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

From 2013.igem.org

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  <h2 class="faq" title="Click to open the FAQ">July 2013</h2>
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  <h2 class="faq" title="Click to open the FAQ">August 2013</h2>
   <div class="infos">
   <div class="infos">
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   <ul class="circlearrow">
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   <li><span class="spantitle2">Week ...</span><br>
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   <li><span class="spantitle2">Week 9 (5-11 August)</span><br>
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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.  
 +
<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>6/09
 +
<br>overnight culture of the Keio Del(EnvZ) Strain in LB and CaCl2
 +
<br>
 +
<br>7/09
 +
<br>dilution of the overnight culture of del(envZ) strain until DO=1
 +
<br>a mix of bacterias with P1 phages is spread on soft LB agar for the night at 30°
 +
<br>overnight culture of  the MG1655 strain
 +
<br>
 +
<br>8/09
 +
<br>pick up of phage with bacterias
 +
<br>dilution of the overnight culture of MG1655 in LB and CaCl2 until DO=1
 +
<br>mix of bacterias (MG1655) with phages
 +
<br>incubation a night at 37°c
 +
<br>
 +
<br>9/09
 +
<br>verification of lysis plaque
 +
<br>
 +
<br><img style="width :340px;" src="https://static.igem.org/mediawiki/2013/7/74/MG16phage.png" class="imgcontent" />
 +
<br>
</li>
</li>
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   <li><span class="spantitle2">Week ….</span><br>
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   <li><span class="spantitle2">Week 10 (12-18 August)</span><br>
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+
Then we needed to remove the Kn cassette with the FLIP recombinase, in order to integrate with a selection factor Kn.
 +
<br>
 +
<br>12/09
 +
<br>Transformation with the pFLP plasmid.
 +
<br>One night on LB agar plate at 30°C
 +
<br>
 +
<br>13/09
 +
<br>Pick 4 clones and cultivate in liquid 2hours at 30°C then 3 hours at 42°C.
 +
<br>Dilution and spread on LB Agar.
 +
<br>
 +
<br>14/09
 +
<br>Pick isolated clones on LB Agar, LB Cm, LB Ap and LB Kn.
 +
<br>Let one night at 42°C.
 +
<br>Right clones are Cm S, Ap S and Kn S.
 +
<br>
 +
<br>15/08
 +
<br>Clones are ok.
 +
<br>Waiting for the primers to verify with PCR.
 +
<br>
</li>
</li>
-
  </ul></li>
+
  <li><span class="spantitle2">Week 11 (19-25 August) </span><br>
-
  </ul>
+
19/08
 +
<br>PCR verification: ok ! The del(envZ) deFRT Kn is constructed!! Thanks to the wonderful help of Caroline Schiavon
 +
<br><img style=”width:600px;” src=”https://static.igem.org/mediawiki/2013/d/d8/PCR_MG16.png “ class=”imgcontent />
 +
<br>
 +
<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>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>Spread on LB Str 50ng during a night at 37°c.
 +
<br>
 +
<br>21/08
 +
<br>Several clones have grown but the right Strepto concentration was 200ng/µL.
 +
<br>Spread on LB Str 200ng during a night at 37°C
 +
<br>
 +
<br>22/08
 +
<br>One clone on the plate
 +
<br>Subcloned the clone on spread on LB Str 200ng over the night at 37°C
 +
<br>
 +
<br>23/08
 +
<br>Few other clones have grown
 +
<br> Subcloned of these clones and spread on LB Str 200ng at 37°C
 +
<br>
 +
<br>25/08
 +
<br>Competent cells of the new strain: MG1655 del(envZ) deFRT Kn StrpR.
 +
<br>Transformation with the plasmid pMS58
 +
<br>Spread on LB Cm over the night at 30°C
 +
<br>
 +
</li>
 +
  <li><span class="spantitle2">Week 12 (26-1 September)</span><br>
 +
26/08
 +
<br>streak 4 clones of the previous transformation on LB Cm at 42°C one night
 +
<br>
 +
<br>27/08
 +
<br>Streak 4 clones of the first integration on LB Cm at 42°C one night
 +
<br>
 +
<br>28/08
 +
<br>Streak 4 clones of the second integration on LB Strp 42°C one night
 +
<br>
 +
<br>29/08
 +
<br>Sub-cloned  few clones on LB Cm, LB Str and LB Kn at 42°C one night
 +
<br>Right clone are StR CmS et KnR
 +
<br>
 +
<br>30/08
 +
<br>Our clones were StrR, but unfortunately they were CmR and KnS…
 +
<br>Restart of the integration from the beginning:
 +
<br>Making competent cells
 +
<br>In parallele, spread of the MG1655 del(envZ) deFRT Kn StrpR strain on LB Cm, LB Str and LB Kn at 42°C
 +
<br>
</li>
</li>
-
  </div>
 
-
</div>
 
-
<div class="clear"></div>
 
-
 
-
<div class="accordeon">
 
-
<h2 class="faq" title="Click to open the FAQ">August 2013</h2>
 
-
  <div class="infos">
 
-
  <ul class="circlearrow">
 
-
  <li><span class="spantitle2">Week ...</span></li>
 
-
  <li><span class="spantitle2">Week ...</span></li>
 
   </ul></li>
   </ul></li>
   </ul>
   </ul>

Revision as of 21:02, 2 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.
    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.

    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.
    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

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