Team:Lyon-INSA/notebook

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<body>
<body>
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<div id="projectBar" class="menuBar">
 
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    <div class="boutonMenu" onclick="window.location='menu';">Menu</div>
 
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    <div id="ongletBar">
 
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        <div class="boutonOnglet"  onclick="window.location='project';">Project</div>
 
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        <div class="boutonOnglet"  onclick="window.location='modelling';">Modelling</div>
 
-
        <div class="boutonOnglet"  onclick="window.location='safety';">Safety</div>
 
-
        <div class="boutonOnglet BOClicked">Notebook</div>
 
-
        <div class="boutonOnglet" onclick="window.location='protocol';">Protocols</div>
 
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        <div class="boutonOnglet"  onclick="window.location='datapage';">Data Page</div>
 
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        <div class="boutonOnglet" onclick="window.location='Achievements';">Achievements</div>
 
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    </div>
 
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<div id="xml" style="display:none;">
<div id="xml" style="display:none;">
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<titre>For all purposes</titre>
<titre>For all purposes</titre>
<date> Monday, July 2nd 2012</date>
<date> Monday, July 2nd 2012</date>
-
<description>Liquid culture (5 mL LB media) of NM 522 cells and overnight incubation under agitation at 37°C for later transformation.</description>
+
<description>Liquid culture (5 mL LB medium) of NM 522 cells and overnight incubation under agitation at 37°C for later transformation.</description>
</jour>
</jour>
                 <jour nb="3">
                 <jour nb="3">
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<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 medium + 500 µL Chloramphenicol + 500 µL liquid culture of transformed bacteria ).</li>
     <li>The 3 genes coding for lysostaphin, dispersin and <i>lacI</i> repressor in pUC57 Ampicillin resistant plasmids were delivered.</li>
     <li>The 3 genes coding for lysostaphin, dispersin and <i>lacI</i> repressor in pUC57 Ampicillin resistant plasmids were delivered.</li>
     <li>Transformation of NM522 strain with pUC57-Lysostaphin, pUC57-Dispersin and pUC57-<i>sfp</i>.</li>
     <li>Transformation of NM522 strain with pUC57-Lysostaphin, pUC57-Dispersin and pUC57-<i>sfp</i>.</li>
Line 501: Line 487:
     <li>Extractions with a miniprep kit are made :
     <li>Extractions with a miniprep kit are made :
     <ul>
     <ul>
-
             <li>4 clones containing the pLac promoter (1,2,3,4);</li>
+
             <li>4 clones containing the P<sub>lac</sub> promoter (1,2,3,4);</li>
             <li>4 clones containing the <i>Bacillus subtilis</i> RBS (1,2,3,4);</li>
             <li>4 clones containing the <i>Bacillus subtilis</i> RBS (1,2,3,4);</li>
             <li>3 clones containing theTerminator (1,2,3);</li>
             <li>3 clones containing theTerminator (1,2,3);</li>
Line 694: Line 680:
     <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 <i>gfp</i>, <i>cfp</i>, <i>yfp</i> ;  test → OK : put in storage.</li>
     <li>Extraction of <i>gfp</i>, <i>cfp</i>, <i>yfp</i> ;  test → OK : put in storage.</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>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 LB+Ampicillin medium : 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 medium under the reference BK15.</li>
</ul>
</ul>
</description>
</description>
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<titre>For all purposes</titre>
<titre>For all purposes</titre>
<description>
<description>
-
<ul>
+
<i>Bacillus subtilis</i> transformation with the 2 shuttle vectors pBK35 and pBK36.
-
 
+
-
<li>Bacillus subtilis transformation with the 2 shuttle vectors pBK35 and pBK36.</li>
+
-
</ul>
+
</description>
</description>
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<titre>For all purposes</titre>
<titre>For all purposes</titre>
<description>
<description>
-
<ul>
+
Screening of 12 clones per <i>Bacillus</i> transformation.
-
 
