Team:Lyon-INSA/notebook

From 2012.igem.org

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                       </jour>
                       </jour>
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<jour nb="29">
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                        <date>Wednesday, August 29th 2012</date>
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Wednesday, 29th August
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                        <titre>Kill</titre>
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For all :
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<description>
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Kill:
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<ul>
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- Transformation of the previous ligation promoter-disp-pBK25 in E. Coli NM522.
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<li>Transformation of the previous ligation [promoter-dispersin-pBK25] in <i>E. coli</i> NM522.</li>
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- Results of the second transformation of BS 168 strain with the pBK28 (1) in the shuttle vector : the negative control is always full of bacteria whereas the erythromycin concentration was higher than the previous try (0,5 µg/mL the first time, 5 and 10 µg/mL the second time).
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<li>Results of the second transformation of BS 168 strain with the pBK28 (1) in the shuttle vector : the negative control is still full of bacteria even though the erythromycin concentration was higher than the previous trial (0,5 µg/mL the first time, 5 and 10 µg/mL the second time).</li>
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We made (GL+Ery) plates with different erythromycin concentrations in order to make different tests.
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<li>We made (GL+Ery) plates with different erythromycin concentrations in order to make different tests.</li>
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</ul>
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Surfactant :
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</description>
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                      <titre>Surfactant</titre>
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only one of the 24 screened colonies concerning the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios ) did not turn up to be red (LB+Cm plate) and did not grow on a LB+Amp plate. The clone was used to inoculate a liquid culture for an extraction.
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<description>
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<ul>
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miniprep of the two clones containing the genes sfp and abrB. The gel electrophoresis did not turn out as expected, probably because the bacteria used for inoculating the liquid cultures were contaminated or did not come from the same colony. We decided to transform the low purity plasmids into the NM522 strain;
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<li>Only one of the 24 screened colonies concerning the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios ) did not turn up to be red (LB+Cm plate) and did not grow on a LB+Amp plate. The clone was used to inoculate a liquid culture for an extraction.</li>
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given the fact we were not very optimistic concerning the 3A ligation of pBK24, pBK4 and pBK29, we decided to do two more essays, this time using  two ligated genes (sfp and abrB) coming from different clones other than the one containing pBK22. However, the miniprep of the bacteria which were supposed to contain the two plasmids was unsatisfying, so we decided to do the 3A ligation with the low purity plasmids, isolated with the classical protocol because we were running out of time and we were behind the schedule.
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<li>Miniprep of the two clones containing the genes sfp and abrB. The gel electrophoresis did not turn out as expected, probably because the bacteria used for inoculating the liquid cultures were contaminated or did not come from the same colony. We decided to transform the low purity plasmids into the NM522 strain.</li>
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in parellel, we decided to do a standard ligation in order to transfer the lacI gene into a plasmid having an antibiotic resistance other than Ampicillin. This way, in case the 3A ligations fails, we would not be forced to do again a 3A ligation because the plasmids would have different antibiotic resistances.
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<li>Given the fact that we were not very optimistic concerning the 3A ligation of pBK24, pBK4 and pBK29, we decided to do two more trials, this time using  two ligated genes (sfp and abrB) coming from different clones other than the one containing pBK22. However, the miniprep of the bacteria which were supposed to contain the two plasmids was unsatisfying, so we decided to do the 3A ligation with the low purity plasmids, isolated with the classical protocol because we were running out of time and we were behind the schedule.</li>
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<li>In parellel, we decided to do a standard ligation in order to transfer the lacI gene into a plasmid having an antibiotic resistance other than Ampicillin. This way, in case the 3A ligations fails, we would not be forced to do again a 3A ligation because the plasmids would have different antibiotic resistances.</li>
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Stick :
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</ul>
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</description>
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                      </jour>
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Thursday, 30th August
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For all :
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Kill:
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Transformation results, too much clones on the control digest vector not liguate so the transformation can’t be used for the transformation Lyso+Dsp in PSB1C3
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12 clones put in culture from the transformation plate Lyso+Dsp in the shuttle navette treated by BamHI
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Preparation of tubes for sequencing, launch of miniprep and midiprep
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- Standard ligation between the shuttle vector pBK26 (PHT315 modified) and the pBK23 plasmid (= Lysostaphine in pSB1C3). Transformation of the ligation product in NM522 strain.
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Surfactant :
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all 5 transformations were successful. White colonies are selected (pBK24 is a plasmid containing RFP in the pSB1C3 backbone, so we can exclude the colonies having integrated the religated backbone) and tested on LB+Cm and LB+Amp plates.
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<jour nb="30">
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the miniprep of the one clone that had the expected phenotype (obtained after the transformation of the 3A ligation of pBK24, pBK4, pBK29) was digested, but the gel electrophoresis din not turn out as expected.  
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                        <date>Thursday, August 30th 2012</date>
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Stick :
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                        <titre>Kill</titre>
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<description>
 +
<ul>
 +
<li>Transformation results : too many clones on the control digestion vector not ligated so the transformation can’t be used for the transformation [Lysostaphin+Dispersin] in pSB1C3.</li>
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<li>12 clones put in culture from the transformation plate [Lysostaphin + Dispersin] in the shuttle navette treated by BamHI.</li>
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<li>Preparation of tubes for sequencing, launch of miniprep and midiprep.</li>
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<li>Standard ligation between the shuttle vector pBK26 (PHT315 modified) and the pBK23 plasmid (= Lysostaphin in pSB1C3). Transformation of the ligation product in NM522 strain.</li>
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</ul>
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</description>
 +
                      <titre>Surfactant</titre>
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<description>
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All 5 transformations were successful. White colonies are selected (pBK24 is a plasmid containing RFP in the pSB1C3 backbone, so we can exclude the colonies having integrated the religated backbone) and tested on LB+Cm and LB+Amp plates.<br />
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The miniprep of the one clone that had the expected phenotype (obtained after the transformation of the 3A ligation of pBK24, pBK4, pBK29) was digested, but the gel electrophoresis did not turn out as expected.
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</description>
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                      </jour>
   
