Team:Lyon-INSA/notebook

From 2012.igem.org

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<jour>
<jour>
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<date> Monday, July 13th 2012</date>
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<date> Monday, July 16th 2012</date>
                         <titre>Kill</titre>
                         <titre>Kill</titre>
<description>
<description>
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     <li>Gel electrophoresis of lysostaphin.</li>
     <li>Gel electrophoresis of lysostaphin.</li>
</ul>
</ul>
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</description>
 
                         <titre>For all purposes</titre>
                         <titre>For all purposes</titre>
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<description>
 
<ul>
<ul>
       <li>Transformation of B0015 terminator.</li>
       <li>Transformation of B0015 terminator.</li>
       <li>Strains BK13 (Bs arbB), BK10 (Bs 168 GFP) and BK11 (Bs 168 mCherry) are put in storage.</li>
       <li>Strains BK13 (Bs arbB), BK10 (Bs 168 GFP) and BK11 (Bs 168 mCherry) are put in storage.</li>
</ul>
</ul>
 +
</description>
 +
</jour>
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 +
<jour>
 +
<date> Tuesday, July 17th 2012</date>
 +
                        <titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
    <li>Long morning meeting to discuss results and to write some posters in a frenetic psychopath way in order to remember our tasks.</li>
 +
    <li>Ligation of promoter-rbs-GFP in plasmid.</li>
 +
    <li>Bs’ genomic DNA extraction: buffers preparation.</li>
 +
    <li>Failure of B0015 transformation.</li>
 +
</ul>
 +
                        <titre>Kill</titre>
 +
<ul>
 +
      <li>Cloning of the constitutive promoter in front of the disp part (the gene coding for Dispersin) and RBS/GFP (of Ecoli) using a 3A ligation followed by transformation of the ligation in the NM522 strain;</li>
 +
      <li>Extraction of pUC57-lysostaphin. Gel electrohporesis showed a shaded DNA → forgetting of RNase during the extraction.</li>
 +
</ul>
 +
</description>
 +
</jour>
 +
 +
<jour>
 +
<date> Wednesday, July 18th 2012</date>
 +
                        <titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
    <li>Extraction and digestion (E & P) of strain Bs 168 GFP’s plasmid. Gel electrohporesis showed absence of non digested plasmid → extraction failure.</li>
 +
    <li>Bs 168’s genomic DNA extraction (Kit Genomic DNA from tissue) .</li>
 +
    <li>Transformation of pBK5 in <i>B. subtilis</i> abrB failed.</li>
 +
</ul>
 +
                        <titre>Kill</titre>
 +
<ul>
 +
      <li>The transformation results of the 3A ligation ([disp+RBS/GFP+pSB1T3]) was unsuccessful, so it was repeated, this time with both a positive control (strain 39) and a negative one with NM522 culture ;</li>
 +
      <li>Addition of RNase to the miniprep from pUC57-lysostaphin clones. Gel electrophoresis: there is still a layered cut. However, the bands are as expected.</li>
 +
</ul>
 +
</description>
 +
</jour>
 +
 +
<jour>
 +
<date> Thursday, July 19th 2012</date>
 +
                        <titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
    <li>TSS tests are also made to be sure that all TSS solutions that we have are ok because some transformations did not work;</li>
 +
    <li>A 50ug/mL erythromycin solution is made in order to test the strains’ resistance;</li>
 +
    <li>Transformation of yfp, gfp et cfp (from iGEM plates) in NM522;</li>
 +
    <li>B0015 transformation in NM522.</li>
 +
</ul>
 +
                        <titre>Kill</titre>
 +
<ul>
 +
      <li>The transformation of the 3A ligation ([disp+RBS/GFP+pSB1T3]) was successful, so 6 clones were tested on  a plate containing BL media supplemented with Tetracyclin;</li>
 +
      <li>The fluorescence test of the transformed bacteria containing [disp+RBS/GFP+pSB1T3]  confirm that the promotor is functional</li>
 +
      <li>Ligation of the promoter in a Cm resistant iGEM plasmid was made in order to send it to the registry.</li>
 +
</ul>
 +
</description>
 +
</jour>
 +
 +
<jour>
 +
<date> Friday, July 20th 2012</date>
 +
                        <titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
    <li>Transformation results : all TSS work (except maybe 1 and 2 with a smaller yield) and prom+pSB1C3 OK → 6 clones are isolated on a GL+Cm plate.</li>
 +
    <li>Quantification and gel electrophoresis of B.subtilis’ genomic DNA.</li>
 +
    <li>Antibiotic testing of <i>B. thuringensis</i> 407 gfp strain and other strains in BL+Ery growth medium.</li>
 +
    <li>Failure of transforming pBK5 in <i>B. subtilis</i> 168 with the same protocol as previously. New task: find a new protocol.</li>
 +
</ul>
 +
                        <titre>Kill</titre>
 +
<ul>
 +
      <li>Transformation of NM522 strain with the part BBa_K606030 (pBK6) and promoter+dispersin;</li>
 +
      <li>Ligation Prom+Dsp in P23 and transformation;</li>
 +
      <li>Ligation Lysostaphin in pSB1C3 and transformation;</li>
 +
      <li>Isolation of 6 clones of the Term transformation;</li>
 +
      <li>Transformation results of fluorescent genes: ok, except yfp </li>
 +
      <li>NM522 strain containing pUC57-lysostaphin is put in storage under the name of BK12.</li>
 +
</ul>
 +
</description>
 +
</jour>
 +
 +
<jour>
 +
<date> Monday, July 23rd 2012</date>
 +
                        <titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
    <li>Selected clones (NM522+pBK6) are red, meaning that pVeg promoter and RBS from subtilis are recognized by <i>E. coli</i>’s ribosome. 4 clones are streaked in LB+Cm.</li>
 +
    <li>GL+Ery and TSB+Tet Petri plates are made.</li>
 +
    <li>YFP transformation was successful.</li>
 +
    <li>GFP and CFP plasmids’ extraction showed a failure in ligation.</li>
 +
    <li><i>B. subtilis</i> 168 is put in storage (BK14) in TSB medium.</li>
 +
    <li>Extraction of B0015 terminator. Gel electrophoresis showed 4 good clones.</li>
 +
</ul>
 +
                        <titre>Kill</titre>
 +
<ul>
 +
      <li>Transformation results</li>
 +
      <ul>
 +
              <li>Prom+Dsp in p23 : nothing on the plate;</li>
 +
              <li>Lysostaphin, negative witness contains bacteria so the plate is put in junk.</li>
 +
      </ul>
 +
      <li>New digestions, ligations,transformations:</li>
 +
      <ul>
 +
              <li>Lyso+Dsp in pUc57;</li>
 +
              <li>Dsp in PSB1C3;</li>
 +
              <li>Lyso in PSB1C3;</li>
 +
              <li>Prom+Dsp in p23.</li>
 +
      </ul>
 +
      <li>streaking of 4 clones of the transformed strain NM522/pBK6 on a Cm + LB plate. All of them formed red colonies which shows that the constitutive promotor and the RBS specific to the Bacillus subtilis strain are recognized by the RNA polymerase of <i>E coli</i>.</li>
 +
</ul>
 +
                      <titre>Stick</titre>
 +
PCR of XylR. Gel electrophoresis: the PCR didn’t work.
 +
</description>
</description>
</jour>
</jour>

Revision as of 04:09, 30 August 2012


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