+
-
<li>Screening of 12 clones per Bacillus transformation;</li>
+
-
</ul>
+
</description>
</description>
<titre>Surfactant</titre>
<titre>Surfactant</titre>
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<titre>For all purposes</titre>
<titre>For all purposes</titre>
<description>
<description>
-
<ul>
+
Erythromycin at different concentrations was spread on LB plates with in order to test the Erythromycin resistance of the <i>B. Subtilis</i> strain transformed with the shuttle vectors.
-
 
+
-
<li>LB plates were spread  with Erythromycin at different concentration in order to test the Erythromycin resistance of the <i>B. Subtilis</i> strain transformed with the shuttle vectors</li>
+
-
</ul>
+
</description>
</description>
<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
-
<li>Unfortunately the screened clones (containing the ligations pBK42+pBK35 and pBK42+pBK36) did not have the right plasmid. Another ligation of the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] is attempted, this time after gel extraction and purification of the digested pBK42 plasmid with EcoRI and PstI. This way, the backbone is eliminated and the chances of ligating the construction instead of the plasmid are higher.</li>
+
Unfortunately the screened clones (containing the ligations pBK42+pBK35 and pBK42+pBK36) did not have the right plasmid. Another ligation of the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] is attempted, this time after gel extraction and purification of the digested pBK42 plasmid with EcoRI and PstI. This way, the backbone is eliminated and the chances of ligating the construction instead of the plasmid are higher.
</description>
</description>
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titre>For all purposes</titre>
titre>For all purposes</titre>
<description>
<description>
-
<ul>
+
Multiple tubes containing LB medium supplemented with Erythromycin at different concentrations are inoculated with the <i>B. subtilis</i> strains containing the shuttle vectors pBK35 and pBK36 in order to test the antibiotic resistance. For the same purpose LB + Ery plates are spotted with the same cultures at different dilution ratios.
-
 
+
-
<li>Multiple tubes containing LB media supplemented with Erythromycin at different concentrations are inoculated with the <i>B. subtilis</i> strains containing the shuttle vectors pBK35 and pBK36 in order to test the antibiotic resistance. For the same purpose LB + Ery plates are spotted with the same cultures at different dilution ratios.</li>
+
-
</ul>
+
</description>
</description>
<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
-
<li>30 clones containing the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the shuttle vector are screened
+
30 clones containing the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the shuttle vector are screened.
-
</li>
+
</description>
</description>
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<titre>For all purposes</titre>
<titre>For all purposes</titre>
<description>
<description>
-
<ul>
+
The strains that were supposed to contain the shuttle vectors did not grow, so we suspect that the clones are in fact mutants.
-
 
+
-
<li>The strains that were supposed to contain the shuttle vectors did not grow, so we suspect that the clones are in fact mutants</li>
+
-
</ul>
+
</description>
</description>
<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
-
<li> Miniprep of the transformed clones with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>]. The gel electrophoresis shows that only two of them have the expected fragments.  
+
Miniprep of the transformed clones with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>]. The gel electrophoresis shows that only two of them have the expected fragments.
-
</li>
+
</description>
</description>
Line 2,161: Line 2,130:
<titre>For all purposes</titre>
<titre>For all purposes</titre>
<description>
<description>
-
<ul>
+
<i>Bacillus subtilis</i> transformation with the shuttle vectors pBK35 and pBK36.
-
 
+
-
<li><i>Bacillus subtilis</i> transformation with the shuttle vectors pBK35 and pBK36</li>
+
-
</ul>
+
</description>
</description>
<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
-
<li> Transformation of <i>B. subtilis</i> 168 and <i>B. subtilis abrB</i> strains with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the pBK35 shuttle vector.  
+
Transformation of <i>B. subtilis</i> 168 and <i>B. subtilis abrB</i> strains with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the pBK35 shuttle vector.  
-
</li>
+
</description>
</description>
Line 2,195: Line 2,160:
<titre>For all purposes</titre>
<titre>For all purposes</titre>
<description>
<description>
-
<ul>
+
12 clones transformed with the shuttle vectors are screened.
-
 
