Team:Lyon-INSA/notebook

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(Difference between revisions)
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     <li>Gel electrophoresis of lysostaphin.</li>
     <li>Gel electrophoresis of lysostaphin.</li>
</ul>
</ul>
-
                         <titre>For all purposes</titre>
+
                         <div class='titreExp'>For all purposes</div>
<ul>
<ul>
       <li>Transformation of B0015 terminator.</li>
       <li>Transformation of B0015 terminator.</li>
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     <li>Failure of B0015 transformation.</li>
     <li>Failure of B0015 transformation.</li>
</ul>
</ul>
-
                         <titre>Kill</titre>
+
                         <div class='titreExp'>Kill</div>
<ul>
<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>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>
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</jour>
</jour>
 +
<jour>
 +
<date> Tuesday, July 24th 2012</date>
 +
                        <titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
    <li>Plasmid extraction from Bacillus strain (BT407 GFP). We tested 2 methods in parallel. However, only the one using the extraction kit seems to work.</li>
 +
    <li>Extraction of gfp, cfp, yfp ;  test => OK : put in storage.</li>
 +
    <li>YFP transformation was successful.</li>
 +
    <li>Extraction of the pHT 315 GFP plasmid of <i>B. thuringiensis</i> 407 GFP to build a shuttle vector <i>B. subtilis</i> ⇔ <i>E. coli</i>. Transformation in NM522 strain and selection on Ampicillin media : ok.</li>
 +
    <li><i>B. subtilis</i> 168 is put in storage (BK14) in TSB medium.</li>
 +
    <li><i>S. epidermidis</i> is put in storage in TSB media under the reference BK15.</li>
 +
</ul>
 +
                        <titre>Kill</titre>
 +
<ul>
 +
      <li>Transformation results:
 +
      <ul>
 +
              <li>Lyso+Dsp in pUc57 : the plate is covered of clones;</li>
 +
              <li>Promoter+Dsp : nothing on the plate;</li>
 +
              <li>Dsp in PSB1C3 : a lot of clones;</li>
 +
              <li>Lyso in PSB1C3 : a lot of clones.</li>
 +
      </ul>
 +
      Selection of 4 clones of each lyso+igem plasmid and dsp+igem plasmid on Cm plate and in a liquid culture. Isolation of lyso+dsp (because there are too many clones!)</li>
 +
 +
      <li>Extraction of the plasmidic DNA [Promoter in PSB1C3].</li>
 +
      <li>Extraction of pBK6 from the transformed strain.</li>
 +
      <li>Extraction of the [Promoter+RBS+GFP] in pBK 23 (Tet R) by miniprep and check by electrophoresis after digesting by EcoRI and PstI : gel ok. Congrats (pBK12) -> 122,6 ng/µL.</li>
 +
      <li>Extraction of the pBK6 plasmid (PVeg+RBS+RFP in psB1C3). The electrophoresis confirms the plasmid’s structure.</li>
 +
      <li>- Liquid culture of Bs168 pWG100 overnight in order to do the first lysostaphin tests on plates.</li>
 +
</ul>
 +
                      <titre>Stick</titre>
 +
New PCR of XylR with some modifications in the protocol : the PCR didn’t work.
