
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Sequencing results : construct 23 has the expected sequence. Concerning construct 22, the sequence is completely different. Construction 22 was abandoned.
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Revision as of 04:48, 21 September 2011


Grenoble 2011, Mercuro-Coli iGEM

August 4th to 10th

BiologyMarion

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

BiologyEric


Reception of two synthesized primers which are hybridized to obtain magLS. Construction from LS PCR product and linearized pSB1C3:

  • rpoSLS-pSB1C3 (construction 19)
  • magLS-pSB1C3 (construction 21)

Construction from LS PCR product and I13401 (standard assembly):

  • fhaLS-I13401-pTOPO (construction 24)
  • pSB1A2-rpoSLS-I13401 (construction 22)
  • pSB1A2-magLS-I13401 (construction 23)

Colony PCR was performed for constructs 19 and 21 with primer VF and VR. Some clones were retained as positive. Digestion of retained clones by EcoRI (E) and PstI (P): Four clones of construct 19 and two of construct 21 were OK and sent to sequencing.

We try to set up a different PCR protocol to screen for the presence of an insert in pTOPO, using either two primers that hybridize on the plasmid, or one on the plasmid and one on the insert. None of the conditions we try work. We definitely give up with the use of this plasmid.

Having to use a new Taq (Pfu polymerase hight fidelity from Invitrogen) in a different lab, we set up the PCR for rposLS and rsmA again.

Production of those two sequences to be cloned into PSB1C3. Adaptation of the IGEM protocol for plasmid production to the Pfu polymerase.

August 11th to 17th

BiologyMarion

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.

BiologyFélix


Screening by enzymatic restriction (E and P) of constructs 19 and 21. Clone 19A,19B, 19E, 21A and 21B were sent to sequencing. Sequencing results shows expected constructs for all clones. Consequently they were stored at -80°C in 40% glycerol.

Screening of construction 22, 23 were performed first by colony PCR. Four clones for constructs 22 and 23 were retaineds for a restriction digest screening. Later experiments confirm colony PCR results for every clone tested.

They were all sent for sequencing.

Construction 24 was abandoned because two EcoRI restriction sites exist in pTOPO around 5’ and 3’ of blunt restriction sites (where standard rpoSLS was ligated). Construction with fhaLS-pTOPO (SpeII and Pst I) and I13401(XbaI and PstI) in standard assembly. fhaSL-I13401-pTOPO (construction 25) Construction 25 gave colonie but all contained empty plasmids as tested by colony PCR.

August 18th to 25th

BiologyEric


Construction of construct 26 using fhaLS-pSB1AT3 constructed by (SpeI and Pst I) and I13401 (XhoI and PstI)

  • fhaSL-I13401-pSB1AT2 (construct 26).

No colonies were obtained.

Sequencing results : construct 23 has the expected sequence. Concerning construct 22, the sequence is completely different. Construction 22 was abandoned.

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