Team:Lyon-INSA/notebook

From 2012.igem.org

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<jour nb="10">
<jour nb="10">
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<date>Wednesday, October 10th 2012</date>
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                        <date>Wednesday, October 10th 2012</date>
<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
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<ul>
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<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>
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</ul>
</description>
</description>
<titre>Stick</titre>
<titre>Stick</titre>
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<description>
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<description>Ligation of the PCR product <i>xylR</i> gene with the part pBK9 (P<sub><i>lac</i></sub> gene)and NM522 transformation.
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</description>
</description>
</jour>
</jour>
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<jour nb="10">
 
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<date>Wednesday, October 10th 2012</date>
 
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<titre>Surfactant</titre>
 
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<description>
 
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</description>
 
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<titre>Stick</titre>
 
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<description>
 
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</description>
 
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</jour>
 
<jour nb="11">
<jour nb="11">
<date>Thursday, October 11th 2012</date>
<date>Thursday, October 11th 2012</date>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
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Screening of 15 clones per transformation (antibiotic resistance test).
</description>
</description>
<titre>Stick</titre>
<titre>Stick</titre>
<description>
<description>
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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>
</description>
</jour>
</jour>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
 +
Liquid cultures are inoculated with colonies having the expected antibiotic phenotype.
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</br>
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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>
</description>
<titre>Stick</titre>
<titre>Stick</titre>
<description>
<description>
 +
<ul>
 +
<li>(24h plate)The same inoculation as previously is prepared.</li>
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<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>
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</ul>
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The plate is incubated at 37°C for 48h
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(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>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
 +
Miniprep of the screened clones. Unfortunately, the gel electrophoresis was disappointing and the extracted plasmids did not contain the ligated genes.
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</br>
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A 24-well plate is incubated with filtered supernatant of the saturated cultures with BK52 and BK49 at room temperature for 24 hours.
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</description>
</description>
<titre>Stick</titre>
<titre>Stick</titre>
<description>
<description>
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(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 %.
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The plate is incubated at 37°C for 48h.
</description>
</description>
</jour>
</jour>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<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>
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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>
</description>
<titre>Stick</titre>
<titre>Stick</titre>
<description>
<description>
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(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.
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Plates are incubated for 36h at 30°C.
</description>
</description>
</jour>
</jour>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
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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>
</description>
<titre>Stick</titre>
<titre>Stick</titre>
<description>
<description>
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(For both) Now let's observe using the confocal microscope !
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Results : It works ! Our positive <i>Bacillus subtilis</i> biofilm inhibits the stick of other bacteria !
</description>
</description>
</jour>
</jour>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
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Liquid cultures were inoculated with the strains BK49 and BK52 and then incubated at 37C for 48 hours.
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</description>
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<titre>Stick</titre>
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-
<description>
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</description>
</description>
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</jour>
</jour>
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<jour nb="18">
 
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<date>Thursday, October 18th 2012</date>
 
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<titre>Surfactant</titre>
 
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<description>
 
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</description>
 
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<titre>Stick</titre>
 
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<description>
 
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</description>
 
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</jour>
 
<jour nb="19">
<jour nb="19">
<date>Saturday, October 19th 2012</date>
<date>Saturday, October 19th 2012</date>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<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.
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</description>
 
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<titre>Stick</titre>
 
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<description>
 
</description>
</description>
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</jour>
</jour>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
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Ligation of pBK40 and pBK16 followed by transformation into the NM522 strain.
</description>
</description>
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<titre>Stick</titre>
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<description>
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</description>
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</jour>
</jour>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<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>
</description>
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<titre>Stick</titre>
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<description>
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</description>
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</jour>
</jour>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
 +
Screening of 12 clones.
</description>
</description>
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<titre>Stick</titre>
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-
<description>
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</description>
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</jour>
</jour>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
 +
Miniprep of the transformed clones. The gel electrophoresis confirmed the expected insert. (<i>abrB</i> gene in pSB1C3 backbone)
</description>
</description>
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<titre>Stick</titre>
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<description>
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</description>
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</jour>
</jour>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
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The plasmids pBK42 and pBK46 were submitted to the Registry.
</description>
</description>
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<titre>Stick</titre>
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-
<description>
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</description>
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</jour>
</jour>

Latest revision as of 00:15, 27 October 2012

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