 +
 +
</description>
 +
</jour>
 +
 +
<jour>
 +
<date> Wednesday, July 25th 2012</date>
 +
                        <titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
    <li>Strains are put in storage :</li>
 +
    <ul>
 +
            <li><i>S. epidermidis</i> on TSB+Tet (BK17);</li>
 +
            <li><i>B. thuringensis</i> 407 GFP on GL+Ery (BK16).</li>
 +
    </ul>
 +
    <li>Minipreps to extract the plasmidic DNA of the clones NM522 + pHT 315 GFP and check by electrophoresis : plasmid ok (digested by E, X, S, P and no digested).</li>
 +
    <li>Transformation trial of Bs 168 by pBK5 with a new procedure : Failure ⇒ the fault of the plasmid which seems not to be the plasmid waited...Try again with the shuttle vector pHT 315 (GFP or not).</li>
 +
    <li>The plasmid extraction protocol from <i>B. subtilis</i> was tested once again, with a few modifications. The results were unsatisfying. After electrophoresis, only genomic DNA was observed.</li>
 +
</ul>
 +
                        <titre>Kill</titre>
 +
<ul>
 +
      <li>Check of the plasmidic DNA extracted of the clones transformed by [Promoter in pSB1C3] : none of the 4 clones is ok.</li>
 +
      <li>Selection of 4 clones transformed by [lyso+dsp] on LB+Amp plates and on liquid cultures.</li>
 +
      <li>Miniprep to extract [Lyso+Cm] and [Disp+Cm] → Digestion.</li>
 +
      <li>3A ligation between : lysostaphin in puc57 + Terminator in pSB1K3 + pSB1C3. Check by electrophoresis : Lysostaphin, terminator and vector are ok but not the ligation (no DNA).</li>
 +
      <li>Lysostaphin tests (Bs 168 pWG100 supernatant) on antibiograms (diameter : 6mm) applied on GL+Tet plates having a biofilm already etablished and in development of <i>S. epidermidis.</i></li>
 +
</ul>
 +
                      <titre>Stick</titre>
 +
PCR on the <i>B. subtilis</i> 168 genome using universal primers of Phil Oger, and positive control with <i>Bulkhoderia cepacia</i> genome provided by Phil Oger. Goal : determine if the PCR problems come from the primers or the low concentration of genomic DNA of <i>B. subtilis</i> 168.
 +
</description>
 +
</jour>
 +
 +
<jour>
 +
<date> Thursday, July 26th 2012</date>
 +
                        <titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
    <li>Transformation of NM522 strain with pHT304 and pHT315 (2 shuttle vectors for <i>B. subtilis</i> and <i>E. coli</i>).</li>
 +
    <li>gDNA extraction with 2 differents protocols : the first for <i>Bacillus subtilis</i> 168 and the second for Gram- bacteria with some modifications. The first protocol gives a better yield.</li>
 +
    <li>PCR of ARNr 16s on B.s. 168 to test if PCR works in the bacteria without being lysed : it works.</li>
 +
    <li>NM522 + gfp, cfp and yfp in storage.</li>
 +
    <li>pHT 315 GFP put in storage under the reference pBK 18.</li>
 +
    <li>NM522 + pHT 315 GFP strain put in storage under the reference BK 21.</li>
 +
</ul>
 +
                        <titre>Kill</titre>
 +
<ul>
 +
      <li>Other clones transformed with the Constitutive Promoter in pSB1C3 are selected in order to do other check by extracting their plasmidic DNA. ( bur they are not good again).</li>
 +
      <li>Extraction of the plasmidic DNA of the clones transformed with Lyso+Disp in pBK2 and check by electrophoresis gel. The clones are not right.</li>
 +
</ul>
 +
                      <titre>Surfactant</titre>
 +
3A assembly rbs-gfp (pbk7-pbk14) in psb1K3 = L1. Transformation of L1 in NM522.
 +
</description>
 +
</jour>
 +
 +
<jour>
 +
<date> Friday, July 27th 2012</date>
 +
                        <titre>For all purposes</titre>
 +
<description>
 +
Extraction of transformed clones (NM522/pHT304 et NM522/pHT315). The electrophoresis showed that the plasmids had approximately 7kb  and the restriction sites XbaI, EcorI and PstI as expected. However, there is a SpeI site which was not mapped.
