Team:Lyon-INSA/notebook

From 2012.igem.org

(Difference between revisions)
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<titre>For all purposes</titre>
<titre>For all purposes</titre>
<date> Tuesday, July 3rd 2012</date>
<date> Tuesday, July 3rd 2012</date>
-
<description><p>Dilution of 100 &micro;L saturated culture in  5 mL LB medium. </p><br />
+
<description><p>Dilution of 100 µL saturated culture in  5 mL LB medium. </p><br />
<p>Incubation time : 2 hours (until OD<sub>600</sub> = 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)  
+
Transformation of the NM522 strain (this experiment was repeated 3 times).
<ul>
<ul>
       <li>For the positive control the pSB1C3 plasmid was used;</li>
       <li>For the positive control the pSB1C3 plasmid was used;</li>
-
       <li>For the transformation, the pBBA_I742123 was used (well A2, 5th iGEM 2012 kit plate).</li>
+
       <li>For the transformation, the pBBA_I742123 was used (well A2, iGEM 2012 kit plate 5).</li>
</ul>
</ul>
The transformed bacteria were selected on chloramphenicol (Cm) plates.
The transformed bacteria were selected on chloramphenicol (Cm) plates.
Line 390: Line 390:
     <li>Positive control : lots of colonies;</li>
     <li>Positive control : lots of colonies;</li>
-
   <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>
+
   <li>Negative control : one of the three negative controls was contaminated (the correspondent transformed colonies were not used). No colonies were observed on the two other negative control plates;</li>
     <li>Test plate : between 1 and 8 were observed.</li></ul>
     <li>Test plate : between 1 and 8 were observed.</li></ul>
<br/>
<br/>
-
4 liquid cultures (5mL LB medium + 50 &micro;L 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/>
-
The antibiotic resistance (Ampicillin, Tetracyclin, Chloramphenicol and Kanamycin ) of the following strains was tested: BS 168, BS 168 MCherry, BS 168 GFP, BS 168 Lysostaphin, <i>Staphylococcus epidermidis</i>, BS Abrb.
+
The antibiotic resistance (Ampicillin, Tetracyclin, Chloramphenicol and Kanamycin) of the following strains was tested : BS 168, BS 168 MCherry, BS 168 GFP, BS 168 Lysostaphin, <i>Staphylococcus epidermidis</i>, BS Abrb.
</description>
</description>
</jour>
</jour>
Line 403: Line 403:
<date> Thursday, July 5th 2012</date>
<date> Thursday, July 5th 2012</date>
<description>
<description>
-
 
Antibiotics resistance tests :<br/><br/>
Antibiotics resistance tests :<br/><br/>
<ul>
<ul>
-
     <li>Bs 168 : no resistance</li>
+
     <li>Bs 168 : no resistance;</li>
-
 
+
     <li>Bs 168 M cherry : no resistance;</li>
-
     <li>Bs 168 M cherry : no resistance</li>
+
     <li>Bs 168 GFP : no resistance;</li>
-
 
+
     <li>Bs abrB : Cm resistant;</li>
-
     <li>Bs 168 GFP : no resistance</li>
+
     <li>Bs 168 lysostaphin PWG100 : no resistance;</li>
-
 
+
     <li><i>S. epidermidis</i> : Tet resistant.</li>
-
     <li>Bs abrB : Cm resistant</li>
+
-
 
+
-
     <li>Bs 168 lysostaphin PWG100 : no resistance</li>
+
-
 
+
-
     <li><i>S. epidermidis</i> : Tet resistant</li>
+
</ul>
</ul>
<br/>
<br/>
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<description>
<description>
-
Telephonic conference with Romain Briandet.<br/><br/>
+
Phone conference with Romain Briandet.<br/><br/>
-
Terminator was retrieved from the plate 1 well 13D.<br/><br/>
+
Terminator was retrieved from plate 1 well 13D.<br/><br/>
-
Long meeting
+
Long meeting.
</description>
</description>
Line 440: Line 434:
<description>
<description>
<ul>
<ul>
-
     <li>pBBa_I742123 was put in storage (under the reference pBK1);</li>
+
     <li>pBBa_I742123 was put in storage (under the reference pBK1).</li>
-
 
+
     <li>A liquid culture of NM522/pBK1 transformed cells was made so we could extract more plasmid DNA (50 mL LB media + 500 µL Chloramphenicol + 500 µL liquid culture of transformed bacteria ).</li>
-
     <li>A liquid culture of NM522/pBK1 transformed cells was made so we could extract more plasmid DNA (50 mL LB media + 500 &micro;L Cloramphenicol + 500 &micro;L liquid culture of transformed bacteria );</li>
+
     <li>The 3 genes coding for lysostaphin, dispersin and lacI repressor in pUC57 Ampicillin resistant plasmids were delivered.</li>
-
 
+
     <li>Transformation of NM522 strain with pUC57-Lysostaphin, pUC57-Dispersin and pUC57-sfp.</li>
-
     <li>We had the 3 genes coding for lysostaphin, dispersin and lacI repressor in pUC57 Ampicillin resistant plasmid delivered;</li>
+
     <li>200 µL of each transformed strains were spread on LB + Ampicillin plates.</li>
-
 
+
     <li>Liquid cultures of the following strains were made : Bs 168 in LB, Bs abrB in LB + Chloramphenicol, <i>S. epidermidis</i> in LB + Tetracyclin</li></ul>
-
     <li>Transformation of NM522 strain with pUC57-Lyso, pUC57-Disp and pUC57-sfp;</li>
+
-
 
+
-
     <li>200 &micro;L of each transformed strains were spread on LB media Ampicillin resistant plates
+
-
     liquid cultures of the following strains were made: Bs 168 in BL, Bs abrB in BL media supplemented with Chloramphenicol, S. epidermidis in BL media supplemented with Tetracyclin</li></ul>
+
</description>
</description>
Line 461: Line 451:
<ul>
<ul>
         <li>Lysostaphin in pUC57 Amp resistant (pBK2);</li>
         <li>Lysostaphin in pUC57 Amp resistant (pBK2);</li>
-
 
         <li>Dispersin in pUC57 Amp resistant (pBK3);</li>
         <li>Dispersin in pUC57 Amp resistant (pBK3);</li>
-
 
+
         <li>Surfactin part 2 (RBS-lacI-terminator) in pUC57 Amp resistant (pBK4).</li>
-
         <li>Surfactin part 2 (RBS-lacI-terminator) dans pUC57 Amp resistant (pBK4).</li>
+
</ul>
</ul>
-
 
+
  <li> and the following strains (in LB broth supplemented with required antibiotic) :</li>
-
  <li> and the following strains :</li>
+
<ul>   
<ul>   
-
         <li>S. epidermidis on BL + Tet (BK1);</li>
+
         <li><i>S. epidermidis</i> = BK1 (Tet<sup>R</sup>);</li>
-
 
+
         <li>Bs abrB = BK2 (Cm<sup>R</sup>);</li>
-
         <li>Bs abrB on BL + Cm (BK2);</li>
+
         <li>Bs 168 = BK3.</li>
-
 
+
-
         <li>Bs 168 on BL (BK3).</li>
+
</ul>
</ul>
-
  <li>   Ligation of Dispersin and Lysostaphin biobricks :</li>
+
  <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>
-
 
         <li>digestion of pBK3 with the restriction enzymes PstI and XbaI;</li>
         <li>digestion of pBK3 with the restriction enzymes PstI and XbaI;</li>
-
 
         <li>digestion of pBK4 with the restriction enzymes EcoRI and PstI;</li>
         <li>digestion of pBK4 with the restriction enzymes EcoRI and PstI;</li>
-
 
         <li>3A ligation of the digested parts;</li>
         <li>3A ligation of the digested parts;</li>
-
 
+
         <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>
-
         <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>
+
     <li>Plasmid A2 extraction with a midiprep (pBK5) and digestion → 3 stripes are observed : <strong>PROBLEM. Digestion is made again with E, P and E+P → There are 2 PstI 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>
     <li>Transformation of NM522 strain with the part BBa_K606061 Chloramphenicol resistant.</li>
-
 
     <li>Transformation of NM522 strain with the part BBa_B0010 Ampicillin resistant.</li>
     <li>Transformation of NM522 strain with the part BBa_B0010 Ampicillin resistant.</li>
-
 
     <li>Transformation of NM522 strain with the part BBa_K606040 Chloramphenicol resistant.</li>
     <li>Transformation of NM522 strain with the part BBa_K606040 Chloramphenicol resistant.</li>
-
     <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 promoter (part BBa_K143012).</li>
</ul>
</ul>
</description>
</description>
</jour>
</jour>
 +
<jour nb="11">
<jour nb="11">
<titre>For all purposes</titre>
<titre>For all purposes</titre>
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<description>
<description>
<ul>
<ul>
-
       <li>Extraction of part BBa_K606061 (RBS) Chloramphenicol resistant from transformed NM522 strain;</li>
+
       <li>Extraction of part BBa_K606061 (RBS) Chloramphenicol resistant from transformed NM522 strain.</li>
-
       <li>Extraction of part BBa_B0010 (terminator) Ampicillin resistant from transformed NM522 strain; <i>(nb : the clones were pink which means that the part contains a RFP gene)</i></li>
+
       <li>Extraction of part BBa_B0010 (terminator) Ampicillin resistant from transformed NM522 strain. <i>(NB : the clones were pink which means that the part contains a RFP gene)</i></li>
-
       <li>Extraction of part BBa_K606040 (pLacI) from transformed NM522 strain;</li>
+
       <li>Extraction of part BBa_K606040 (pLacI) from transformed NM522 strain.</li>
-
       <li>Extraction of  pUC57-Lyso/pUC57-Disp/pUC57-lacI from transformed NM522 strains;</li>
+
       <li>Extraction of  pUC57-Lysostaphin/pUC57-Dispersin/pUC57-lacI from transformed NM522 strains.</li>
-
       <li>Transformation of NM522 strain with the part BBa_0010 Ampicillin resistant. The observed phenotype does not correspond to the description found in the registry (the colour of the colonies is pink). We decided to make another transformation with the same part, only this time the 2011 kit was used.</li>
+
       <li>Transformation of NM522 strain with the part BBa_0010 Ampicillin resistant. The observed phenotype does not correspond to the description found in the registry (the color of the colonies is pink). We decided to make another transformation with the same part, only this time the 2011 kit was used.</li>
</ul>
</ul>
</description>
</description>
Line 516: Line 496:
<description>
<description>
<ul>
<ul>
-
     <li>PCR product electrophoresis of Promoter PCR : the product is not the good one.</li>
+
     <li>PCR product electrophoresis of Promoter PCR : the product is not the good one.</li>
-
     <li>Reception and storage of the primers (for the amplification of the BBa_K143012 part);</li>
+
     <li>Reception and storage of the primers (for the amplification of the BBa_K143012 part).</li>
-
     <li>The transformed NM522 strain with  the part  BBa_0010 Ampicillin resistant from the 2011 iGEM kit shows the same phenotype as the part found in the 2012 kit;</li>
+
     <li>The transformed NM522 strain with  the part  BBa_0010 Ampicillin resistant from the 2011 iGEM kit shows the same phenotype as the part found in the 2012 kit.</li>
     <li>Extractions with a miniprep kit are made :
     <li>Extractions with a miniprep kit are made :
     <ul>
     <ul>
-
             <li>4 clones containing the pLac promotor (1,2,3,4)</li>
+
             <li>4 clones containing the pLac promoter (1,2,3,4);</li>
-
             <li>4 clones containing the Bacillus subtilis RBS (1,2,3,4)</li>
+
             <li>4 clones containing the Bacillus subtilis RBS (1,2,3,4);</li>
-
             <li>3 clones containing theTerminator 1,2,3</li>
+
             <li>3 clones containing theTerminator (1,2,3);</li>
-
             <li>4 clones containing the gene for Dispersin</li>
+
             <li>4 clones containing the gene for Dispersin;</li>
-
             <li>4 clones containing the gene for Lysostaphin</li>
+
             <li>4 clones containing the gene for Lysostaphin;</li>
-
             <li>4 clones containing the gene for lacI</li>
+
             <li>4 clones containing the gene for lacI.</li>
     </ul>
     </ul>
Then a digestion is made on a 0.7% agarose gel.</li>
Then a digestion is made on a 0.7% agarose gel.</li>
-
 
