Team:Lyon-INSA/notebook

From 2012.igem.org

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</description>
</description>
                       </jour>
                       </jour>
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 +
<jour nb="20">
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                        <date>Monday, August 20th 2012</date>
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                        <titre>For all purposes</titre>
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<description>
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<ul>
 +
<li>The pBK19 with no SpeI site was put in storage under the reference pBK25.</li>
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<li>The pBK20 with no SpeI site was put in storage under the reference pBK26.</li>
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</ul>
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</description>
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                      <titre>Kill</titre>
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<description>
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<ul>
 +
<li>Launch of 8 [Promoter+Dispersin] cultures.</li>
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<li>Results of the transformation of NM522 by [pSB1T3 + pXyl] : the plate with bacteria transformed by [pSB1T3 + pXyl] contains few clones and the controls are standard. 3 clones are selected to do liquid culture and extract their DNA : no plasmid.</li>
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<li>Results of the transformation of NM522 by [pSB1T3 + rbs-abrB] : no clones.</li>
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</ul>
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</description>
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                      <titre>Stick</titre>
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<description>
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<ul>
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<li>Results of the transformation of NM522 by [pSB1K3 + XylR] : the plate with bacteria transformed by [pSB1K3 + XylR] contains a lot of clones and the controls are standard. 3 clones are selected to do liquid culture and extract their DNA : they have just the vector without XylR.</li.
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<li>A new PCR of xylR is made, in order to increase the stock.</li>
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</ul>
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</description>
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                      </jour>
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 +
<jour nb="21">
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                        <date>Tuesday, August 21st 2012</date>
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                        <titre>For all purposes</titre>
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<description>
 +
<ul>
 +
<li>Meeting at 9 o’clock.</li>
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<li>Midiprep of the modified pBK25 and pBK26 shuttle plasmids (without the SpeI site). The extracted DNA was verified by electrophoresis.</li>
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</ul>
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</description>
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                      <titre>Kill</titre>
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<description>
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<ul>
 +
<li>Standard ligation of pBK23 (= Constitutive Promoter + RBS + Lysostaphin in pSB1C3) with pBK25 (the shuttle vector <i>E. coli</i> - <i>B. subtilis</i>). Different ligations are made with differents proportions of insert and vector. Transformation in NM522 strain.</li>
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<li>Digestion of Lysostaphin in pSB1C3 and Dsp in pUC57.</li>
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<li>Check of 8 [promoter-dispersin] clones : clones happen to be nonstandard.</li>
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</ul>
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</description>
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                      <titre>Surfactant</titre>
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<description>
 +
<ul>
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<li>Extraction of the plasmid containing the ligation [sfp+abrB+pSB1A3] from a liquid culture of transformed bacteria.</li>
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<li>Results of the second transformation of NM522 by pSB1T3 and rbs-abrB : no clones.</li>
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</ul>
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</description>
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                      <titre>Stick</titre>
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<description>
 +
<ul>
 +
<li>Two standard ligations are done :</li>
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      <ul>
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    <li>pBK7-xylR (xylR cut in X and P sites);</li>
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            <li>pBK24-xylR (xylR cut in E and S sites).</li>
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      </ul>
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<li>Transformation of pBK7-xylR into NM522 strain.</li>
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</ul>
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</description>
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                      </jour>
 +
 +
___________________________________________________________________________________
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Wednesday, 22nd August:
 +
For all :
 +
 +
Kill :
 +
- Result of the transformation of NM522 with [Constitutiv Promoter + RBS + Lysostaphin] in pBK25 : all the controls are right, and there are a lot of clones on the different plates with the transformed bacteria. 14 clones are selected to do liquid cultures and extract their DNA.
 +
- New try of clonage : Digestion, Ligation ,transformation to construct :
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Lyso+Dsp in PSB1C3 and Lyso+Dspin the shuttle vector (vector for E.coli and B.subtilis)
 +
 +
Surfactant :
 +
-  6 clones pXyl were tested, but none had integrated the plasmid
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Stick :
 +
- checking of 6 clones XylR-psB1K3 : They have just the vector.
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- Transformation of pBK24-xylR into NM522 strain.
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- Results of pBK7-xylR transformation: lots of clones and no clones in the negative witness. 12 clones are put in liquid culture for extraction.
 +
 +
___________________________________________________________________________________
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Thursday, 23rd August:
 +
 +
cloning of the iGEM linker into the shuttle vector (pBK25 and pBK26) and transformation in the NM522 strain;
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 +
Kill:
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-Transformations results : All controls are okay
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Too much clones on Lyso+Dsp in shuttle vector transformation plate, so some clones are selected and spread and a new LB plate with the appropriate antibiotic
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12 liquid culture in LB are launched for the clones Lyso+Dsp in PSB1C3
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- Miniprep of 14 clones transformed with the ligation product [Constitutiv Promoter + RBS + Lysostaphin] in pBK25 and electrophoresis to analyse the extracted DNA : the transformation is succesful for 3 clones =)
 +
- The first plasmid  [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (1).
 +
- The second plasmid  [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (2).
 +
- The third plasmid  [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (3).
 +
Surfactant :
 +
- Transformation of pXyl+pSB1T3 in NM522.
 +
Stick :
 +
- Results of pBK24-xylR transformation: most of the clones are red, however a few of them are white, which is good. 7 white clones are put in liquid culture for extraction.
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- Extraction of pBK7-xylR from 12 clones, and then a gel electrophoresis is run: 2 clones seem interesting. We ran a second gel to verify these 2 clones, but it showed us that they were not good.
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------------------------------------------------------------------------------------------------------------------
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Friday 24th August
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For all :
 +
 +
the transformations of the cloned shuttle vectors was successful, so 12 clones are put in liquid culture for further tests;
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Kill:
 +
Surfactant :
 +
 +
3A ligation of pBK4 (pUC57 containing the lacI gene) and pBK29 (pSB1A3 containing the sfp and abrB genes) in pBK24 backbone (pSB1C3 containing a RFP gene). The cloned fragment was transformed in the NM522 strain;
 +
 +
Stick :
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- After the bad results with xylR, we decided to cut pBK10 plasmid in Sma1 site, and ligate with xylR, doing a blunt ligation. We also decided to ligate xylR with xylR, creating a big polymer, which will be like a “pre-ligation” molecule.
 +
- Extraction of pBK24-xylR from 7 clones. Gel electrophoresis is run → ONE CLONE IS GOOD!! meaning that it’s the first time we manage to ligate xylR successfully.
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------------------------------------------------------------------------------------------------------------------
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Saturday, 25th August
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For all :
 +
 +
miniprep of 12 clones selected from transformed bacteria with the cloned shuttle vectors; we were out of SpeI enzyme, so we could not digest the extracted plasmids.
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Kill:
 +
Surfactant :
 +
 +
Stick :
 +
-
 +
------------------------------------------------------------------------------------------------------------------
 +
 +
Sunday, 26th August
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For all :
 +
Kill:
 +
Surfactant :
 +
 +
miniprep of 6 clones (3A ligation: pBK24, pBK4, pBK29). The electrophoresis showed that none of the tested clones had integrated the right plasmid.
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Stick :

Revision as of 17:09, 10 September 2012


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