 +
 +
                        <titre>Kill</titre>
 +
<ul>
 +
      <li>Extraction of plasmidic DNA : clones Lysostaphin in PSB1C3, Promoter in pSB1C3.</li>
 +
      <li>Plasmidic DNA check by digestion and electrophoresis : Clones Lysostaphin okay and Promoter clones are not okay.</li>
 +
      <li>New 3A ligation with Lyso/Terminator/pSB1C3 and transformation in NM522 strain.</li>
 +
      <li>Results of the lysostaphin tests on plates : no effect (regular biofilms)</li>
 +
</ul>
 +
                      <titre>Surfactant</titre>
 +
<ul>
 +
      <li>sfp (surfactin part 1) and abrB put in storage : pBK16 et pBK17.</li>
 +
      <li>Failure of transformation of L1 in NM522.</li>
 +
      <li>3A assembly of RBS-CFP (pBK7-pBK13) in pSB1C3 = L2. (!! the part2 construction already has a terminator)</li>
 +
      <li>3A assembly of sfp-part2(lacI)-term (pBK4-pBK10) in pSB1C3 = IV.</li>
 +
</ul>
 +
                      <titre>Stick</titre>
 +
<ul>
 +
      <li>PCR of xylR from gDNA extracted yesterday. Test on gel : PCR successful !!</li>
 +
      <li>3A ligation of RBS (pBK7) and XylR (produced by PCR) in pSB1C3 ( ! the vector plamid has the same resistance as the plasmid containing the RBS...).</li>
 +
</ul>
 +
</description>
 +
</jour>
 +
 +
<jour>
 +
<date> Monday, July 30th 2012</date>
 +
                        <titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
      <li>Meeting at 9 o’clock.</li>
 +
      <li><i>It was also a fire day : a man’s hair and a lab bench on fire !!!! As the saying goes, ”everything comes in threes, if it's happened twice, it'll happen a third time ” (we’re still waiting for the third fire...).</i></li>
 +
</ul>
 +
                        <titre>Kill</titre>
 +
<ul>
 +
      <li>Digestion test and gel of pBK2 (prom + lyso) by EcoRI and SpeI, pBK3 (disp + term) by XbaI and PstI and pSB1C3 by EcoRI and Pst1.</li>
 +
      <li>We got 3 clones for the transformation of NM522 bacteria with the ligation’s product Lyso+Terminater-pSB1C3 : the plasmidic DNA of these 3 clones is extracted and tested by electrophoresis gel ⇒ the 3 clones aren’t right.</li>
 +
      <li>Lysostaphin test on plate : this time, by trying with biofilm in formation less dense (OD = 0,5, dilution the culture x100, 1000 and 10 000).</li>
 +
</ul>
 +
                      <titre>Surfactant</titre>
 +
<ul>
 +
      <li>Transformation of L1, L2 et IV in NM522.</li>
 +
      <li>Transformation of sfp and abrB in NM522.</li>
 +
</ul>
 +
                      <titre>Stick</titre>
 +
Purification of XylR, produced by PCR.
 +
</description>
 +
</jour>
 +
 +
<jour>
 +
<date> Tuesday, July 31st 2012</date>
 +
                        <titre>Kill</titre>
 +
<description>
 +
Results of the lysostaphin tests on plates : negative. There is no biofilm : too diluated but even though, the colonies do not seem to be affected by the presence of Bs 168 pWG100 supernatant ⇒ the Bs 168 pWG100 have lost their plasmide ? (are cultivated without selection pressure, that is whitout erythromycin).
 +
                      <titre>Surfactant</titre>
 +
<ul>
 +
      <li>Given the fact that the previous transformation didn’t work, we made an other transformation of NM522 strain with the abrB and sfp parts.</li>
 +
      <li>Results of the transformations of L1, L2 and IV : ok. Selection of some clones in order to do minipreps.</li>
 +
</ul>
 +
                      <titre>Stick</titre>
 +
Ligation of RBS and XylR and transformation in NM522 strain.
 +
</description>
 +
</jour>
</month>
</month>
 +
 +
</project>
</project>
   </xml>
   </xml>

Revision as of 04:48, 31 August 2012


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