</ul>
</ul>
</description>
</description>
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<description>
<description>
<ul>
<ul>
-
     <li>PCR of the constitutive promotor (part BBa_K143012); ? the PCR did not work so we ran a new PCR with a more diluted promotor solution.</li>
+
     <li>PCR of the constitutive promoter (part BBa_K143012) the PCR did not work so we ran a new PCR with a more diluted promoter solution.</li>
     <li>The following strains are put in storage:
     <li>The following strains are put in storage:
     <ul>
     <ul>
-
             <li>NM522 containing lacI-pUC57</li>
+
             <li>NM522 containing lacI-pUC57;</li>
-
             <li>NM522 containing rbs-pUC57 </li>
+
             <li>NM522 containing rbs-pUC57;</li>
-
             <li>NM522 containing dsp-pUC57</li>
+
             <li>NM522 containing dispersin-pUC57;</li>
     </ul>
     </ul>
</ul>
</ul>
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<description>
<description>
<ul>
<ul>
-
     <li>PCR of constitutive promoter ? it didn’t work. A new PCR is run with modifications of settings. The annealing temperature was modified from 50°C to 57°C, and 3 solutions with different concentration were tested, however the concentration did not affect the yield.</li>
+
     <li>PCR of constitutive promoter it didn’t work. A new PCR is run with modifications of settings. The annealing temperature was modified from 50°C to 57°C, and 3 solutions with different concentration were tested, however the concentration did not affect the yield.</li>
     <li>Purification of the PCR product.</li>
     <li>Purification of the PCR product.</li>
     <li>Gel electrophoresis of lysostaphin.</li>
     <li>Gel electrophoresis of lysostaphin.</li>
Line 574: Line 553:
<description>
<description>
<ul>
<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>Long morning meeting to discuss results and to write some posters in a frenzied psychopath way in order to remember our tasks.</li>
     <li>Ligation of promoter-rbs-GFP in plasmid.</li>
     <li>Ligation of promoter-rbs-GFP in plasmid.</li>
-
     <li>Bs’ genomic DNA extraction: buffers preparation.</li>
+
     <li>Genomic DNA extraction of <i>B. subtilis</i> 168: buffers preparation.</li>
     <li>Failure of B0015 transformation.</li>
     <li>Failure of B0015 transformation.</li>
</ul>
</ul>
Line 583: Line 562:
<description>
<description>
<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 dispersin part (the gene coding for Dispersin) and RBS/GFP (of <i>E. coli</i>) 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>
+
       <li>Extraction of pUC57-lysostaphin. Gel electrophoresis showed a shaded DNA RNase addition in the extraction kit buffer was forgotten.</li>
</ul>
</ul>
</description>
</description>
Line 594: Line 573:
<description>
<description>
<ul>
<ul>
-
     <li>Extraction and digestion (E &amp; P) of strain Bs 168 GFP’s plasmid. Gel electrohporesis showed absence of non digested plasmid ? extraction failure.</li>
+
     <li>Extraction and digestion (E & P) of Bs 168 strain GFP’s plasmid. Gel electrophoresis showed absence of non digested plasmid extraction failure.</li>
-
     <li>Bs 168’s genomic DNA extraction (Kit Genomic DNA from tissue) .</li>
+
     <li>Genomic DNA extraction of <i>B. subtilis</i> 168 (Kit Genomic DNA from tissue).</li>
     <li>Transformation of pBK5 in <i>B. subtilis</i> abrB failed.</li>
     <li>Transformation of pBK5 in <i>B. subtilis</i> abrB failed.</li>
</ul>
</ul>
Line 602: Line 581:
<description>
<description>
<ul>
<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>The transformation results of the 3A ligation ([dispersin+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>
+
       <li>Addition of RNase to the miniprep from pUC57-lysostaphin clones. Gel electrophoresis : there is still a smear. However, the bands are as expected.</li>
</ul>
</ul>
</description>
</description>
Line 613: Line 592:
<description>
<description>
<ul>
<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>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>A 50µg/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>Transformation of yfp, gfp et cfp (from iGEM plates) in NM522;</li>
     <li>B0015 transformation in NM522.</li>
     <li>B0015 transformation in NM522.</li>
Line 622: Line 601:
<description>
<description>
<ul>
<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 transformation of the 3A ligation ([dispersin+RBS/GFP+pSB1T3]) was successful, so 6 clones were tested on a plate containing LB broth 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>The fluorescence test of the transformed bacteria containing [dispersin+RBS/GFP+pSB1T3]  confirm that the promoter 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>
       <li>Ligation of the promoter in a Cm resistant iGEM plasmid was made in order to send it to the registry.</li>
</ul>
</ul>
Line 634: Line 613:
<description>
<description>
<ul>
<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>Transformation results : all TSS work (except maybe 1 and 2 with a smaller yield) and promoter+pSB1C3 OK 6 clones are isolated on a LB+Cm plate.</li>
     <li>Quantification and gel electrophoresis of B.subtilis’ genomic DNA.</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>Antibiotic testing of <i>B. thuringensis</i> 407 gfp strain and other strains in LB+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>
+
     <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>
</ul>
</description>
</description>
Line 643: Line 622:
<description>
<description>
<ul>
<ul>
-
       <li>Transformation of NM522 strain with the part BBa_K606030 (pBK6) and promoter+dispersin;</li>
+
       <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 Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection) and transformation.</li>
-
       <li>Ligation Lysostaphin in pSB1C3 and transformation;</li>
+
       <li>Ligation Lysostaphin in pSB1C3 and transformation.</li>
-
       <li>Isolation of 6 clones of the Term transformation;</li>
+
       <li>Isolation of 6 clones of the Terminator transformation.</li>
-
       <li>Transformation results of fluorescent genes: ok, except yfp </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>
       <li>NM522 strain containing pUC57-lysostaphin is put in storage under the name of BK12.</li>
</ul>
</ul>
Line 658: Line 637:
<description>
<description>
<ul>
<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>Selected clones (NM522+pBK6) are red, meaning that P<sub>veg</sub> promoter and RBS from <i>B. subtilis</i> 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>LB+Ery and TSB+Tet Petri plates are made.</li>
     <li>YFP transformation was successful.</li>
     <li>YFP transformation was successful.</li>
     <li>GFP and CFP plasmids’ extraction showed a failure in ligation.</li>
     <li>GFP and CFP plasmids’ extraction showed a failure in ligation.</li>
Line 671: Line 650:
       <li>Transformation results</li>
       <li>Transformation results</li>
       <ul>
       <ul>
-
               <li>Prom+Dsp in p23 : nothing on the plate;</li>
+
               <li>Promoter+Dispersin in pIG23 : nothing on the plate;</li>
-
               <li>Lysostaphin, negative witness contains bacteria so the plate is put in junk.</li>
+
               <li>Lysostaphin, negative control contains bacteria so the plate is put in junk.</li>
       </ul>
       </ul>
       <li>New digestions, ligations,transformations:</li>
       <li>New digestions, ligations,transformations:</li>
       <ul>
       <ul>
-
               <li>Lyso+Dsp in pUc57;</li>
+
               <li>Lysostaphin+Dispersin in pUC57;</li>
-
               <li>Dsp in PSB1C3;</li>
+
               <li>Dispersin in pSB1C3;</li>
-
               <li>Lyso in PSB1C3;</li>
+
               <li>Lysostaphin in pSB1C3;</li>
-
               <li>Prom+Dsp in p23.</li>
+
               <li>Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection).</li>
       </ul>
       </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>
+
       <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 promoter and the RBS specific to the <i>Bacillus subtilis</i> strain are recognized by the RNA polymerase of <i>E. coli</i>.</li>
</ul>
</ul>
</description>
</description>
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
-
PCR of XylR. Gel electrophoresis: the PCR didn’t work.
+
PCR of xylR. Gel electrophoresis : the PCR didn’t work.
-
 
