Team:INSA Toulouse/contenu/lab practice/notebook/calendar/MG1655

From 2013.igem.org

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In order to use this strain for light sensors characterization, we need to delete the <i>envZ</i> gene. For this we did a transduction of the strain with phages previously mixed with the del(EnvZ) strain from the Keio collection.  
In order to use this strain for light sensors characterization, we need to delete the <i>envZ</i> gene. For this we did a transduction of the strain with phages previously mixed with the del(EnvZ) strain from the Keio collection.  
<br>The strains from this collection have individual kan cassette replacements of non-essential genes flanked by FRT sites allowing excision using the FLP recombinase.
<br>The strains from this collection have individual kan cassette replacements of non-essential genes flanked by FRT sites allowing excision using the FLP recombinase.
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<br> <a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/integration" target="_blank"> View Protocols </a>
<br>6/09
<br>6/09
<br>overnight culture of the Keio Del(EnvZ) Strain in LB and CaCl2
<br>overnight culture of the Keio Del(EnvZ) Strain in LB and CaCl2
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   <li><span class="spantitle2">Week 10 (12-18 August)</span><br>
   <li><span class="spantitle2">Week 10 (12-18 August)</span><br>
Then we needed to remove the Kn cassette with the FLIP recombinase, in order to integrate with a selection factor Kn.
Then we needed to remove the Kn cassette with the FLIP recombinase, in order to integrate with a selection factor Kn.
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<br> <a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/integration" target="_blank"> View Protocols </a>
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<br>12/09
<br>12/09
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19/08
19/08
<br>PCR verification: ok ! The del(envZ) deFRT Kn is constructed!! Thanks to the wonderful help of Caroline Schiavon
<br>PCR verification: ok ! The del(envZ) deFRT Kn is constructed!! Thanks to the wonderful help of Caroline Schiavon
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<br>20/08
<br>20/08
<br>Then in order to integrate modules in the genome with integration plasmids, the strain needs to be Streptomycine resistant for selection.
<br>Then in order to integrate modules in the genome with integration plasmids, the strain needs to be Streptomycine resistant for selection.
<br>The strain has the Streptomycine resistance gene but with a mutation. With several spread on LB agar Strp, the strain can recover the resistance.
<br>The strain has the Streptomycine resistance gene but with a mutation. With several spread on LB agar Strp, the strain can recover the resistance.
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<br> <a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/integration" target="_blank"> View Protocols </a>
<br>Spread on LB Str 50ng during a night at 37°c.
<br>Spread on LB Str 50ng during a night at 37°c.
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<h2 class="faq" title="Click to open the FAQ">September 2013</h2>
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  <li><span class="spantitle2">Week 13 (2-8 September)</span><br>
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2/09
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<br>Stop of the integration: the MG1655 del(envZ) deFRT Kn StrpR strain was already CmR…
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<br>Because of a lack of time, we need to concentrate the force on the wiki, there is few weeks left…
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Latest revision as of 18:17, 3 October 2013

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Calendar

MG1655 del(EnvZ) Strain

August 2013

  • Week 9 (5-11 August)
    In order to use this strain for light sensors characterization, we need to delete the envZ gene. For this we did a transduction of the strain with phages previously mixed with the del(EnvZ) strain from the Keio collection.
    The strains from this collection have individual kan cassette replacements of non-essential genes flanked by FRT sites allowing excision using the FLP recombinase.
    View Protocols
    6/09
    overnight culture of the Keio Del(EnvZ) Strain in LB and CaCl2

    7/09
    dilution of the overnight culture of del(envZ) strain until DO=1
    a mix of bacterias with P1 phages is spread on soft LB agar for the night at 30°
    overnight culture of the MG1655 strain

    8/09
    pick up of phage with bacterias
    dilution of the overnight culture of MG1655 in LB and CaCl2 until DO=1
    mix of bacterias (MG1655) with phages
    incubation a night at 37°c

    9/09
    verification of lysis plaque


  • Week 10 (12-18 August)
    Then we needed to remove the Kn cassette with the FLIP recombinase, in order to integrate with a selection factor Kn.
    View Protocols

    12/09
    Transformation with the pFLP plasmid.
    One night on LB agar plate at 30°C

    13/09
    Pick 4 clones and cultivate in liquid 2hours at 30°C then 3 hours at 42°C.
    Dilution and spread on LB Agar.

    14/09
    Pick isolated clones on LB Agar, LB Cm, LB Ap and LB Kn.
    Let one night at 42°C.
    Right clones are Cm S, Ap S and Kn S.

    15/08
    Clones are ok.
    Waiting for the primers to verify with PCR.
  • Week 11 (19-25 August)
    19/08
    PCR verification: ok ! The del(envZ) deFRT Kn is constructed!! Thanks to the wonderful help of Caroline Schiavon


    20/08
    Then in order to integrate modules in the genome with integration plasmids, the strain needs to be Streptomycine resistant for selection.
    The strain has the Streptomycine resistance gene but with a mutation. With several spread on LB agar Strp, the strain can recover the resistance.
    View Protocols
    Spread on LB Str 50ng during a night at 37°c.

    21/08
    Several clones have grown but the right Strepto concentration was 200ng/µL.
    Spread on LB Str 200ng during a night at 37°C

    22/08
    One clone on the plate
    Subcloned the clone on spread on LB Str 200ng over the night at 37°C

    23/08
    Few other clones have grown
    Subcloned of these clones and spread on LB Str 200ng at 37°C

    25/08
    Competent cells of the new strain: MG1655 del(envZ) deFRT Kn StrpR.
    Transformation with the plasmid pMS58
    Spread on LB Cm over the night at 30°C
  • Week 12 (26-1 September)
    26/08
    streak 4 clones of the previous transformation on LB Cm at 42°C one night

    27/08
    Streak 4 clones of the first integration on LB Cm at 42°C one night

    28/08
    Streak 4 clones of the second integration on LB Strp 42°C one night

    29/08
    Sub-cloned few clones on LB Cm, LB Str and LB Kn at 42°C one night
    Right clone are StR CmS et KnR

    30/08
    Our clones were StrR, but unfortunately they were CmR and KnS…
    Restart of the integration from the beginning:
    Making competent cells
    In parallele, spread of the MG1655 del(envZ) deFRT Kn StrpR strain on LB Cm, LB Str and LB Kn at 42°C

September 2013

  • Week 13 (2-8 September)
    2/09
    Stop of the integration: the MG1655 del(envZ) deFRT Kn StrpR strain was already CmR…

    Because of a lack of time, we need to concentrate the force on the wiki, there is few weeks left…

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