   
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<jour nb="31">
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Friday, 31th August
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                        <date>Friday, August 31st 2012</date>
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For all :
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                        <titre>Kill</titre>
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Kill:
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<description>
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Check of clones Lysostaphin-Dispersine in the shuttle vector (pBK26): clones are not good
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<ul>
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Check of the miniprep for sequencing (Prom+Dsp in PSB1C3): DNA is okay
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<li>Verification of [Lysostaphin-Dispersin] in the shuttle vector (pBK26) clones : clones are not good.</li>
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- Result of the transformation of NM 522 strain with the Lysostaphin inPBK26 :  
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<li>Verification of the miniprep for sequencing ([Promoter + Dispersin] in pSB1C3) : DNA is okay.</li>
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          - the negativ control is ok.
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<li>Result of the transformation of NM 522 strain with the Lysostaphin in PBK26 :</li>
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          - the positiv control is full of colonies.
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      <ul>
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        - the transformation is full of colonies : 24 clones are selected (on plates and in liquid culture) in order to extract their DNA and check it.  
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            <li>The negative control is ok;</li>
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Surfactant :
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            <li>The positive control is full of colonies;</li>
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            <li>The transformation is full of colonies : 24 clones are selected (on plates and in liquid culture) in order to extract their DNA and check it. </li>
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      </ul>
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</ul>
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</description>
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                      <titre>Surfactant</titre>
miniprep of the saturated cultures with transformed bacteria containing the ligation of the sfp and abrB genes. The electrophoresis confirmed that this time the bacteria contained the rigth plasmids.
miniprep of the saturated cultures with transformed bacteria containing the ligation of the sfp and abrB genes. The electrophoresis confirmed that this time the bacteria contained the rigth plasmids.

Revision as of 15:17, 11 September 2012


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