+
</description>
</description>
</jour>
</jour>
Line 697: Line 675:
<ul>
<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>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>
-
     <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 pHT315 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>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>
Line 707: Line 685:
       <li>Transformation results:
       <li>Transformation results:
       <ul>
       <ul>
-
               <li>Lyso+Dsp in pUc57 : the plate is covered of clones;</li>
+
               <li>Lysostaphin+Dispersin in pUC57 : the plate is covered of clones;</li>
-
               <li>Promoter+Dsp : nothing on the plate;</li>
+
               <li>Promoter+Dispersin : nothing on the plate;</li>
-
               <li>Dsp in PSB1C3 : a lot of clones;</li>
+
               <li>Dispersin in pSB1C3 : a lot of clones;</li>
-
               <li>Lyso in PSB1C3 : a lot of clones.</li>
+
               <li>Lysostaphin in pSB1C3 : a lot of clones.</li>
       </ul>
       </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>
+
       Selection of 4 clones of each lysostaphin+iGEM plasmid and dispersin+iGEM plasmid on Cm plate and in a liquid culture. Isolation of lysostaphin+dispersin (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/&micro;L.</li>
+
       <li>Extraction of the [Promoter+RBS+GFP] in pBK23 (Tet R) by miniprep and verification by electrophoresis after digestion 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>Extraction of the pBK6 plasmid (P<sub>veg</sub>+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>
+
       <li>Liquid culture of Bs168 pWG100 overnight in order to do the first lysostaphin tests on plates.</li>
</ul>
</ul>
</description>
</description>
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
-
New PCR of XylR with some modifications in the protocol : the PCR didn’t work.
+
New PCR of xylR with some modifications in the protocol : the PCR didn’t work.
</description>
</description>
Line 735: Line 713:
     <li>Strains are put in storage :</li>
     <li>Strains are put in storage :</li>
     <ul>
     <ul>
-
             <li><i>S. epidermidis</i> on TSB+Tet (BK17);</li>
+
             <li><i>S. epidermidis</i> in TSB+Tet (BK17);</li>
-
             <li><i>B. thuringensis</i> 407 GFP on GL+Ery (BK16).</li>
+
             <li><i>B. thuringensis</i> 407 GFP in LB+Ery (BK16).</li>
     </ul>
     </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>Minipreps to extract the plasmidic DNA of the clones NM522 + pHT315 GFP and verification by electrophoresis : plasmid ok (digested by E, X, S, P and not digested).</li>
-
     <li>Transformation attempt 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>Transformation attempt of Bs 168 by pBK5 with a new procedure : Failure → blaming the plasmid which seems not to be the expected plasmid... Try again with the shuttle vector pHT315 (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>
     <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>
</ul>
Line 747: Line 725:
<ul>
<ul>
       <li>Verification of the extracted plasmidic DNA 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 [lysostaphin+dispersin] on LB+Amp plates and in liquid cultures.</li>
-
       <li>Miniprep to extract [Lyso+Cm] and [Disp+Cm] ? Digestion.</li>
+
       <li>Miniprep to extract [Lysostaphin+Cm] and [Dispersin+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>3A ligation between : lysostaphin in pUC57 + Terminator in pSB1K3 + pSB1C3. Verification 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>
+
       <li>Lysostaphin tests (Bs 168 pWG100 supernatant) on antibiograms (diameter : 6mm) applied on LB+Tet plates having a biofilm already established and in development of <i>S. epidermidis.</i></li>
</ul>
</ul>
</description>
</description>
Line 765: Line 743:
<ul>
<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>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>gDNA extraction with 2 different 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>PCR of rRNA 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>NM522 + gfp, cfp and yfp in storage.</li>
-
     <li>pHT 315 GFP put in storage under the reference pBK 18.</li>
+
     <li>pHT 315 GFP put in storage under the reference pBK18.</li>
-
     <li>NM522 + pHT 315 GFP strain put in storage under the reference BK 21.</li>
+
     <li>NM522 + pHT315 GFP strain put in storage under the reference BK21.</li>
</ul>
</ul>
</description>
</description>
Line 775: Line 753:
<description>
<description>
<ul>
<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>Other clones transformed with the Constitutive Promoter in pSB1C3 are selected in order to do other verification by extracting their plasmidic DNA (but 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>
+
       <li>Extraction of the plasmidic DNA of the clones transformed with Lysostaphin+Dispersin in pBK2 and verification by electrophoresis gel. The clones are not right.</li>
</ul>
</ul>
</description>
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
-
3A assembly rbs-gfp (pbk7-pbk14) in psb1K3 = L1. Transformation of L1 in NM522.
+
3A assembly rbs-gfp (pBK7-pBK14) in pSB1K3 = L1. Transformation of L1 in NM522.
</description>
</description>
</jour>
</jour>
Line 794: Line 772:
<description>
<description>
<ul>
<ul>
-
       <li>Extraction of plasmidic DNA : clones Lysostaphin in PSB1C3, Promoter in pSB1C3.</li>
+
       <li>Extraction of plasmidic DNA : Lysostaphin in pSB1C3 clones, Promoter in pSB1C3 clones.</li>
-
       <li>Plasmidic DNA check by digestion and electrophoresis : Clones Lysostaphin okay and Promoter clones are not okay.</li>
+
       <li>Plasmidic DNA verification by digestion and electrophoresis : Lysostaphin clones okay and Promoter clones are not okay.</li>
-
       <li>New 3A ligation with Lyso/Terminator/pSB1C3 and transformation in NM522 strain.</li>
+
       <li>New 3A ligation with Lysostaphin/Terminator/pSB1C3 and transformation in NM522 strain.</li>
       <li>Results of the lysostaphin tests on plates : no effect (regular biofilms)</li>
       <li>Results of the lysostaphin tests on plates : no effect (regular biofilms)</li>
</ul>
</ul>
Line 806: Line 784:
       <li>Failure of transformation of L1 in NM522.</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 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>
+
       <li>3A assembly of sfp-part2(lacI)-terminator (pBK4-pBK10) in pSB1C3 = IV.</li>
</ul>
</ul>
</description>
</description>
Line 812: Line 790:
<description>
<description>
<ul>
<ul>
-
       <li>PCR of xylR from gDNA extracted yesterday. Test on gel : PCR successful !!</li>
+
       <li>PCR of xylR from gDNA extracted yesterday. Test on gel : successful PCR !!</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>
+
       <li>3A ligation of RBS (pBK7) and xylR (produced by PCR) in pSB1C3 ( ! the vector plasmid has the same resistance as the plasmid containing the RBS...).</li>
</ul>
</ul>
</description>
</description>
Line 824: Line 802:
<ul>
<ul>
       <li>Meeting at 9 o’clock.</li>
       <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>
+
       <li><strong>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…).</strong></li>
</ul>
</ul>
</description>
</description>
Line 830: Line 808:
<description>
<description>
<ul>
<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>Digestion test and gel of pBK2 (promoter + lysostaphin) by EcoRI and SpeI, pBK3 (dispersin + terminator) by XbaI and PstI and pSB1C3 by EcoRI and PstI.</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>We got 3 clones for the transformation of NM522 bacteria with the ligation’s product Lysostaphin+Terminator-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>
+
       <li>Lysostaphin test on plate : this time, by trying with biofilm in formation less dense (OD<sub>600</sub> = 0.5, dilution the culture x100, 1000 and 10 000).</li>
</ul>
</ul>
</description>
</description>
Line 844: Line 822:
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
-
Purification of XylR, produced by PCR.
+
Purification of xylR, produced by PCR.
</description>
</description>
</jour>
</jour>
Line 852: Line 830:
                         <titre>Kill</titre>
                         <titre>Kill</titre>
<description>
<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).
+
Results of the lysostaphin tests on plates : negative. There is no biofilm : too diluted but even though, the colonies do not seem to be affected by the presence of Bs 168 pWG100 supernatant → have the Bs 168 pWG100 lost their plasmid? (cultivated without selection pressure, that is without erythromycin).
</description>
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
<ul>
<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>Given the fact that the previous transformation didn’t work, we made another 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>
       <li>Results of the transformations of L1, L2 and IV : ok. Selection of some clones in order to do minipreps.</li>
</ul>
</ul>
Line 863: Line 841:
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
-
Ligation of RBS and XylR and transformation in NM522 strain.
+
Ligation of RBS and xylR and transformation in NM522 strain.
</description>
</description>
</jour>
</jour>
Line 885: Line 863:
               <li>Lysostaphin+dispersin : a lot of clones.</li>
               <li>Lysostaphin+dispersin : a lot of clones.</li>
       </ul>
       </ul>
-
       Cultures in liquid LB of Lyso+Dsp are launched.</li>
+
       Cultures in LB broth of Lysostaphin+Dispersin are launched.</li>
-
       <li>Digestion of Promoter clones and lysostaphin clones followed by an electophoresis : lysostaphin clones are okay, promoter clones are not okay.</li>
+
       <li>Digestion of Promoter clones and lysostaphin clones followed by an electrophoresis : lysostaphin clones are okay, promoter clones are not okay.</li>
-
       <li>pUC57 with Dsp put in storage : pBK3.</li>
+
       <li>pUC57 with Dispersin put in storage : pBK3.</li>
-
       <li>Lysostaphin test : the come back ! This time, we used more <i>S. epidermidis</i> (OD<sub>600</sub> = 0.75 diluted 1000 times) and  a control for the stability of the pWG100 plasmid (spreading of 200 &micro;L on GL + Ery plates), after the liquid culture without selective pressure. Spreading on LB plates without Tetracycline (but addition of Tetracycline into the Bs 168 pWG100 supernatant).</li>
+
       <li>Lysostaphin test returns ! This time, we used more <i>S. epidermidis</i> (OD<sub>600</sub> = 0.75 diluted 1000 times) and  a control for the stability of pWG100 plasmid (spreading of 200 µL on LB + Ery plates), after the liquid culture without selective pressure. Spreading on LB plates without Tetracycline (but addition of Tetracycline into the Bs 168 pWG100 supernatant).</li>
</ul>
</ul>
</description>
</description>
Line 896: Line 874:
       <li>The strain NM522/pBK6 was put in storage under the reference BK22.</li>
       <li>The strain NM522/pBK6 was put in storage under the reference BK22.</li>
       <li>Transformations of the NM522 strain with the ordered constructions (sfp and abrB in pUC57 AmpR). The transformation was unsuccessful, so we did it again.</li>
       <li>Transformations of the NM522 strain with the ordered constructions (sfp and abrB in pUC57 AmpR). The transformation was unsuccessful, so we did it again.</li>
-
       <li>Quantification of Sfp and abrB constructions provided by Genecust using the Nanodrop.</li>
+
       <li>Quantification of sfp and abrB constructions provided by Genecust using the Nanodrop.</li>
       <li>Miniprep of the L1, L2 and IV ligations : it didn’t work.</li>
       <li>Miniprep of the L1, L2 and IV ligations : it didn’t work.</li>
</ul>
</ul>
Line 907: Line 885:
<description>
<description>
<ul>
<ul>
-
       <li>The transformation of the Bacillus strain failed because of contaminated LB media, so a new transformation was attempted.</li>
+
       <li>The transformation of the Bacillus strain failed because of contaminated LB broth, so a new transformation was attempted.</li>
-
       <li>pHT 304 and pHT 315 plasmids supernumerary site SpeI deletion attempt by digestion, filling-in, ligation and transformation in NM522.</li>
+
       <li>pHT304 and pHT315 plasmids supernumerary site SpeI deletion attempt by digestion, filling-in, ligation and transformation in NM522.</li>
</ul>
</ul>
</description>
</description>
Line 914: Line 892:
<description>
<description>
<ul>
<ul>
-
       <li>Extraction of plasmidic DNA from the Lysostaphin + Dispersin clones in Cm iGEM plasmid (pSB1C3) and from the BK12 strain clones. Plasmidic DNA is checked by digestions.<br /> Lysostaphin+Dsp clones digestion : clones not okay.</li>
+
       <li>Extraction of plasmidic DNA from the Lysostaphin + Dispersin clones in Cm iGEM plasmid (pSB1C3) and from the BK12 strain clones. Plasmidic DNA is checked by digestions.<br /> Lysostaphin+Dispersin clones digestion : clones not okay.</li>
       <li>Ligations of :
       <li>Ligations of :
       <ul>
       <ul>
Line 921: Line 899:
       </ul>
       </ul>
       Ligation were verified by electrophoresis.</li>
       Ligation were verified by electrophoresis.</li>
-
       <li>Results of the Lysostaphin tests : some Bs 168 pWG100 contaminated the plate. However, we noticed a halo of 2-3 mm around the Bacillus colonies  where the <i>S. epidermidis</i> didn’t grow due to lysostaphin activity.</li>
+
       <li>Results of the Lysostaphin tests : some Bs 168 pWG100 contaminated the plate. However, we noticed a halo of 2-3 mm around the Bacillus colonies  where <i>S. epidermidis</i> didn’t grow due to lysostaphin activity.</li>
-
       <li>New Lysostaphin test on LB+Tet plate with a negative control (10 &micro;L of Ampicillin).</li>
+
       <li>New Lysostaphin test on LB+Tet plate with a negative control (10 µL of Ampicillin).</li>
</ul>
</ul>
</description>
</description>
Line 929: Line 907:
<ul>
<ul>
       <li>Miniprep of 5 clones of the transformed NM522 strain with RBS-xylR and 2 clones of the NM522 transformed with the abrB construction. Given the fact that the NM522 strain used for the transformations was contaminated, the electrophoresis showed unexpected results.</li>
       <li>Miniprep of 5 clones of the transformed NM522 strain with RBS-xylR and 2 clones of the NM522 transformed with the abrB construction. Given the fact that the NM522 strain used for the transformations was contaminated, the electrophoresis showed unexpected results.</li>
-
       <li>New ligation of RBS and XylR in pSB1A3 and transformation in NM522 strain.</li>
+
       <li>New ligation of RBS and xylR in pSB1A3 and transformation in NM522 strain.</li>
</ul>
</ul>
</description>
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
-
The transformation of Sfp and abrB in NM522 strain didn’t work.
+
Transformation of sfp and abrB in NM522 strain didn’t work.
</description>
</description>
</jour>
</jour>
Line 944: Line 922:
<ul>
<ul>
       <li>Meeting at 9 o’clock.</li>
       <li>Meeting at 9 o’clock.</li>
-
       <li>Purification of the NM 522 strain (because of the problems on the negative control during the transformation) and resistance tests on the purified clones.</li>
+
       <li>Purification of the NM522 strain (because of the problems on the negative control during the transformation) and resistance tests on the purified clones.</li>
       <li>The results of Bacillus transformation are inconclusive : there are only a few clones on the plate. However, the negative control has a few colonies too. Despite this result, we decided to verify six clones and extract their DNA.</li>
       <li>The results of Bacillus transformation are inconclusive : there are only a few clones on the plate. However, the negative control has a few colonies too. Despite this result, we decided to verify six clones and extract their DNA.</li>
-
       <li>A lot of clones for the transformation of the pHT 304 and pHT 315 plasmids (without SpeI site) on LB+Amp plates ? Purification of 12 clones of each.</li>
+
       <li>A lot of clones for the transformation of the pHT304 and pHT315 plasmids (without SpeI site) on LB+Amp plates Purification of 12 clones each.</li>
</ul>
</ul>
</description>
</description>
Line 952: Line 930:
<description>
<description>
<ul>
<ul>
-
       <li>Replication with velvet of the Lysostaphin+Dispersin in pSB1C3 transformation plate on LB + Amp.</li>
+
       <li>Velvet replication of Lysostaphin+Dispersin in pSB1C3 transformation plate on LB + Amp.</li>