+
-
<li>12 clones transformed with the shuttle vectors are screened</li>
+
-
</ul>
+
</description>
</description>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
-
<li> 24 clones transformed with the construction [pXyl-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>] are screened;
+
24 clones transformed with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>] are screened.
-
</li>
+
</description>
</description>
</jour>
</jour>
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<titre>For all purposes</titre>
<titre>For all purposes</titre>
<description>
<description>
-
<ul>
+
Multiple tubes containing  2 mL liquid LB medium are supplemented with Erythromycin at various concentrations in order to test the antibiotic resistance of the two shuttle vectors. Also, LB+Ery plates are spotted with liquid cultures with the <i>B. subtilis </i> strains containing the shuttle vectors.
-
 
+
-
<li>Multiple tubes containing  2 mL liquid LB media are supplemented with Erythromycin at various concentrations in order to test the antibiotic resistance of the two shuttle vectors. Also, LB+Ery plates are spotted with liquid cultures with the <i>B. subtilis </i> strains containing the shuttle vectors</li>
+
-
</ul>
+
</description>
</description>
Line 2,230: Line 2,188:
<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
-
<li> 6 of the 24 clones are put in liquid culture for further testing in LB medium supplemented with xylose at a concentration of 2%;
+
6 of the 24 clones are put in liquid culture for further testing in LB medium supplemented with xylose at a concentration of 2%.
-
</li>
+
</description>
</description>
</jour>
</jour>
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<titre>For all purposes</titre>
<titre>For all purposes</titre>
<description>
<description>
-
<ul>
+
Both strains grew in all tubes. However, there is a difference concerning OD<sub>600</sub> between the two strains. The test is repeated (with antibiotic concentrations ranging from 0 to 1.5 mg/mL) in order to make sure there is a notable difference between the two strains. </li>
-
 
+
-
<li> Both strains grew in all tubes. However, there is a difference concerning the OD between the two strains. The test is repeated (with antibiotic concentrations ranging from 0 to 1,5 mg/mL) in order to make sure there is a notable difference between the two strains. </li>
+
-
</ul>
+
</description>
</description>
<titre>Surfactant</titre>
<titre>Surfactant</titre>
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<titre>For all purposes</titre>
<titre>For all purposes</titre>
 +
<description>
 +
The results of the antibiotic resistance in liquid culture show that the <i>B. subtilis</i> strain containing pBK35 is less resistant than the one containing pBK36.</li>
 +
</description>
 +
<titre>Surfactant</titre>
 +
<description>
 +
The emulsion test and the biofilm formation test confirm the presence of <i>sfp</i> and <i>abrB</i> genes;
 +
</description>
 +
</jour>
 +
 +
</month>
 +
 +
<month name="October" size="31">
 +
 +
<jour nb="10">
 +
                        <date>Wednesday, October 10th 2012</date>
 +
 +
<titre>Surfactant</titre>
<description>
<description>
<ul>
<ul>
 +
<li>Ligation of the part pBK14 containing the <i>gfp</i> gene with the part pBK7 (containing a <i>B. subtilis</i> RBS).</li>
 +
<li>Ligation of the part pBK13 containing the <i>rfp</i> gene with the part pBK7 (containing a <i>B. subtilis</i> RBS).</li>
 +
<li>Transformation of the NM522 <i>E. coli</i> strain with the two ligations.</li>
 +
</ul>
 +
</description>
 +
<titre>Stick</titre>
 +
<description>Ligation of the PCR product <i>xylR</i> gene with the part pBK9 (P<sub><i>lac</i></sub> gene)and NM522 transformation.
 +
</description>
 +
</jour>
-
<li>The results of the antibiotic resistance in liquid culture show that the <i>B. subtilis</i> strain containing pBK35 is less resistant than the one containing pBK36.</li>
+
<jour nb="11">
 +
<date>Thursday, October 11th 2012</date>
 +
 +
<titre>Surfactant</titre>
 +
<description>
 +
Screening of 15 clones per transformation (antibiotic resistance test).
 +
 
