Team:Lyon-INSA/notebook

From 2012.igem.org

(Difference between revisions)
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<date> Tuesday, July 3rd 2012</date>
<date> Tuesday, July 3rd 2012</date>
<description><p>Dilution of 100 µL saturated culture in  5 mL LB medium. </p><br />
<description><p>Dilution of 100 µL saturated culture in  5 mL LB medium. </p><br />
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<p>Incubation time : 2 hours (until O.D =0,3). </p><br />
+
<p>Incubation time : 2 hours (until OD<sub>600</sub> = 0.3). </p><br />
-
Transformation of the NM522 strain  (this experiment was made 3 times) <br />
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Transformation of the NM522 strain  (this experiment was made 3 times)  
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<ul>
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For the positive control the pSB1C3 plasmid was used ;
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      <li>For the positive control the pSB1C3 plasmid was used;</li>
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For the transformation, the pBBA_I742123 was used (well A2, 5th iGEM 2012 kit plate)
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      <li>For the transformation, the pBBA_I742123 was used (well A2, 5th iGEM 2012 kit plate).</li>
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The transformed bacteria were selected on chloramphenicol plates.
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</ul>
 +
The transformed bacteria were selected on chloramphenicol (Cm) plates.
</description>
</description>
</jour>
</jour>
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Transformation analysis :<br/><br/>
Transformation analysis :<br/><br/>
<ul>
<ul>
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     <li>Positive control : lots of colonies</li>
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     <li>Positive control : lots of colonies;</li>
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   <li>Negative control : one of the three negative controls was contaminated (the correspondent transformed colonies were not used). No colonies were observed on the other two negative control plates.</li>
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   <li>Negative control : one of the three negative controls was contaminated (the correspondent transformed colonies were not used). No colonies were observed on the other two negative control plates;</li>
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     <li>Test plate: between 1 and 8 were observed.</li></ul>
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     <li>Test plate : between 1 and 8 were observed.</li></ul>
<br/>
<br/>
4 liquid cultures (5mL LB medium + 50 µL chloramphenicol) and 4 streaked chloramphenicol plates were made using isolated colonies likely to contain transformed bacteria.<br/><br/>
4 liquid cultures (5mL LB medium + 50 µL chloramphenicol) and 4 streaked chloramphenicol plates were made using isolated colonies likely to contain transformed bacteria.<br/><br/>
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<description>
<description>
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Telephonic conference with Romain Briandet<br/><br/>
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Telephonic conference with Romain Briandet.<br/><br/>
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Terminator was retrieved from the plate 1 well 13D<br/><br/>
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Terminator was retrieved from the plate 1 well 13D.<br/><br/>
Long meeting
Long meeting
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<date> Tuesday, July 10th 2012</date>
<date> Tuesday, July 10th 2012</date>
<description>
<description>
-
    the following parts were put in storage:
+
<li> The following parts were put in storage :</li>
<ul>
<ul>
         <li>Lysostaphin in pUC57 Amp resistant (pBK2);</li>
         <li>Lysostaphin in pUC57 Amp resistant (pBK2);</li>
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         <li>Dispersin in pUC57 Amp resistant (pBK3);</li>
         <li>Dispersin in pUC57 Amp resistant (pBK3);</li>
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         <li>Surfactin part 2 (RBS-lacI-terminator) dans pUC57 Amp resistant (pBK4)</li>
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         <li>Surfactin part 2 (RBS-lacI-terminator) dans pUC57 Amp resistant (pBK4).</li>
</ul>
</ul>
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  and the following strains:
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<li> and the following strains :</li>
<ul>   
<ul>   
-
         <li>S epi on BL + Tet (BK1)</li>
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         <li>S. epidermidis on BL + Tet (BK1);</li>
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         <li>Bs abrB on BL + Cm (BK2)</li>
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         <li>Bs abrB on BL + Cm (BK2);</li>
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         <li>Bs 168 on BL (BK3)</li>
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         <li>Bs 168 on BL (BK3).</li>
</ul>
</ul>
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    ligation of Dispersin and Lysostaphin biobricks:
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  <li>  Ligation of Dispersin and Lysostaphin biobricks :</li>
<ul>
<ul>
         <li>digestion of pBK2 with the restriction enzymes EcoRI and SpeI;</li>
         <li>digestion of pBK2 with the restriction enzymes EcoRI and SpeI;</li>
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         <li>3A ligation of the digested parts;</li>
         <li>3A ligation of the digested parts;</li>
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         <li>electrophoresis control showed the expected fragment for lysostaphin and dispersin (2,1 kb). However, the digested backbone plasmid had 3 fragments instead of 2.<br/>
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         <li>electrophoresis control showed the expected fragment for lysostaphin and dispersin (2,1 kb). However, the digested backbone plasmid had 3 fragments instead of 2.</li></ul>
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     <strong>Plasmid A2 extraction with a midiprep (pBK5) and digestion ? 3 stripes are observed : PROBLEM. Digestion is made again with E, P and E+P ? There are 2 Pst1 sites !!! WRONG PLASMID</strong></li>
+
     <li><strong>Plasmid A2 extraction with a midiprep (pBK5) and digestion 3 stripes are observed : PROBLEM. Digestion is made again with E, P and E+P There are 2 Pst1 sites !!! WRONG PLASMID</strong></li>
-
     <li>transformation of NM522 strain with the part BBa_K606061 Chloramphenicol resistant;</li>
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     <li>Transformation of NM522 strain with the part BBa_K606061 Chloramphenicol resistant;</li>
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     <li>transformation of NM522 strain with the part BBa_B0010 Ampicillin resistant;</li>
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     <li>Transformation of NM522 strain with the part BBa_B0010 Ampicillin resistant;</li>
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     <li>transformation of NM522 strain with the part BBa_K606040 Chloramphenicol resistant;</li>
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     <li>Transformation of NM522 strain with the part BBa_K606040 Chloramphenicol resistant;</li>
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     <li>design and order of the primers for the constitutive promotor (part BBa_K143012)</li>
+
     <li>Design and order of the primers for the constitutive promotor (part BBa_K143012)</li>
</ul>
</ul>
</description>
</description>
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     <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>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>Extraction of gfp, cfp, yfp ;  test => OK : put in storage.</li>
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    <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>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>
     <li><i>S. epidermidis</i> is put in storage in TSB media under the reference BK15.</li>
</ul>
</ul>
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       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>
       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 the plasmidic DNA [Promoter in pSB1C3].</li>
       <li>Extraction of pBK6 from the transformed strain.</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 [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>
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<description>
<description>
<ul>
<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>Verification of the extracted plasmidic DNA 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>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>Miniprep to extract [Lyso+Cm] and [Disp+Cm] ? Digestion.</li>

Revision as of 22:19, 21 September 2012


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