-
       <li>Antibiotic resistance checked for 5 clones Lysostaphin+Dispersin in iGEM plasmid (clones 1 to 5).</li>
+
       <li>Antibiotic resistance checked for 5 Lysostaphin+Dispersin in iGEM plasmid clones (clones 1 to 5).</li>
-
       <li>BK12 strain check : spread on LB ampicillin.</li>
+
       <li>BK12 strain verification : spreading on LB + Amp plate.</li>
-
       <li>Ligation Promoter+Dispersin in Cm iGEM plasmid followed by transformation of the 3 ligations.</li>
+
       <li>Ligation of Promoter+Dispersin in Cm iGEM plasmid followed by transformation of the 3 ligations.</li>
       Ligation were verified by electrophoresis.</li>
       Ligation were verified by electrophoresis.</li>
       <li>Transformation of Promoter+Dispersin ligation.</li>
       <li>Transformation of Promoter+Dispersin ligation.</li>
-
       <li>PCR to check the presence of the promoter in the clones Promoter in Cm iGEM plasmid.</li>
+
       <li>PCR to check the presence of the promoter in the Promoter in Cm iGEM plasmid clones.</li>
-
       <li>Preliminary test of the liquid lysostaphin (10 &micro;L of tetracycline to kill <i>Bacillus</i>, 500 &micro;L of BS 168 pWG100 supernatant, 500 &micro;L of <i>S. epidermidis</i> with OD=1,2 (the strains are cultivated overnight at 30°C). This test shows a possible effect of lysostaphin (decrease of the OD with the time, during 3h30). To be repeated with a negative control without Bs 168 pWG100.</li>
+
       <li>Preliminary test of the liquid lysostaphin (10 µL of tetracycline to kill <i>Bacillus</i>, 500 µL of Bs 168 pWG100 supernatant, 500 µL of <i>S. epidermidis</i> with OD<sub>600</sub>=1.2 (the strains are cultivated overnight at 30°C). This test shows a possible effect of lysostaphin (decrease of OD<sub>600</sub> with the time, during 3h30). To be repeated with a negative control without Bs 168 pWG100.</li>
</ul>
</ul>
</description>
</description>
Line 967: Line 945:
       <li>Ligation (3A protocol) of pBK7, pBK13 and iGEM backbone pSB1K3 (=RBS+CFP+pSB1K3).</li>
       <li>Ligation (3A protocol) of pBK7, pBK13 and iGEM backbone pSB1K3 (=RBS+CFP+pSB1K3).</li>
       <li>Ligation (3A protocol) of pBK7, pBK14 and iGEM backbone pSB1K3 (=RBS+GFP+pSB1K3).</li>
       <li>Ligation (3A protocol) of pBK7, pBK14 and iGEM backbone pSB1K3 (=RBS+GFP+pSB1K3).</li>
-
       <li>Because of the difficulties to transform the bacteria with the plasmids containing Sfp and abrB, we decided to do an electrophoresis directly on the constructions sent by Genecust. Result : the genes were not inserted in a pUC57 plasmid.</li>
+
       <li>Because of the difficulties to transform the bacteria with the plasmids containing sfp and abrB, we decided to do an electrophoresis directly on the constructions sent by Genecust. Result : the genes were not inserted in a pUC57 plasmid.</li>
       <li>Ligation of abrB and sfp in pSB1K3 and transformation in NM522.</li>
       <li>Ligation of abrB and sfp in pSB1K3 and transformation in NM522.</li>
</ul>
</ul>
Line 973: Line 951:
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
-
New ligation between RBS and XylR in pSB1A3 and transformation in NM522 strain.
+
New ligation between RBS and xylR in pSB1A3 and transformation in NM522 strain.
</description>
</description>
</jour>
</jour>
Line 987: Line 965:
               <li>There are clones on every transformation plates (Promoter in pSB1C3, Dispersin in pSB1C3, Promoter+Dispersin in pSB1C3).</li>
               <li>There are clones on every transformation plates (Promoter in pSB1C3, Dispersin in pSB1C3, Promoter+Dispersin in pSB1C3).</li>
       </ul>
       </ul>
-
       8 clones per type of transformation are chosen and spread on LB+Cm and LB+Amp plates.</li>
+
       8 clones for each transformation are chosen and spread on LB+Cm and LB+Amp plates.</li>
</ul>
</ul>
</description>
</description>
Line 993: Line 971:
<description>
<description>
<ul>
<ul>
-
       <li>Transormations of abrB and sfp are a success !! :D</li>
+
       <li>Transformations of abrB and sfp are a success !! :D</li>
       <li>Liquid cultures of abrB and sfp clones are launched to do plasmid extractions.</li>
       <li>Liquid cultures of abrB and sfp clones are launched to do plasmid extractions.</li>
</ul>
</ul>
Line 999: Line 977:
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
-
Sorting of the RBS + xylR clones to eliminate the clones having two plasmids religated.
+
Sorting of the RBS + xylR clones to eliminate the clones containing two relegated plasmids.
</description>
</description>
</jour>
</jour>
Line 1,007: Line 985:
                         <titre>For all purposes</titre>
                         <titre>For all purposes</titre>
<description>
<description>
-
Liquid culture of 6 NM522 clones with pHT 304 S and 6 clones pHT 315 S.
+
Liquid culture of 6 NM522 clones with pHT304 S and 6 clones pHT315 S.
</description>
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
Line 1,015: Line 993:
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
-
Liquid cultures of RBS+XylR clones are launched to do plasmid extractions.
+
Liquid cultures of RBS+xylR clones are launched to do plasmid extractions.
</description>
</description>
</jour>
</jour>
Line 1,024: Line 1,002:
<description>
<description>
<ul>
<ul>
-
<li>Plasmid extraction from 6 clones BS 168 transformed by pH315 GFP : the electrophoresis of the digested plasmids showed that the clones had the right plasmid. The <i>Bacillus</i> transformation also worked, but the yield must be improved.</li>
+
<li>Plasmid extraction from 6 clones Bs 168 transformed by pH315 GFP : the electrophoresis of the digested plasmids showed that the clones had the right plasmid. The <i>Bacillus</i> transformation also worked, but the yield must be improved.</li>
-
<li>Miniprep and digestions by SpeI of the plasmid extracted from the 6 clones of NM522 with pHT 304 S and NM 522 with pHT 315 S : Failure ! The SpeI site is still on the plasmids :’( </li>
+
<li>Miniprep and digestions by SpeI of the plasmid extracted from the 6 clones of NM522 with pHT304 S and NM522 with pHT315 S : Failure ! The SpeI site is still in the plasmids :’( </li>
</ul>
</ul>
</description>
</description>
Line 1,031: Line 1,009:
<description>
<description>
<ul>
<ul>
-
<li>Selection of clones growing on LB Cm and not on LB Amp:
+
<li>Selection of clones growing on LB+Cm plates and not on LB+Amp plates :
       <ul>
       <ul>
               <li>4 clones Dispersin in pSB1C3;</li>
               <li>4 clones Dispersin in pSB1C3;</li>
Line 1,038: Line 1,016:
       </ul>
       </ul>
       Liquid cultures in LB+Cm are made with these clones. Then extraction of plasmidic DNA.</li>
       Liquid cultures in LB+Cm are made with these clones. Then extraction of plasmidic DNA.</li>
-
<li>8 clones Lysostaphin + Dispersin in pSB1C3 are spread on GL+Cm and GL+Amp to test their resistance.</li>
+
<li>8 clones Lysostaphin + Dispersin in pSB1C3 are spread on LB+Cm and LB+Amp to test their resistance.</li>
<li>Transformation of pSB1C3 and pSB1T3 in order to obtain a clone from the PCR matrix (red clone).</li>
<li>Transformation of pSB1C3 and pSB1T3 in order to obtain a clone from the PCR matrix (red clone).</li>
</ul>
</ul>
Line 1,050: Line 1,028:
             <li>pBK7+pBK14+pSB1K3 (RBS+GFP in Kanamycin resistant backbone).</li>
             <li>pBK7+pBK14+pSB1K3 (RBS+GFP in Kanamycin resistant backbone).</li>
       </ul>
       </ul>
-
<li>PCR of RBS-abrB and pXyl and purification of these PCR products.</li>
+
<li>PCR of RBS-abrB and P<sub>xyl</sub> and purification of these PCR products.</li>
</ul>
</ul>
</description>
</description>
Line 1,057: Line 1,035:
<ul>
<ul>
       <li>Purification of the xylR gene.</li>
       <li>Purification of the xylR gene.</li>
-
       <li>Miniprep of RBS-xylR and electrophoresis test the ligation didn’t work again...</li>
+
       <li>Miniprep of RBS-xylR and electrophoresis test the ligation failed again...</li>
</ul>
</ul>
</description>
</description>
Line 1,067: Line 1,045:
<description>
<description>
<ul>
<ul>
-
<li>There is a little plasmid pHT 304 S and pHT 315 S post filling-in, new digestion by Spel enzyme to eliminate the plasmids which are not full on the SpeI site in order to increase the amount of plasmids without SpeI site.</li>
+
<li>There is a little plasmid pHT304 S and pHT315 S post filling-in, new digestion by Spel enzyme to eliminate the plasmids which are not full on the SpeI site in order to increase the amount of plasmids without SpeI site.</li>
-
<li>Transformation in NM522 and spreading on LB Amp.</li>
+
<li>Transformation in NM522 and spreading on LB+Amp plates.</li>
</ul>
</ul>
</description>
</description>
Line 1,074: Line 1,052:
<description>
<description>
<ul>
<ul>
-
<li>8 new clones Lysostaphin+Dsp in PSB1C3 are screened on Ampicillin. Ampicillin sensitive clones are put in liquid culture.</li>
+
<li>8 new clones Lysostaphin+Dispersin in pSB1C3 are screened on Ampicillin. Ampicillin sensitive clones are put in liquid culture.</li>
-
<li>Checking of plasmids extract the previous day and electrophoresis : Dsp in pSB1C3 (clones not okay), Promoter+Dsp in pSB1C3 (Clones not okay).</li>
+
<li>Verification of plasmids extracted the previous day and electrophoresis : Dispersin in pSB1C3 (clones not okay), Promoter+Dispersin in pSB1C3 (Clones not okay).</li>
<li>The transformation of pSB1C3 and pSB1T3 didn’t work.</li>
<li>The transformation of pSB1C3 and pSB1T3 didn’t work.</li>
-
<li>New Lysostaphin liquid test with 3 measures and a negativ control (Bs 168 supernatant instead of Bs 168 pWG 100). This time, the cultures grew up one and a half day at 37°C (efficiency of the lysostaphin in stationary phase ?) the test is positive, lysostaphin has a right effect on the <i>S. epidermidis</i> cultures.</li>
+
<li>New Lysostaphin liquid test with 3 measurements and a negative control (Bs 168 supernatant instead of Bs 168 pWG 100). This time, the cultures grew over 36 hours at 37°C (efficiency of the lysostaphin in stationary phase ?) the test is positive, lysostaphin has a right effect on the <i>S. epidermidis</i> cultures.</li>
</ul>
</ul>
</description>
</description>
Line 1,083: Line 1,061:
<description>
<description>
<ul>
<ul>
-
<li>3A ligation : [pXyl-RBS-Sfp] + [RBS-abrB] + [psB1T3].</li>
+
<li>3A ligation : [P<sub>xyl</sub>-RBS-sfp] + [RBS-abrB] + [psB1T3].</li>
-
<li>Transformation of the ligation [pXyl-RBS-Sfp] + [RBS-abrB] + pSB1T3 in NM522 strain and LB+Tet medium.</li>
+
<li>Transformation of the ligation [P<sub>xyl</sub>-RBS-sfp] + [RBS-abrB] + pSB1T3 in NM522 strain and spreading LB+Tet plates.</li>
-
<li>Results of the transformation by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : Failure! The negativ control has few colonies : the LB+Kan plate was contaminated and the used LB medium too! The transformation is done again...</li>
+
<li>Results of the transformation by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : Failure! The negative control has a few colonies : the LB+Kan plate was contaminated and the used LB medium too! The transformation is done again...</li>
-
<li>Purification of pXyl produced by PCR.</li>
+
<li>Purification of P<sub>xyl</sub> produced by PCR.</li>
</ul>
</ul>
</description>
</description>
Line 1,096: Line 1,074:
<description>
<description>
<ul>
<ul>
-
<li>Result of the transformation of NM522 by pHT 304 S et pHT 315 S : 0 clone and 1 clone... this only clone is tested but the test is negative : the SpeI site is always here at 311 bp). The cultures of the transformation are concentrated and spread on GL plate : 0 clone for pHT 304 S and 18 for PHT 315 S but none of the clones is good. We must do the filling-in again...</li>
+
<li>Result of the transformation of NM522 by pHT304 S et pHT315 S : 0 clone and 1 clone... this only clone is tested but the test is negative : the SpeI site is always here at 311 bp. The cultures of the transformation are concentrated and spread on LB plate : 0 clone for pHT304 S and 18 for pHT315 S but none of the clones is good. We must do the filling-in again...</li>
-
<li>Transformation of the pSB1C3 + RFP and T3 + RFP plasmids extracts of the iGEM plates to use them as tool of cloning after extraction.</li>
+
<li>Transformation of the pSB1C3 + RFP and pSB1T3 + RFP plasmids extracts of the iGEM plates to use them as tool of cloning after extraction.</li>
</ul>
</ul>
</description>
</description>
Line 1,103: Line 1,081:
<description>
<description>
<ul>
<ul>
-
<li>Plasmidic extraction of clones Lyso+Dsp, then digestion and electrophoresis : clones not okay.</li>
+
<li>Plasmidic extraction of Lysostaphin+Dispersin clones, then digestion and electrophoresis : clones not okay.</li>
<li>New ligations :  
<li>New ligations :  
       <ul>
       <ul>
-
               <li>Promoter+Dsp in pSB1C3;</li>
+
               <li>Promoter+Dispersin in pSB1C3;</li>
-
               <li>Lyso+Dsp in pSB1C3.</li>
+
               <li>Lysostaphin+Dispersin in pSB1C3.</li>
       </ul>
       </ul>
       Then transformation in NM522.</li>
       Then transformation in NM522.</li>
-
<li>Plasmidic extraction of the strain BK12 : plasmid okay.</li>
+
<li>Plasmidic extraction of the BK12 strain : plasmid okay.</li>
</ul>
</ul>
</description>
</description>
Line 1,117: Line 1,095:
Results of the transformations :
Results of the transformations :
       <ul>
       <ul>
-
             <li>Transformations of NM522 by BK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : ok.</li>
+
             <li>Transformations of NM522 by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : ok.</li>
-
             <li>Transformation by Sfp-abrB-pSB1T3 : the negative control isn’t good, the transformation is done again...</li>
+
             <li>Transformation by sfp-abrB-pSB1T3 : the negative control isn’t good, the transformation is done again...</li>
-
             <li>Miniprep of other clones transformed with the plasmid RBS + XylR : the electrophoresis shows that the ligation isn’t still good…</li>
+
</ul>
 +
             <li>Miniprep of other clones transformed with the plasmid RBS + xylR : the electrophoresis shows that the ligation still isn’t good…</li>
       </ul>
       </ul>
</description>
</description>
Line 1,137: Line 1,116:
Transformation results :  
Transformation results :  
<ul>
<ul>
-
         <li>Witness + : okay;</li>
+
         <li>Positive control : okay;</li>
-
         <li>Witness - : okay;</li>
+
         <li>Negative control : okay;</li>
-
         <li>Lyso+Dsp : 8 clones;</li>
+
         <li>[Lysostaphin + Dispersin] : 8 clones;</li>
-
         <li>Prom+Dsp : more than 30.</li>
+
         <li>[Promoter + Dispersin]  : more than 30 clones.</li>
</ul>
</ul>
-
Clones are screened on Ampicillin and Chloramphenicol.
+
Clones are screened on LB + Amp and LB + Cm plates.
</description>
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
<ul>
<ul>
-
<li>The transformation of NM522 strain by Sfp-abrB didn’t work again : although the positive control contains many clones (plasmid A2) and the negative control contains nothing, the true transformation contains nothing too ! We decided to do the ligation Sfp-abrB again...</li>
+
<li>The transformation of NM522 strain by sfp-abrB didn’t work again : although the positive control contains many clones (plasmid A2) and the negative control contains nothing, the real transformation contains nothing either ! We decided to do the sfp-abrB ligation again...</li>
<li>Plasmid extraction of 4 clones NM522 transformed by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : the electrophoresis of the digested plasmids showed that 3 out of 4 clones had the good fragment.</li>
<li>Plasmid extraction of 4 clones NM522 transformed by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : the electrophoresis of the digested plasmids showed that 3 out of 4 clones had the good fragment.</li>
 +
<li><strong>PROBLEM : WE RAN OUT OF LIGASE!!</strong>
</ul>
</ul>
</description>
</description>
Line 1,158: Line 1,138:
<description>
<description>
<ul>
<ul>
-
<li>Deletion of the site SpeI from pHT315 and pHT304 plasmids and filling-in using the Pfu polymerase. This time we used gel electrophoresis to verify the plasmids after each purification.</li>
+
<li>Deletion of the SpeI site from pHT315 and pHT304 plasmids and filling-in using the Pfu polymerase. This time we used gel electrophoresis to verify the plasmids after each purification.</li>
<li>Extraction of the plasmid containing the RFP gene in the pSB1C3 iGEM backbone (midiprep).</li>
<li>Extraction of the plasmid containing the RFP gene in the pSB1C3 iGEM backbone (midiprep).</li>