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
Inoculation of 5 mL of LB medium with chloramphenicol with 1 colony of <i>Bacillus subtilis</i> 168 <i>ΔabrB</i> and 5 mL of LB medium with 1 colony of <i>Bacillus subtilis</i> 168. Incubation overnight at 37°C. (This is for the 48h positive <i>Bacillus subtilis</i> biofilm test plate)
 +
</description>
 +
</jour>
 +
<jour nb="12">
 +
<date>Friday, October 12th 2012</date>
 +
 +
<titre>Surfactant</titre>
 +
<description>
 +
Liquid cultures are inoculated with colonies having the expected antibiotic phenotype.
 +
</br>
 +
To test the surfactant properties of the surfactin produced by the <i>B. subtilis</i> BK52 strain, a biofilm test was made in a 24-well plate. Liquid cultures are seeded with BK52 and BK49.
 +
 
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
<ul>
 +
<li>(24h plate)The same inoculation as previously is prepared.</li>
 +
<li>(48h plate)<i>Bacillus subtilis</i> 168 <i>ΔabrB</i> or <i>Bacillus subtilis</i> 168 are added in the 12 well plate with MgSO4  1mM and Glucose 0.1 %.</li>
</ul>
</ul>
 +
The plate is incubated at 37°C for 48h
 +
 +
(For both) Inoculation of 5 mL of LB medium with 1 colony of <i>E coli</i> (<i>ompR++</i> GFP, curlis overproduction). Incubation overnight at 37°C.
</description>
</description>
 +
</jour>
 +
<jour nb="13">
 +
<date>Saturday, October 13th 2012</date>
 +
<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
-
<li> The emulsion test and the biofilm formation test confirm the presence of <i>sfp</i> and <i>abrB</i> genes;
+
Miniprep of the screened clones. Unfortunately, the gel electrophoresis was disappointing and the extracted plasmids did not contain the ligated genes.
-
</li>
+
</br>
 +
A 24-well plate is incubated with filtered supernatant of the saturated cultures with BK52 and BK49 at room temperature for 24 hours.
 +
 
 +
 
</description>
</description>
-
</jour>
+
<titre>Stick</titre>
 +
<description>
 +
(24h plate)<i>Bacillus subtilis</i> 168 <i>ΔabrB</i> or <i>Bacillus subtilis</i> 168 are added in a 12-well plate with MgSO4  1mM and Glucose 0.1 %.
 +
The plate is incubated at 37°C for 48h.
 +
</description>
 +
</jour>
 +
<jour nb="14">
 +
<date>Sunday, October 14th 2012</date>
 +
 +
<titre>Surfactant</titre>
 +
<description>
 +
The wells are carefully rinsed with M63 medium two times after having previously discarded the supernatant. Then, each well is inoculated with a saturated adherent <i>E. coli</i> strain diluted 100 times in LB medium diluted 2 times.</br>
 +
Moreover, another 24-well plate assay was made using the same supernatant, but this time the plate was incubated only 4 hours before <i>E. coli</i> inoculation.
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
(For both) The supernatant is removed, wells are washed and LB is added.To see if <i>E coli</i> forms a biofilm over the <i>Bacillus subtilis</i> biofilm, the overnight culture of <i>E coli</i> is added into each well.
 +
Plates are incubated for 36h at 30°C.
 +
</description>
 +
</jour>
 +
<jour nb="16">
 +
<date>Tuesday, October 16th 2012</date>
 +
 +
<titre>Surfactant</titre>
 +
<description>
 +
Unfortunately, the observation of the plates under confocal microscope is not possible, so the experiment must be repeated. The only difference is that this time 12-well plates  with glass lamellae are used so the observation of the biofilm would be possible.
 +
</description>
 +
<titre>Stick</titre>
 +
<description>
 +
(For both) Now let's observe using the confocal microscope !
 +
Results : It works ! Our positive <i>Bacillus subtilis</i> biofilm inhibits the stick of other bacteria !
 +
</description>
 +
</jour>
 +
<jour nb="17">
 +
<date>Wednesday, October 17th 2012</date>
 +
 +
<titre>Surfactant</titre>
 +
<description>
 +
Liquid cultures were inoculated with the strains BK49 and BK52 and then incubated at 37C for 48 hours.
 +
</description>
 +
 +
</jour>
 +
<jour nb="19">
 +
<date>Saturday, October 19th 2012</date>
 +
 +
<titre>Surfactant</titre>
 +
<description>
 +
The supernatant from the plate is discarded. Each well is inoculated with an <i>E. coli</i> saturated culture diluted 50 times in LB medium diluted 2 times.
-
</month>
 