</ul>
</ul>
Line 1,165: Line 1,145:
<description>
<description>
<ul>
<ul>
-
<li>New Lysostaphin tests are planned (on plates) : in this purpose, a culture of 60 mL of Bs 168 pWG100 is inoculated and then incubated over night at 28°C.</li>
+
<li>New Lysostaphin tests are planned (on plates) : in this purpose, a culture of 60 mL of Bs 168 pWG100 is inoculated and then incubated overnight at 28°C.</li>
-
<li>None of the clones Lyso+Dsp grew on Ampicillin so LB cultures were inoculated.</li>
+
<li>None of the clones Lysostaphin+Dispersin grew in LB + Amp so LB cultures were inoculated.</li>
-
<li>Out of 29 clones, 18 clones Prom+Dsp are Ampicillin sensitive, LB cultures are launched with these clones.</li>
+
<li>Out of 29 clones, 18 Promoter+Dispersin clones are Ampicillin sensitive, LB cultures are launched with these clones.</li>
<li>Plasmidic extractions of these clones : clones not okay.</li>
<li>Plasmidic extractions of these clones : clones not okay.</li>
-
<li>PCR to obtain pSB1C3 : the electrophoresis showed that there was no DNA, so another essay was done, but it was unsuccessful.</li>
+
<li>PCR to obtain pSB1C3 : the electrophoresis showed that there was no DNA, so another trial was done, but it was unsuccessful.</li>
-
<li>The strain NM522 with Constitutive Promotor + pSB1C3 was put in storage under the reference BK27.</li>
+
<li>The strain NM522 with Constitutive Promoter + pSB1C3 was put in storage under the reference BK27.</li>
</ul>
</ul>
</description>
</description>
Line 1,177: Line 1,157:
<ul>
<ul>
<li>Ligation of the sfp and abrB parts in an iGEM backbone.</li>
<li>Ligation of the sfp and abrB parts in an iGEM backbone.</li>
-
<li>The strain NM522 with abrB-pSB1K3 was put in storage under the reference BK25.</li>
+
<li>The NM522 strain with abrB-pSB1K3 was put in storage under the reference BK25.</li>
-
<li>The strain NM522 with sfp-pSB1K3 was put in storage under the reference BK26.</li>
+
<li>The NM522 strain with sfp-pSB1K3 was put in storage under the reference BK26.</li>
</ul>
</ul>
</description>
</description>
Line 1,204: Line 1,184:
<description>
<description>
<ul>
<ul>
-
<li>We identified the reason why the deletion of the site SpeI from pHT315 and pHT304 plasmids and filling-in was not successful : the Pfu enzyme requires Mg2+ and the buffer we used did not contain any, so we decided to give it another try, this time with the proper buffer.</li>
+
<li>We identified the reason why the deletion of the site SpeI from pHT315 and pHT304 plasmids and filling-in was not successful : the Pfu enzyme requires Mg<sup>2+</sup> and the buffer we used did not contain any, so we decided to give it another try, this time with the proper buffer.</li>
-
<li>The extracted plasmids containing the RFP gene in the pSB1C3 backbone were digested and verified by electrophoresis.</li>
+
<li>The extracted plasmids containing the RFP gene in the pSB1C3 backbone were digested and verified by electrophoresis.</li>
</ul>
</ul>
</description>
</description>
Line 1,211: Line 1,191:
<description>
<description>
<ul>
<ul>
-
<li>Lysostaphin tests : Flasks of 25 mL of Bs 168 pWG 100 filtered supernatant were cultivated Friday and frozen at -20°C Sunday (2 flasks of each). The control flask is filled with 25 mL of LB media.  These flasks are then frozen at -80°C and freeze-dried overnight.</li>
+
<li>Lysostaphin tests : Flasks of 25mL of Bs 168 pWG 100 filtered supernatant were cultivated on Friday and frozen at -20°C on Sunday (2 flasks of each). The control flask is filled with 25mL of LB broth.  These flasks are then frozen at -80°C and freeze-dried overnight.</li>
-
<li>The gel electrophoresis of the digested plasmid [Lysostaphin + Dispersin] in pSB1C3 doesn’t correspond to the expected fragments. Similarly, the second electrophoresis shows that the clones with [Constitutive promotor + Dispersin] in pSB1C3 do not have the right plasmid : the digested fragments do not have  the right size.</li>
+
<li>The gel electrophoresis of the digested plasmid [Lysostaphin + Dispersin] in pSB1C3 doesn’t correspond to the expected fragments. Similarly, the second electrophoresis shows that the clones with [Constitutive promoter + Dispersin] in pSB1C3 do not have the right plasmid : the digested fragments do not have  the right size.</li>
</ul>
</ul>
</description>
</description>
Line 1,230: Line 1,210:
<description>
<description>
<ul>
<ul>
-
<li>Ligation of the Constitutive Promoter (extracted by PCR) in the plasmid pSB1C3 containing the RFP gene (purified by midiprep) in order to obtain the Constitutive Promoter in the pSB1C3.</li>
+
<li>Ligation of the Constitutive Promoter (extracted by PCR) in the pSB1C3 plasmid containing the RFP gene (purified by midiprep) in order to obtain the Constitutive Promoter in the pSB1C3.</li>
-
<li>Lysostaphin tests on plates : the come back ! The freeze-dries products are put in five times less water than at first. Volumes up to 100 &micro;L of concentrated supernatant are applied on antibiograms which are used for tests on the <i>S. epidermidis</i> biofilm.</li>
+
<li>Lysostaphin tests on plates return again ! The freeze-dried products are put in five times less water than at first. Volumes up to 100 µL of concentrated supernatant are applied on antibiograms which are used for tests on the <i>S. epidermidis</i> biofilm.</li>
</ul>
</ul>
</description>
</description>
Line 1,240: Line 1,220:
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
-
Standard ligation between pBK7 (=RBS in pSB1C3) and XylR (obtained by PCR) with 1&micro;L of insert for 5&micro;L of vector. The ligation was transformed into the NM522 strain.  
+
Standard ligation between pBK7 (=RBS in pSB1C3) and xylR (obtained by PCR) with 1µL of insert for 5µL of vector. The ligation was transformed into the NM522 strain.  
</description>
</description>
                       </jour>
                       </jour>
Line 1,253: Line 1,233:
<description>
<description>
<ul>
<ul>
-
<li>The lysostaphin test didn’t work : Val&eacute;rie did the test again by applying first the supernatant on the paper disks before putting them on the plates, and by using TSM media instead of LB media.</li>
+
<li>The lysostaphin test didn’t work : Valérie did the test again by applying first the supernatant on the paper disks before putting them on the plates, and by using TSM medium instead of LB broth.</li>
<li>Transformation of the ligation [pSB1C3 + constitutive promoter] in NM 522 strain.</li>
<li>Transformation of the ligation [pSB1C3 + constitutive promoter] in NM 522 strain.</li>
</ul>
</ul>
Line 1,259: Line 1,239:
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
-
None of the 4 clones tested had the attended fragments, so we screened another 6.
+
None of the 4 clones tested had the expected fragments, so we screened 6 others.
</description>
</description>
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
<ul>
<ul>
-
<li>Results of the transformation of NM522 by pBK7 and XylR : the negative control contains nothing, the positive control (transformed with pBK5) is full of bacteria and the plate with NM522 transformed by pBK7 + XylR contains a lot of clones too. 8 clones are selected to do liquid culture and extract their DNA.</li>
+
<li>Results of the transformation of NM522 by pBK7 and xylR : the negative control contains nothing, the positive control (transformed with pBK5) is full of bacteria and the plate with NM522 transformed by pBK7 + xylR contains a lot of clones too. 8 clones are selected to do liquid culture and extract their DNA.</li>
-
<li>Second standard ligation between pBK7 (=RBS in pSB1C3) and XylR (producted by PCR) with more insert (3&micro;L of insert for 2&micro;L of vector). Transformation into NM522 strains. </li>
+
<li>Second standard ligation between pBK7 (=RBS in pSB1C3) and xylR (produced by PCR) with more insert (3µL of insert for 2µL of vector). Transformation into NM522 strains. </li>
</ul>
</ul>
</description>
</description>
Line 1,274: Line 1,254:
                         <titre>For all purposes</titre>
                         <titre>For all purposes</titre>
<description>
<description>
-
The electrophoresis after digestion with EcoRI and SpeI showed that the SpeI site was eliminated. 4 clones were chosen for further tests. However, the DNA concentration was too low so the enzymes did not digest the plasmids.
+
<ul>
 +
<li>Miniprep of the clones having integrated the modified  shuttle vector (after filling-in). 4 clones seem to have the right plasmid.</li>
 +
<li>The electrophoresis after digestion with EcoRI and SpeI showed that the SpeI site was eliminated. 4 clones were chosen for further tests. However, the DNA concentration was too low so the enzymes did not digest the plasmids.</li>
 +
</ul>
</description>
</description>
                       <titre>Kill</titre>
                       <titre>Kill</titre>
<description>
<description>
<ul>
<ul>
-
<li>Ligation of the Constitutive promoter (pVeg) produced by PCR in an iGEM plasmid.</li>
+
<li>Ligation of the Constitutive promoter (P<sub>veg</sub>) produced by PCR in an iGEM plasmid.</li>
-
<li>Result of the lysostaphin tests : didn’t work again. The Bs 168 pWG100 supernatant doesn’t contain any lysostaphin : the lysostaphin is probably degraded in one way or another (sensitivity to the defrosting ?).</li>
+
<li>Result of the lysostaphin tests : didn’t work again. The Bs 168 pWG100 supernatant doesn’t contain any lysostaphin : the lysostaphin is probably degraded in one way or another (sensitivity to defrosting ?).</li>
<li>Result of the transformation with [pSB1C3 + promoter] : a lot of red clones, some white clones (20 - 30). Isolation of 6 white clones and plasmid extractions by miniprep.</li>
<li>Result of the transformation with [pSB1C3 + promoter] : a lot of red clones, some white clones (20 - 30). Isolation of 6 white clones and plasmid extractions by miniprep.</li>
</ul>
</ul>
Line 1,287: Line 1,270:
<description>
<description>
<ul>
<ul>
-
<li>Two of the 6 [sfp+abrB] clones had the expected fragments.</li>
+
<li>Miniprep of 4 clones with the transformed 3A ligation (pBK22, gene abrB and pSB1A3 backbone). The electrophoresis did not turn out as expected for any of the 4 clones, so we decided to screen 6 others.</li>
-
<li>Ligation of pXyl produced by PCR in an iGEM plasmid.</li>
+
<li>Ligation of P<sub>xyl</sub> produced by PCR in an iGEM plasmid.</li>
</ul>
</ul>
</description>
</description>
Line 1,294: Line 1,277:
<description>
<description>
<ul>
<ul>
-
<li>Ligation of XylR produced by PCR in an iGEM plasmid (pSB1K3).</li>
+
<li>Ligation of xylR produced by PCR in an iGEM plasmid (pSB1K3).</li>
-
<li>Results of the transformation of NM522 by [pBK7 + XylR] (for the second ligation) : the plate with bacteria transformed by [pBK7 + XylR] contains a lot of clones and the controls are standard. 14 clones are selected to do liquid culture and extract their DNA.</li>
+
<li>Results of the transformation of NM522 by [pBK7 + xylR] (for the second ligation) : the plate with bacteria transformed by [pBK7 + xylR] contains a lot of clones and the controls are standard. 14 clones are selected to do liquid culture and extract their DNA.</li>
-
<li>Plasmid extractions from the 8 clones transformed by [pBK7 + XylR]. The electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they have just the vector without XylR.</li>
+
<li>Plasmid extractions from the 8 clones transformed by [pBK7 + xylR]. The electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they have just the vector without xylR.</li>
</ul>
</ul>
</description>
</description>
Line 1,305: Line 1,288:
                         <titre>For all purposes</titre>
                         <titre>For all purposes</titre>
<description>
<description>
-
The plasmids (pBK19 and pBK20) from 2 clones were re-extracted using columns. This time the digestion was successful. The tests showed that there was no SpeI site. However, the pHT315 plasmid also had no XbaI site.
+
The plasmids (pBK19 and pBK20) from 2 clones were re-extracted using columns. This time, the digestion was successful. The tests showed that there was no SpeI site. However, the pHT315 plasmid also had no XbaI site. We digested the two plasmids with the XbaI enzyme and this time we incubated the digestion during one hour, instead of 10 minutes. We also tested the enzyme on another plasmid which had a XbaI site. The digestion of the control plasmid was partial.
 +
</description>
 +
<titre>Surfactant</titre>
 +
<description>
 +
Miniprep of 6 clones with the transformed 3A ligation (pBK22, gene abrB and pSB1A3 backbone). The gel verification showed that there were 3 clones having the expected profile. The plasmid was put in storage under the reference pBK29.
</description>
</description>
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
-
Plasmid extractions from the 14 clones transformed by [pBK7 + XylR] (with the second ligation). Like for the first transformation, the electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they have just the vector without XylR.
+
Plasmid extractions from the 14 clones transformed by [pBK7 + xylR] (with the second ligation). As for the first transformation, the electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they only have the vector without xylR.
</description>
</description>
                       </jour>
                       </jour>
Line 1,318: Line 1,305:
<description>
<description>
<ul>
<ul>
-
<li>The pBK19 with no SpeI site was put in storage under the reference pBK25.</li>
+
<li>pBK19 with no SpeI site was put in storage under the reference pBK25.</li>
-
<li>The pBK20 with no SpeI site was put in storage under the reference pBK26.</li>
+
<li>pBK20 with no SpeI site was put in storage under the reference pBK26.</li>
</ul>
</ul>
</description>
</description>
Line 1,326: Line 1,313:
<ul>
<ul>
<li>Launch of 8 [Promoter+Dispersin] cultures.</li>
<li>Launch of 8 [Promoter+Dispersin] cultures.</li>
-
<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>
+
<li>Results of the transformation of NM522 by [pSB1T3 + P<sub>xyl</sub>] : the plate with bacteria transformed by [pSB1T3 + P<sub>xyl</sub>] contains few clones and the controls are standard. 3 clones are selected to do liquid cultures and extract their DNA : no plasmid.</li>
<li>Results of the transformation of NM522 by [pSB1T3 + rbs-abrB] : no clones.</li>
<li>Results of the transformation of NM522 by [pSB1T3 + rbs-abrB] : no clones.</li>
</ul>
</ul>
Line 1,333: Line 1,320:
<description>
<description>
<ul>
<ul>
-
<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.
+
<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.
<li>A new PCR of xylR is made, in order to increase the stock.</li>
<li>A new PCR of xylR is made, in order to increase the stock.</li>
</ul>
</ul>
Line 1,351: Line 1,338:
<description>
<description>
<ul>
<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>
+
<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 different proportions of insert and vector. Transformation in NM522 strain.</li>
-
<li>Digestion of Lysostaphin in pSB1C3 and Dsp in pUC57.</li>
+
<li>Digestion of Lysostaphin in pSB1C3 and Dispersin in pUC57.</li>
-
<li>Check of 8 [promoter-dispersin] clones : clones happen to be nonstandard.</li>
+
<li>Verification of 8 [promoter-dispersin] clones : clones happen to be nonstandard.</li>
</ul>
</ul>
</description>
</description>
Line 1,382: Line 1,369:
<ul>
<ul>
<li>Result of the transformation of NM522 with [Constitutive 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.</li.
<li>Result of the transformation of NM522 with [Constitutive 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.</li.
-
<li>New trial of clonage : Digestion, Ligation, transformation to construct :</li>
+
<li>New trial of cloning : Digestion, Ligation, transformation to construct :</li>
       <ul>
       <ul>
             <li>[Lysostaphin + Dispersin] in pSB1C3;</li>
             <li>[Lysostaphin + Dispersin] in pSB1C3;</li>
Line 1,391: Line 1,378:
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
-
6 pXyl clones were tested, but none had integrated the plasmid.
+
6 P<sub>xyl</sub> clones were tested, but none had integrated the plasmid.
</description>
</description>
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
<ul>
<ul>
-
<li>Check of 6 [XylR-pSB1K3] clones : they only have the vector.</li>
+
<li>Verification of 6 [xylR-pSB1K3] clones : they only have the vector.</li>
<li>Transformation of [pBK24-xylR] into NM522 strain.</li>
<li>Transformation of [pBK24-xylR] into NM522 strain.</li>
-
<li>Results of [pBK7-xylR] transformation : lots of clones and no clones in the negative witness. 12 clones are put in liquid culture for extraction.</li>
+
<li>Results of [pBK7-xylR] transformation : lots of clones and no clones in the negative control. 12 clones are put in liquid culture for extraction.</li>
</ul>
</ul>
</description>
</description>
Line 1,415: Line 1,402:
<li>Too many clones on [Lysostaphin + Dispersin] in shuttle vector transformation plate, so some clones are selected and spread and a new LB plate with the appropriate antibiotic.</li>
<li>Too many clones on [Lysostaphin + Dispersin] in shuttle vector transformation plate, so some clones are selected and spread and a new LB plate with the appropriate antibiotic.</li>
<li>12 liquid culture in LB are launched for the clones [Lysostaphin + Dispersin] in pSB1C3.</li>
<li>12 liquid culture in LB are launched for the clones [Lysostaphin + Dispersin] in pSB1C3.</li>
-
<li>Miniprep of 14 clones transformed with the ligation product [Constitutive Promoter + RBS + Lysostaphin] in pBK25 and electrophoresis to analyse the extracted DNA : the transformation is succesful for 3 clones =)</li>
+
<li>Miniprep of 14 clones transformed with the ligation product [Constitutive Promoter + RBS + Lysostaphin] in pBK25 and electrophoresis to analyze the extracted DNA : the transformation is successful for 3 clones =)</li>
       <ul>
       <ul>
             <li>The first clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (1);</li>
             <li>The first clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (1);</li>
Line 1,425: Line 1,412:
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
-
Transformation of [pXyl+pSB1T3] in NM522.
+
Transformation of [P<sub>xyl</sub>+pSB1T3] in NM522.
</description>
</description>
                       <titre>Stick</titre>
                       <titre>Stick</titre>
Line 1,440: Line 1,427:
                         <titre>For all purposes</titre>
                         <titre>For all purposes</titre>
<description>
<description>
-
The transformation of the cloned shuttle vectors was successful, so 12 clones are put in liquid culture for further tests.
+
The transformation of the cloned shuttle vectors was successful, so 12 clones are put in liquid cultures for further tests.
</description>
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
-
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.
+
3A ligation of pBK4 (pUC57 containing the lacI gene) and pBK29 (pSB1A3 containing the sfp and abrB genes) in pBK24 (pSB1C3 containing a RFP gene). The cloned fragment was transformed in the NM522 strain.
</description>
</description>
                       <titre>Stick</titre>
                       <titre>Stick</titre>
Line 1,450: Line 1,437:
<ul>
<ul>
<li>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.</li>
<li>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.</li>
-
<li>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.</li>
+
<li>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.</li>
</ul>
</ul>
</description>
</description>
Line 1,460: Line 1,447:
<description>
<description>
Miniprep of 12 clones selected from transformed bacteria with the cloned shuttle vectors; we ran out of SpeI enzyme, so we could not digest the extracted plasmids.
Miniprep of 12 clones selected from transformed bacteria with the cloned shuttle vectors; we ran out of SpeI enzyme, so we could not digest the extracted plasmids.
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
6 white clones from the ligation pBK24-xylR are purified and put in liquid cultures.
</description>
</description>
                       </jour>
                       </jour>
Line 1,465: Line 1,456:
<jour nb="26">
<jour nb="26">
                         <date>Sunday, August 26th 2012</date>
                         <date>Sunday, August 26th 2012</date>
 +
<titre>Kill</titre>
 +
<description>
 +
Extraction from 12 clones of lysostaphin and dispersin in pBK26 (shuttle vector) ligation and gel electrophoresis. The gel didn’t show any good clone.
 +
</description>
                         <titre>Surfactant</titre>
                         <titre>Surfactant</titre>
<description>
<description>
Miniprep of 6 clones (3A ligation: pBK24, pBK4, pBK29). The electrophoresis showed that none of the tested clones had integrated the right plasmid.
Miniprep of 6 clones (3A ligation: pBK24, pBK4, pBK29). The electrophoresis showed that none of the tested clones had integrated the right plasmid.
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
Extraction from the 6 white clones (containing xylR-pBK26). Gel electrophoresis is run, but it didn’t show any good clone.
</description>
</description>
                       </jour>
                       </jour>
Line 1,476: Line 1,475:
<description>
<description>
<ul>
<ul>
-
<li>Transformation of BS 168 strain with the pBK28 (1) plasmid which contains the [Constitutive Promoter + RBS + Lysostaphin] in the shuttle vector.</li>
+
<li>Transformation of Bs 168 strain with pBK28 (1) plasmid which contains the [Constitutive Promoter + RBS + Lysostaphin] in the shuttle vector.</li>
-
<li>Midiprep : Extraction of pveg-disp-pSB1C3 of clone 22.</li>
+
<li>Midiprep : Extraction of P<sub>veg</sub>-dispersin-pSB1C3 of clone 22.</li>
<li>BK33 strain was put in storage.</li>
<li>BK33 strain was put in storage.</li>
</ul>
</ul>
Line 1,485: Line 1,484:
<ul>
<ul>
<li>Given the fact that the digested plasmids extracted from transformed NM522 <i>E. coli</i> with the 3A ligation (pBK24, pBK4, pBK29) had unexpected fragments (and impossible to explain), the ligation was done again, this time changing the vector/insert ratio (1/1 and 1/3, as opposed to the first trial with a ⅙ ratio ).</li>
<li>Given the fact that the digested plasmids extracted from transformed NM522 <i>E. coli</i> with the 3A ligation (pBK24, pBK4, pBK29) had unexpected fragments (and impossible to explain), the ligation was done again, this time changing the vector/insert ratio (1/1 and 1/3, as opposed to the first trial with a ⅙ ratio ).</li>
-
<li>Liquid cultures of two potentially good clones of the NM522 strain transformed with a 3A ligation (pBK22(promotor pXyl, RBS and sfp gene), abrB gene and pSB1A3 backbone) were made for miniprep (the two plasmids were extracted using the classical protocol, with extraction solutions that we made, so the yield and the purity of the extractions were poor; that’s why we decided to extract the plasmids again, this time using an extraction kit).</li>
+
<li>Liquid cultures of two potentially good clones of the NM522 strain transformed with a 3A ligation (pBK22(promoter P<sub>xyl</sub>, RBS and sfp gene), abrB gene and pSB1A3 backbone) were made for miniprep (the two plasmids were extracted using the classic ABC protocol, with extraction solutions that we made, so the yield and the purity of the extractions were poor; that’s why we decided to extract the plasmids again, this time using an extraction kit).</li>
</ul>
</ul>
</description>
</description>
                       <titre>Stick</titre>
                       <titre>Stick</titre>
<description>
<description>
-
No transformation was done, because we ran out of LB growth medium.  
+
No transformation was done, because we ran out of LB broth.  
</description>
</description>
                       </jour>
                       </jour>
Line 1,504: Line 1,503:
<ul>
<ul>
<li>Ligation of [Constitutive promoter + Dispersin] in the shuttle vector pBK25.</li>
<li>Ligation of [Constitutive promoter + Dispersin] in the shuttle vector pBK25.</li>
-
<li>Results of the transformation of BS 168 strain with the pBK28 (1) in the shuttle vector : the negative control is full of bacteria Is the BS 168 strain resistant to erythromycin ? Or is the erythromycin concentration too low ? <br />
+
<li>Results of the transformation of BS 168 strain with the pBK28 (1) in the shuttle vector : the negative control is full of bacteria Is the Bs 168 strain resistant to erythromycin ? Or is the erythromycin concentration too low ? <br />
-
We made another transformation and we spread the transformed bacteria on GL+Ery plates with different erythromycin concentration (from 5 &micro;g/mL to 10 &micro;g/mL).</li>
+
We made another transformation and we spread the transformed bacteria on LB+Ery plates with different erythromycin concentration (from 5 µg/mL to 10 µg/mL).</li>
</ul>
</ul>
</description>
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
-
The transformations with the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios ) was successful. 12 clones per transformation are put in liquid culture and then streaked on LB+Cm plates and LB+Amp plates.
+
The transformations with the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios ) was successful. 12 clones per transformation are put in liquid cultures and then streaked on LB+Cm plates and LB+Amp plates.
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
Transformation of xylR-pBK10 (blunt end ligation) in NM522.
</description>
</description>
                       </jour>
                       </jour>
Line 1,520: Line 1,523:
<ul>
<ul>
<li>Transformation of the previous ligation [promoter-dispersin-pBK25] in <i>E. coli</i> NM522.</li>
<li>Transformation of the previous ligation [promoter-dispersin-pBK25] in <i>E. coli</i> NM522.</li>
-
<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 &micro;g/mL the first time, 5 and 10 &micro;g/mL the second time).</li>
+
<li>Results of the second transformation of Bs 168 strain with 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>
-
<li>We made (GL+Ery) plates with different erythromycin concentrations in order to make different tests.</li>
+
<li>We made (LB+Ery) plates with different erythromycin concentrations in order to make different tests.</li>
</ul>
</ul>
</description>
</description>
Line 1,527: Line 1,530:
<description>
<description>
<ul>
<ul>
-
<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>
+
<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>
-
<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>
+
<li>Miniprep of the two clones containing the sfp and abrB genes. 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>
-
<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>
+
<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 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 classic protocol because we were running out of time and we were behind the schedule.</li>
-
<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>
+
<li>In parallel, 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>
 +
</ul>
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
<ul>
 +
<li>Transformation of xylR-pBK24 in NM522. The transformation of xylR-pBK24 is done in order to see if xylR was successfully ligated or not. If the bacteria are red, then xylR was not ligated.</li>
 +
<li>24 clones containing pBK10-xylR are patched in LB+Amp growth medium, and then incubated.</li>
</ul>
</ul>
</description>
</description>
Line 1,540: Line 1,550:
<description>
<description>
<ul>
<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>
+
<li>Transformation results : too many clones on the control digestion not ligated vector so the transformation can’t be used for the transformation [Lysostaphin+Dispersin] in pSB1C3.</li>
-
<li>12 clones put in culture from the transformation plate [Lysostaphin + Dispersin] in the shuttle navette treated by BamHI.</li>
+
<li>12 clones are put in culture from the transformation of [Lysostaphin + Dispersin] in the shuttle vector treated by BamHI.</li>
<li>Preparation of tubes for sequencing, launch of miniprep and midiprep.</li>
<li>Preparation of tubes for sequencing, launch of miniprep and midiprep.</li>
-
<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>
+
<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>
</ul>
</ul>
</description>
</description>
Line 1,552: Line 1,562:
<li>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.</li>
<li>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.</li>
<li>The miniprep confirmed that the ligation containing lacI was successful.</li>
<li>The miniprep confirmed that the ligation containing lacI was successful.</li>
 +
</ul>
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
<ul>
 +
<li>The patched plate is used as a master for replica-printing. The replica plate contains LB+Kan growth medium. Results : only one clone didn’t grow in the replica plate, meaning that it might be a clone containing xylR-pBK10 ligation. The clone is put in liquid culture overnight.</li>
 +
<li>Another test is run to confirm that pBK34 plasmid (xylR-pBK24) doesn’t contain xylR : we digested the plasmid using NdeI restriction enzyme. Since xylR contains a NdeI restriction site (pBK24 doesn’t contain it), if pBK34 is opened by the enzyme, it means that it contains xylR. Result : pBK34 doesn’t contain xylem.</li>
</ul>
</ul>
</description>
</description>
Line 1,560: Line 1,577:
                         <titre>For all purposes</titre>
                         <titre>For all purposes</titre>
<description>
<description>
-
We have finally received the enzymes, so we tested the minipreps containing the modified shuttle vectors (containing the iGEM linker). The results are promising : 4 plasmids are digested by SpeI, so the ligation was successful. The four plasmids are further tested for the other 3 iGem sites. To make sure that the site SpeI is not the initial site that we eliminated by filling-in and that the plasmid used for transformation and ligation was not contaminated with the original plasmid, a double digested was done (EcoRI and SpeI). There was no fragment at 1000 pb, so the ligation was successful.
+
We have finally received the enzymes, so we tested the minipreps containing the modified shuttle vectors (containing the iGEM linker). The results are promising : 4 plasmids are digested by SpeI, so the ligation was successful. The four plasmids are further tested for the other 3 iGEM sites. To make sure that the site SpeI is not the initial site that we eliminated by filling-in and that the plasmid used for transformation and ligation was not contaminated with the original plasmid, a double digestion was done (EcoRI and SpeI). There was no fragment at 1,000 bp, so the ligation was successful.
</description>
</description>
                         <titre>Kill</titre>
                         <titre>Kill</titre>
Line 1,567: Line 1,584:
<li>Verification of [Lysostaphin-Dispersin] in the shuttle vector (pBK26) clones : clones are not good.</li>
<li>Verification of [Lysostaphin-Dispersin] in the shuttle vector (pBK26) clones : clones are not good.</li>
<li>Verification of the miniprep for sequencing ([Promoter + Dispersin] in pSB1C3) : DNA is okay.</li>
<li>Verification of the miniprep for sequencing ([Promoter + Dispersin] in pSB1C3) : DNA is okay.</li>
-
<li>Result of the transformation of NM 522 strain with the Lysostaphin in PBK26 :</li>
+
<li>Result of the transformation of NM522 strain with the Lysostaphin in pBK26 :</li>
       <ul>  
       <ul>  
             <li>The negative control is ok;</li>
             <li>The negative control is ok;</li>
             <li>The positive control is full of colonies;</li>
             <li>The positive control is full of colonies;</li>
-
             <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>
+
             <li>The transformation is full of colonies : 24 clones are selected (on plates and in liquid culture) in order to extract and check their DNA.  </li>
       </ul>
       </ul>
</ul>
</ul>
Line 1,578: Line 1,595:
<description>
<description>
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 right 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 right plasmids.
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
<ul>
 +
<li>Extraction of the good clone from the replica-printing. Then a electrophoresis gel is run : the blunt end ligation was successful.</li>
 +
<li>The plasmid is digested by EcoRI, XbaI, SpeI and PstI to find out if the problem comes from a bad site sequence. Result : the EcoRI restriction site is not good, therefore is not recognized by EcoRI enzyme.</li>
 +
</ul>
</description>
</description>
                       </jour>
                       </jour>
Line 1,585: Line 1,609:
<jour nb="1">
<jour nb="1">
                       <date>Saturday, September 1st 2012</date>
                       <date>Saturday, September 1st 2012</date>
-
                       <titre>For all purposes</titre>
+
                       <titre>Kill</titre>
-
<description>
+
-
 