 +
</description>
 +
 +
</jour>
 +
 +
<jour nb="20">
 +
<date>Sunday, October 20th 2012</date>
 +
 +
<titre>Surfactant</titre>
 +
<description>
 +
Ligation of pBK40 and pBK16 followed by transformation into the NM522 strain.
 +
 +
</description>
 +
 +
</jour>
 +
 +
<jour nb="21">
 +
<date>Monday, October 21st 2012</date>
 +
 +
<titre>Surfactant</titre>
 +
<description>
 +
The surfactin pre-treated glass lamellae are observed under the confocal microscope. The negative control has a thick biofilm whereas the treated lamella has a few isolated colonies.
 +
 +
</description>
 +
 +
</jour>
 +
 +
<jour nb="22">
 +
<date>Tuesday, October 22nd 2012</date>
 +
 +
<titre>Surfactant</titre>
 +
<description>
 +
Screening of 12 clones.
 +
 +
</description>
 +
 +
</jour>
 +
 +
<jour nb="23">
 +
<date>Wednesday, October 23rd 2012</date>
 +
 +
<titre>Surfactant</titre>
 +
<description>
 +
Miniprep of the transformed clones. The gel electrophoresis confirmed the expected insert. (<i>abrB</i> gene in pSB1C3 backbone)
 +
 +
</description>
 +
 +
</jour>
 +
 +
<jour nb="24">
 +
<date>Tuesday, October 24th 2012</date>
 +
 +
<titre>Surfactant</titre>
 +
<description>
 +
The plasmids pBK42 and pBK46 were submitted to the Registry.
 +
 +
</description>
 +
 +
</jour>
 +
 +
</month>
</project>
</project>
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</div>
</div>
-
<div id="contentPage">
+
<div id="pageContent">
-
<br/>
+
 
<!--[if IE]>
<!--[if IE]>
     <center><span style="font-size:23px;text-decoration:blink;color:red;">Sorry !! Our notebook doesn't work on IE, please use another browser to enjoy it</span></center>
     <center><span style="font-size:23px;text-decoration:blink;color:red;">Sorry !! Our notebook doesn't work on IE, please use another browser to enjoy it</span></center>
<![endif]-->
<![endif]-->
-
<br/>
 
<h1>The Notebook</h1>
<h1>The Notebook</h1>
<br/>
<br/>
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        <div class="month" id="August">August</div>
        <div class="month" id="August">August</div>
                         <div class="month" id="September">September</div>
                         <div class="month" id="September">September</div>
 +
                        <div class="month" id="October">October</div>
 +
</div>
</div>
<br/>
<br/>

Latest revision as of 00:15, 27 October 2012

The Notebook


July
August
September
October


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Our collections

Strains collection
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