+
-
</description>
+
-
                      <titre>Kill</titre>
+
<description>
<description>
<ul>
<ul>
-
<li>Digestion of Lysostaphin in the shuttle vector and dispersine in pUC57 and verification of digestion.</li>
+
<li>Digestion of Lysostaphin in the shuttle vector and dispersin in pUC57 and verification of digestion. Then the vector is treated with CALF protein and a ligation is made using different of insert concentration. Then the ligation is transformed with commercial cells (STLBÉ) according to the  manufacturer protocol and another trial is made with higher concentration.</li>
<li>Verification of midiprep : Midiprep of Lysostaphin in pSB1C3 is okay but not the one of Dispersin in pUC57.</li>
<li>Verification of midiprep : Midiprep of Lysostaphin in pSB1C3 is okay but not the one of Dispersin in pUC57.</li>
<li>Miniprep of 14 clones NM522 transformed with Lysostaphin in pBK26. The digestions and electrophoresis show that 7 clones are ok ! They are put in storage under the references pBK38 (1) to pBK38 (6).</li>
<li>Miniprep of 14 clones NM522 transformed with Lysostaphin in pBK26. The digestions and electrophoresis show that 7 clones are ok ! They are put in storage under the references pBK38 (1) to pBK38 (6).</li>
 +
<li>Extraction of 14 clones transformed with Dispersin in pBK25. Gel electrophoresis showed no good clones.</li>
</ul>
</ul>
</description>
</description>
Line 1,600: Line 1,621:
<description>
<description>
<ul>
<ul>
-
<li>One of the 12 screened clones transformed with the ligation of pBK22 and pBK39 has the expected fragments. Unfortunately, the plasmid is mixed with another one. To separate the two plasmids we decided to transform 1 &micro;L of the miniprep into the NM522 <i>E. coli</i> strain.</li>
+
<li>One of the 12 screened clones transformed with the ligation of pBK22 and pBK39 has the expected fragments. Unfortunately, the plasmid is mixed with another one. To separate the two plasmids we decided to transform 1 µL of the miniprep into <i>E. coli</i> NM522 strain.</li>
-
<li>Another 6 clones with the transformed 3A ligation (done on August 29th) are screened.</li>
+
<li>6 other clones with the transformed 3A ligation (done on August 29th) are screened.</li>
<li>Standard ligation of the abrB gene and the plasmid containing lacI in pSB1C3 backbone (pBK37) and transformation. In parallel, another ligation is attempted : [pBK37 + pBK29].</li>
<li>Standard ligation of the abrB gene and the plasmid containing lacI in pSB1C3 backbone (pBK37) and transformation. In parallel, another ligation is attempted : [pBK37 + pBK29].</li>
</ul>
</ul>
Line 1,611: Line 1,632:
                       <titre>For all purposes</titre>
                       <titre>For all purposes</titre>
<description>
<description>
-
Liquid cultures containing the modified shuttle vectors with the iGEM linker are inoculated and incubated over night.
+
Liquid cultures containing the modified shuttle vectors with the iGEM linker are inoculated and incubated overnight.
</description>
</description>
                       <titre>Kill</titre>
                       <titre>Kill</titre>
<description>
<description>
-
Shuttle vector containing the lysostaphin is treated with an alcaline phosphatase, then a ligation with the dispersin is realised followed by transformation in NM522 <i>E. coli</i> strain.
+
<ul>
 +
<li>Shuttle vector containing lysostaphin is treated with an alkaline phosphatase, then a ligation with dispersin is realized,  followed by transformation in <i>E. coli</i> NM522 strain.</li>
 +
<li>Dispersin is ligated with pBK26 and then digested by BamHI before transformation. BamHI digestion allows not to transform the circularized vectors which doesn’t contain the insert.</li>
 +
</ul>
</description>
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
<ul>
<ul>
-
<li>Plasmids from the 6 clones containing the 3A ligation are extracted and digested. Gel electrophoresis showed that one of the clones has the expected fragments. However, it was mixed with another plasmid. In order to separate them, we decided to transform the NM522 strain with 1&micro;L of the miniprep.</li>
+
<li>Plasmids from the 6 clones containing the 3A ligation are extracted and digested. Gel electrophoresis showed that one of the clones has the expected fragments. However, it was mixed with another plasmid. In order to separate them, we decided to transform the NM522 strain with 1µL of the miniprep.</li>
<li>12 clones for each ligation (done the day before) are screened.</li>
<li>12 clones for each ligation (done the day before) are screened.</li>
</ul>
</ul>
Line 1,630: Line 1,654:
                       <titre>For all purposes</titre>
                       <titre>For all purposes</titre>
<description>
<description>
-
Midiprep of the cloned shuttle vectors (containing the iGEM linker).
+
<ul>
 +
<li>Midiprep of the cloned shuttle vectors (containing the iGEM linker).</li>
 +
<li>Miniprep of pBK26 shuttle vector is done in order to increase the stock.</li>
 +
</ul>
</description>
</description>
                       <titre>Kill</titre>
                       <titre>Kill</titre>
<description>
<description>
-
Transformations of BS 168 :
+
<ul>
 +
<li>None clones are obtained on the plate lysostaphin+dispersin when we have followed manufacturer protocol even on the positive control. But few colonies appeared on plates done with our own protocol.These clones are put in liquid cultures.</li>
 +
<li>Tests are launched to verify manufacturer cells. And other tests are launched to verify that our plates contained the appropriate antibiotic.</li>
 +
<li>A transformation is made with our cells of their positive control.</li>
 +
<li>Transformations of Bs 168 :
<ul>
<ul>
       <li>Trial n°1 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK25 (=pBK28).</li>
       <li>Trial n°1 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK25 (=pBK28).</li>
       <li>Trial n°2 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK26 (=pBK38).</li>
       <li>Trial n°2 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK26 (=pBK38).</li>
</ul>
</ul>
-
The bacteria are spread on GL+Ery plates ([ERY]=1 &micro;g/mL and [ERY]=10 &micro;g/mL).
+
The bacteria are spread on LB+Ery plates ([Ery]=1 µg/mL and [Ery]=10 µg/mL).<br />
 +
The transformation of NM522 with ligation Dispersin in pBK26 was successful.</li>
 +
</ul>
</description>
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
Line 1,655: Line 1,688:
                       <titre>For all purposes</titre>
                       <titre>For all purposes</titre>
<description>
<description>
-
Multiple tubes containing LB media and Ampicillin at different concentrations ranging from 0 to 1,3 mg/mL are inoculated with bacteria containing the shuttle vectors (pBK35 and pBK36).
+
Multiple tubes containing LB medium and Ampicillin at different concentrations ranging from 0 to 1.3 mg/mL are inoculated with bacteria containing the shuttle vectors (pBK35 and pBK36).
</description>
</description>
                       <titre>Kill</titre>
                       <titre>Kill</titre>
<description>
<description>
<ul>
<ul>
-
<li>Results of the transformations of BS 168 : not coherent. However, the problem seems to be identified : it is the erythromycin solution. Indeed, the erythromycin must be diluted in ethanol and not in water and must be kept refrigerated at -20°C.</li>
+
<li>Lots of clones are obtained with our cells.So we have tried a transformation of commercial cells with bigger quantities of DNA.</li>
 +
<li>Miniprep of clones obtained with STLB2 are made. Clones are not okay. A new transformation with the same ligation mix is made but with NM522.</li>
 +
<li>Results of the transformations of Bs 168 : not coherent. However, the problem seems to be identified : it is the erythromycin solution. Indeed, the erythromycin must be diluted in ethanol and not in water and must be kept refrigerated at -20°C.</li>
<li>Standard ligations between :
<li>Standard ligations between :
       <ul>
       <ul>
-
             <li>pLac in pSB1C3 (pBK9) digested by SpeI and PstI and [RBS-GFP] in pSB1T3 digested by XbaI and PstI;</li>
+
             <li>P<sub>lac</sub> in pSB1C3 (pBK9) digested by SpeI and PstI and [RBS-GFP] in pSB1T3 digested by XbaI and PstI;</li>
-
             <li>pXyl (amplified by PCR) digested by SpeI and EcoRI and [RBS-GFP] in pSB1T3 digested by XbaI and EcoRI.</li>
+
             <li>P<sub>xyl</sub> (amplified by PCR) digested by SpeI and EcoRI and [RBS-GFP] in pSB1T3 digested by XbaI and EcoRI.</li>
     </ul>
     </ul>
These two ligation products are transformed in NM522 strain.</li>
These two ligation products are transformed in NM522 strain.</li>
 +
<li>Acrylamide gels and samples are prepared for SDS-PAGE.</li>
</ul>
</ul>
</description>
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
-
Gel electrophoresis showed that 5 clones out of 6 clones screened after the transformation with the miniprep containing two plasmids had the expected fragments. However, we had doubts and we decided to search for restriction sites in the sequences that the plasmid was supposed to contain to make sure we had the right construction. We found out that a digestion with EcoRV would give 5 fragments of different sizes, enough to confirm the presence of the expected genes. Unfortunately, the electrophoresis was disappointing.
+
Gel electrophoresis showed that 5 out of 6 clones screened after the transformation with the miniprep containing two plasmids had the expected fragments. However, we had doubts and we decided to search for restriction sites in the sequences that the plasmid was supposed to contain to make sure we had the right construction. We found out that a digestion with EcoRV would give 5 fragments of different sizes, enough to confirm the presence of the expected genes. Unfortunately, the electrophoresis was disappointing.
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
A PCR is run in order to get RBS-xylR. The PCR failed (not enough DNA-polymerase).
</description>
</description>
                       <titre>Biofilm</titre>
                       <titre>Biofilm</titre>
Line 1,678: Line 1,718:
<li>One plate with <i>S. epidermidis</i> to evaluate the adhesion.</li>
<li>One plate with <i>S. epidermidis</i> to evaluate the adhesion.</li>
<li>One plate with <i>S. epidermidis</i> + supernatant of different cultures (lysostaphin, dispersin...).</li>
<li>One plate with <i>S. epidermidis</i> + supernatant of different cultures (lysostaphin, dispersin...).</li>
-
<li>Same plate as the one above one + crystal violet.</li>
+
<li>Same plate as the one above + crystal violet.</li>
</ul>
</ul>
</description>
</description>
Line 1,685: Line 1,725:
<jour nb="5">
<jour nb="5">
                       <date>Wednesday, September 5th 2012</date>
                       <date>Wednesday, September 5th 2012</date>
 +
<titre>Kill</titre>
 +
<description>
 +
Few clones are obtained with commercial cells, there are not very efficient. A new transformation is made with the same ligation mix but in NM522.
 +
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
-
Standard ligation of pBK22 (sfp gene) and pBK39 (abrB-lacI) and transformation into the NM522 <i>E. coli</i> strain.
+
<ul>
 +
<li>Standard ligation of pBK22 (sfp gene) and pBK39 (abrB-lacI) and transformation into the <i>E. coli</i> NM522 strain.</li>
 +
<li>A SDS-PAGE gel is run to analyze lysostaphin (in BK32 and BK35 strains) and dispersin (BK29 strain).</li>
 +
</ul>
</description>
</description>
                       </jour>
                       </jour>
Line 1,695: Line 1,742:
                       <titre>For all purposes</titre>
                       <titre>For all purposes</titre>
<description>
<description>
-
Ampicillin resistances tests had some surprising results : even at 1.3 mg/mL there is bacterial growth. However, the higher Ampicillin concentration is, the lower is the OD of the liquid cultures. Ampicillin resistance test of the two shuttle vectors did not show any significant difference between the two strains (BK37 and BK38). The result is not surprising, given the fact that both plasmids (pBK36 and pBK35) derive from the shuttle vectors pHT315 and pHT304 which have the same origin replication as pUC19. However, the test must be done again, this time with at least two samples for each Ampicillin concentration in order to be able to estimate an error.
+
Ampicillin resistance tests gave some surprising results : even at 1.3 mg/mL there is bacterial growth. However, the higher Ampicillin concentration is, the lower OD<sub>600</sub> of the liquid cultures is. Ampicillin resistance test of the two shuttle vectors did not show any significant difference between the two strains (BK37 and BK38). The result is not surprising, given the fact that both plasmids (pBK36 and pBK35) derive from the shuttle vectors pHT315 and pHT304 which have the same origin replication as pUC19. However, the test must be done again, this time with at least two samples for each Ampicillin concentration in order to be able to estimate an error.
</description>
</description>
                       <titre>Kill</titre>
                       <titre>Kill</titre>
<description>
<description>
-
Transformation of BS 168 : new try ! To improve our protocol, we took periodic OD600 readings in order to stop the cell growth at the best stage (just between the exponential and the stationary phase). The BS 168 strain is transformed by :
+
<ul>
 +
<li>Clones obtained on lysostaphin-dispersin plates are put in liquid culture.</li>
 +
<li>Transformation of Bs 168 : new trial ! To improve our protocol, we took periodic OD<sub>600</sub>600 measurements in order to stop the cell growth at the best stage (just between the exponential and the stationary phase). The Bs 168 strain is transformed by :
       <ul>
       <ul>
             <li>pBK 25 (= modified shuttle vector pHT 304);</li>
             <li>pBK 25 (= modified shuttle vector pHT 304);</li>
Line 1,707: Line 1,756:
             <li>Dispersin in pBK26.</li>
             <li>Dispersin in pBK26.</li>
       </ul>
       </ul>
-
We spread 200 &micro;L of each transformed cells on GL + Erythromycin ([ERY] = 16 &micro;g/mL) plates.
+
We spread 200 µL of each transformed cells on LB + Erythromycin ([Ery] = 16 µg/mL) plates.</li>
 +
</ul>
</description>
</description>
                       <titre>Surfactant</titre>
                       <titre>Surfactant</titre>
<description>
<description>
12 clones transformed with the ligation pBK22 and pBK39 are screened.
12 clones transformed with the ligation pBK22 and pBK39 are screened.
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
2 new PCR are done to have xylR, but they failed.
</description>
</description>
                       </jour>
                       </jour>
Line 1,720: Line 1,774:
<description>
<description>
<ul>
<ul>
-
<li>Result of the transformation of BS 168 : </li>
+
<li>A miniprep of potential lysostaphin-dispersin clones is made : no clones are okay.</li>
 +
<li>Result of the transformation of Bs 168 : </li>
       <ul>
       <ul>
-
             <li>All the negative controls plates are clear : the erythromycin concentration is high enough to do a selection;</li>
+
             <li>All the negative control plates are clear : the erythromycin concentration is high enough to do a selection;</li>
-
             <li>Some plates with the transformed bacteria are empty but some have from 1 to 4 clones : these clones are put in liquid cultures in order to make miniprep and analyse their DNA;</li>
+
             <li>Some plates with the transformed bacteria are empty but some have from 1 to 4 clones : these clones are put in liquid cultures in order to extract and analyze their DNA;</li>
       </ul>
       </ul>
-
<li>Transformation of BS 168 : new trial by electroporation. As the last transformation, the BS 168 strain is transformed by pBK25, pBK26, pBK28, pBK38 and dispersin in pBK26.</li>
+
<li>Transformation of Bs 168 : new trial by electroporation. As the last transformation, the Bs 168 strain is transformed by pBK25, pBK26, pBK28, pBK38 and dispersin in pBK26.</li>
 +
<li>A new SDS-PAGE is made using supernatants and pellets as samples. The samples were not concentrated enough, so the gels didn’t show any of the expected bands.</li>
</ul>
</ul>
</description>
</description>
Line 1,731: Line 1,787:
<description>
<description>
All the screened clones had the religated vector, so we decided to use commercial competent cells from STLB2 strain to transform the ligation of pBK39 and pBK22 in order to diminish the number of clones so that the chances of finding the recombinant plasmid containing the 3 genes (sfp, abrB and lacI) would be higher.
All the screened clones had the religated vector, so we decided to use commercial competent cells from STLB2 strain to transform the ligation of pBK39 and pBK22 in order to diminish the number of clones so that the chances of finding the recombinant plasmid containing the 3 genes (sfp, abrB and lacI) would be higher.
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
A new RBS-xylR PCR is run, now with different concentrations of genomic DNA and a different annealing temperature. The PCR was successful.
</description>
</description>
                       </jour>
                       </jour>
Line 1,738: Line 1,798:
                       <titre>Kill</titre>
                       <titre>Kill</titre>
<description>
<description>
-
Miniprep of the clones BS 168 transformed by pBK26, pBK28 : we use the same protocol as DNA extraction of <i>E.coli</i> with addition of Lysosyme the buffer A1. Electrophoresis to analyze extracted DNA : there is nothing on the gel => Maybe the DNA isn’t concentrated enough ?<br />
+
Miniprep of the clones BS 168 transformed by pBK26, pBK28 : we use the same protocol as DNA extraction for <i>E.coli</i> with addition of Lysozyme to the buffer A1. Electrophoresis to analyze extracted DNA : there is nothing on the gel Maybe the DNA isn’t concentrated enough ?<br />
New clones are put in liquid cultures in order to make other minipreps.  
New clones are put in liquid cultures in order to make other minipreps.  
</description>
</description>
Line 1,744: Line 1,804:
<description>
<description>
16 clones containing the ligation of pBK22 and pBK39 transformed into STLB2 commercial competent cells are screened.
16 clones containing the ligation of pBK22 and pBK39 transformed into STLB2 commercial competent cells are screened.
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
Ligation of P<sub>lac</sub>-RBS-xylR and then transformation in NM522.
</description>
</description>
                       </jour>
                       </jour>
Line 1,752: Line 1,816:
<description>
<description>
<ul>
<ul>
-
<li>Miniprep of the clones BS 168 transformed by pBK25, pBK26, pBK28, and Dispersin in pBK26. Electrophoresis to analyze extracted DNA :</li>
+
<li>Miniprep of the clones Bs 168 transformed by pBK25, pBK26, pBK28, and Dispersin in pBK26. Electrophoresis to analyze extracted DNA :</li>
       <ul>
       <ul>
-
             <li>BS 168 clone transformed by pBK25 has the right plasmid !! =)</li>
+
             <li>Bs 168 clone transformed by pBK25 has the right plasmid !! =)</li>
-
    <li>BS 168 clone transformed by pBK28 has the right plasmid !! BACILLUS LYSOSTAPHIN IS HERE !! :D :D :D</li>
+
    <li>Bs 168 clone transformed by pBK28 has the right plasmid !! <strong>BACILLUS LYSOSTAPHIN IS HERE !! :D :D :D</strong></li>
-
    <li>BS 168 clones transformed by pBK26 and dispersine in pBK26 : nothing on the gel ... </li>
+
    <li>Bs 168 clones transformed by pBK26 and dispersin in pBK26 : nothing on the gel... </li>
     </ul>
     </ul>
-
<li>Transformation of BS 168 GFP with the same five plasmids as before. The transformed bacteria are spread on GL + ERY plates, with two different concentrations : [ERY]=10 &micro;g/mL and [ERY]=15 &micro;g/mL.</li>
+
<li>Transformation of Bs 168 GFP with the five same plasmids as before. The transformed bacteria are spread on LB + Ery plates, with two different concentrations : [Ery]=10 µg/mL and [Ery]=15 µg/mL.</li>
<li>Given the fact that there was nothing on the miniprep for pBK26 and pBK28, we made a new electrophoresis after using the seeDNA method  to concentrate the DNA of the miniprep. We see very light stripes on the gel, but the results for pBK26 and dispersin in pBK26 have to be confirm again...</li>
<li>Given the fact that there was nothing on the miniprep for pBK26 and pBK28, we made a new electrophoresis after using the seeDNA method  to concentrate the DNA of the miniprep. We see very light stripes on the gel, but the results for pBK26 and dispersin in pBK26 have to be confirm again...</li>
<li>Thanks to the good results, we put in storage the transformed Bacillus under the reference :</li>
<li>Thanks to the good results, we put in storage the transformed Bacillus under the reference :</li>
       <ul>
       <ul>
-
             <li>BK41 : BS 168 + pBK25</li>
+
             <li>BK41 : Bs 168 + pBK25</li>
-
             <li>BK42 : BS 168 + pBK28</li>
+
             <li>BK42 : Bs 168 + pBK28</li>
-
             <li>BK43 : BS 168 + pBK26 (to confirm)</li>
+
             <li>BK43 : Bs 168 + pBK26 (to confirm)</li>
-
             <li>BK44 : BS 168 + Disp in pBK26 (to confirm)</li>
+
             <li>BK44 : Bs 168 + Dispersin in pBK26 (to confirm)</li>
       </ul>
       </ul>
</ul>
</ul>
Line 1,775: Line 1,839:
                       </jour>
                       </jour>
 +
<jour nb="10">
 +
<date>Monday, September 10th 2012</date>
 +
<titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
<li>4 transformations with the same amount of plasmid are done (pBK19,pBK20, pBK35 and pBK36) in order to characterize the transformation efficiency of the shuttle vectors.</li>
 +
<li>Ampicillin resistance test was repeated, this time with 3 tubes per Ampicillin concentration for each plasmid.</li>
 +
</ul>
 +
</description>
 +
<titre>Surfactant</titre>
 +
<description>
 +
We decided to give another try and do the ligation once again, this time using 5 time less vector and a ⅛ ratio vector/insert. Given the fact that we are running out of time, we did two ligations hoping to construct the same biobrick: pBK22+pBK39 and pBK29+pBK37. Both ligations were transformed into the <i>E. coli</i> NM522 strain.
 +
</description>
 +
<titre>Kill</titre>
 +
<description>
 +
<ul>
 +
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>E. coli</i> strains which might produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture.</li>
 +
<li><i>S. epidermidis</i> cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic.</li>
 +
<li>According to the results there is an effect of lysostaphin but we observed an effect due to the initial OD<sub>600</sub> of cultures that were different, so we don’t know if the results can be used. A new protocol is tried out, culture are diluted to have the same OD<sub>600</sub>.</li>
 +
<li>New SDS-PAGE 12% gel is done with just the supernatants. The samples preparation didn’t go well and they were not enough concentrated. Therefore the gel didn’t show anything.</li>
 +
</ul>
 +
</description>
 +
<titre>P<sub>lac</sub> and P<sub>xyl</sub> kinetics</titre>
 +
<description>
 +
<ul>
 +
<li>Measurement of the NM522 + P<sub>lac</sub> kinetics (with Cm) during 12h in a 96 wells plate with and without different IPTG concentration addition. (1 mM; 0.5mM and 0.1mM)</li>
 +
<li>In the same 96 wells plate NM522 + P<sub>xyl</sub> kinetics is also measured during 12h with and without different xylose concentration. (2%; 0.5% and 0.2%)</li>
 +
<li>Results : kinetics decreases so there is a problem. But it can be explained by the fact that the promoters need to be activated by a sigma factor which is active in the exponential phase and 12h is not enough. So this experiment has to be done during 24h.</li>
 +
</ul>
 +
</description>
 +
</jour>
 +
<jour nb="11">
 +
<date>Tuesday, September 11th 2012</date>
 +
<titre>Experiments realized in Massy</titre>
 +
<description>
 +
<ul>
 +
<li>Seeding a 96 wells plate with <i>S. aureus</i> to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+disp in pBK26 and Romain Briandet’s strains (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)</li>
 +
<li>Seeding a 96 wells plate with <i>S. aureus</i> to produce a biofilm (plate 2).</li>
 +
</ul>
 +
</description>
 +
<titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
<li>Design of the primers required to sequence the modified sequences of the shuttle vectors (after elimination of SpeI site by filling-in and cloning of the iGEM linker).</li>
 +
<li>Ampicillin resistance test was a complete failure : the negative control (NM522 strain grown in LB broth supplemented with Ampicillin at the usual selective concentration), so the Ampicillin that was used lost it’s biological activity.</li>
 +
</ul>
 +
</description>
 +
<titre>Surfactant</titre>
 +
<description>
 +
12 clones per transformation are screened.
 +
</description>
 +
<titre>Kill</titre>
 +
<description>
 +
<ul>
 +
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>E. coli</i> strains which might produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. <i>S. epidermidis</i> cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic. According to the results there is an effect of lysostaphin but we observed an effect of dilution due to the protocol, so we don’t know if the results can be used. So a new protocol is worked out.</li>
 +
<li>SDS-PAGE is done using pellets, to find a good concentration.</li>
 +
</ul>
 +
</description>
 +
</jour>
 +
 +
<jour nb="12">
 +
<date>Wednesday, September 12th 2012</date>
 +
<titre>Experiments realized in Massy</titre>
 +
<description>
 +
<ul>
 +
<li>We remove the supernatant in the plate 2 made on Tuesday and added Bs168+dispersin in pBK26 and Bs168+pBK26 with different dilutions. Observation of the plate at the confocal laser scanning microscope after 5 hours of incubation.  Exploitation of the pictures with Imaris software. Dispersin seems to have an effect on the biofilm.</li>
 +
<li>Reading of the plate 1 with a confocal laser scanning microscope (after an overnight incubation). Exploitation of the pictures with Imaris software.</li>
 +
</ul>
 +
</description>
 +
<titre>For all purposes</titre>
 +
<description>
 +
The transformed plates with the modified and unmodified shuttle vectors do not show any significant difference, so we decided to make another essay, this time with 3 transformation for each plasmid.
 +
</description>
 +
<titre>Surfactant</titre>
 +
<description>
 +
Unfortunately, the screened clones did not integrate the expected plasmid.
 +
</description>
 +
<titre>Kill</titre>
 +
<description>
 +
<ul>
 +
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture with the new protocol. Strains used for the test are <i>Bacillus subtilis</i> strains which may produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. There is a problem with the results : the control is not good.</li>
 +
<li>Miniprep of one clone containing dispersin in pBK26 is done. Gel electrophoresis showed that the clone was good. The plasmid is then put in storage (pBK41).</li>
 +
</ul>
 +
</description>
 +
</jour>
 +
 +
<jour nb="13">
 +
<date>Thursday, September 13th 2012</date>
 +
<titre>Experiments realized in Massy</titre>
 +
<description>
 +
<ul>
 +
<li>Seeding of 2 96 wells plate with <i>S. aureus</i> to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+dispersin in pBK26 and Romain Briandet's strain (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)</li>
 +
<li>OD<sub>600</sub> measurements with filtered supernatants.</li>
 +
<li>Mobility test of Bs168  on agar 0.25% plate.</li>
 +
</ul>
 +
</description>
 +
<titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
<li>The transformed plates with the modified and unmodified shuttle vectors do not show any undeniable difference, so we decided to make another essay, this time with 3 transformation for each plasmid.</li>
 +
<li>Ampicillin resistance test is repeated (3 samples for each Ampicillin concentration).</li>
 +
</ul>
 +
</description>
 +
<titre>Surfactant</titre>
 +
<description>
 +
Another 12 clones per transformation were screened (from the transformation made on 10th September).
 +
</description>
 +
<titre>Kill</titre>
 +
<description>
 +
OD<sub>600</sub> test in tanks to test dispersin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>Bacillus subtilis</i> strains which may produce dispersin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. Dispersin has no effect on <i>S. epidermidis</i> culture according to the results.
 +
</description>
 +
</jour>
 +
 +
<jour nb="14">
 +
<date>Friday, September 14th 2012</date>
 +
<titre>For all purposes</titre>
 +
<description>
 +
<ul>
 +
<li>Transformation efficiency tests confirm our supposition : there is no significant difference between the transformation yields of the 4 plasmids (unmodified shuttle vectors, pBK19 and pBK20 and modified shuttle vectors, pBK35 and pBK36).<br />
 +
Moreover, OD<sub>600</sub> of the bacterial cultures in LB medium at various Ampicillin concentrations shows no significant difference between the BK37 and BK38 strains.</li>
 +
<li>TG1 (<i>E. coli</i>) strain containing pJIM2241 shuttle vector is put in storage (BK47).</li>
 +
</ul>
 +
</description>
 +
<titre>Kill</titre>
 +
<description>
 +
<ul>
 +
<li>OD<sub>600s</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. The strains used for the test are <i>Bacillus subtilis</i> strains which may produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture.</li>
 +
<li>SDS-PAGE is done with some changes in the protocol. The gels showed a band that matches with the expected lysostaphin molecular weight.</li>
 +
</ul>
 +
</description>
 +
<titre>Surfactant</titre>
 +
<description>
 +
Gel electrophoresis of the extracted plasmids shows that there are 5 possible plasmids containing the ligated insert (P<sub>xyl</sub>-sfp-abrB-lacI-terminator). The five plasmids are digested by EcoRV and the electrophoresis confirms that, this time, the plasmids have the expected fragments.
 +
</description>
 +
</jour>
 +
 +
<jour nb="15">
 +
<date>Saturday, September 15th 2012</date>
 +
<titre>Kill</titre>
 +
<description>
 +
OD<sub>600</sub> test in 96 well plate to test impact of lysostaphin and dispersin on a culture of <i>S. epidermidis</i> (test during 12 hours).
 +
</description>
 +
</jour>
 +
 +
<jour nb="21">
 +
<date>Friday, September 21st 2012</date>
 +
<titre>P<sub>lac</sub> and P<sub>xyl</sub> kinetics</titre>
 +
<description>
 +
A 7-hour-long kinetics is made with P<sub>lac</sub> and P<sub>xyl</sub> to be sure to have measurement in the exponential phase. Only one IPTG and xylose concentration is used.
 +
P<sub>lac</sub> = IPTG 0.5% and P<sub>xyl</sub> = xylose 0.5%.<br />
 +
Moreover, a 24-hour-long kinetics is also made in a 96 well plate. The conditions are the same as the one done for 12h except that bacteria are inoculated in a 1/100 dilution (instead of 1/50).
 +
</description>
 +
</jour>
</month>
</month>
Line 1,813: Line 2,030:
                    
                    
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