Team:Lyon-INSA/notebook

From 2012.igem.org

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<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>
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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>
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<li>Ligation of the part pBK13 containing the <i>RFP</i> gene with the part pBK7 (containing a <i>B. subtilis</i> RBS);</li>
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<li>Ligation of the part pBK13 containing the <i>rfp</i> gene with the part pBK7 (containing a <i>B. subtilis</i> RBS).</li>
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<li> transformation of the NM522 <i>E. coli</i> strain with the two ligations</li>
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<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>Ligation of the PCR product <i>xylR</i> gene with the part pBK9 (<i>Plac</i> gene)and NM522 transformation.
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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.
</description>
</description>
</jour>
</jour>
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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)  
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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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Inoculate 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. Incubate overnight at 37°C. (This is for the 48h positive <i>Bacillus subtilis</i> biofilm test plate)
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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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Liquid cultures are inoculated with colonies having the expected antibiotic phenotype.
Liquid cultures are inoculated with colonies having the expected antibiotic phenotype.
</br>
</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.
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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>
-
(24h plate)The same inoculation as previously is prepared.
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<ul>
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(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 %.
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<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>
The plate is incubated at 37°C for 48h
The plate is incubated at 37°C for 48h
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(For both)Inoculate 5 mL of LB medium with 1 colony of <i>E coli</i> (<i>ompR++</i> GFP, curlis overproduction).Incubate overnight at 37°C.  
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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>Stick</titre>
<titre>Stick</titre>
<description>
<description>
-
(24h 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 %.
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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 %.
The plate is incubated at 37°C for 48h.
The plate is incubated at 37°C for 48h.
</description>
</description>
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<titre>Surfactant</titre>
<titre>Surfactant</titre>
<description>
<description>
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The wells are carefully rinsed with M63 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 media diluted 2 times.</br>
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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.
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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 Bacillus subtilis biofilm the overnight culture of <i>E coli</i> is added into each wells.
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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.
Plates are incubated for 36h at 30°C.
Plates are incubated for 36h at 30°C.
</description>
</description>
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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.
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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 the Confocal microscopy !
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(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 !
Results : It works ! Our positive <i>Bacillus subtilis</i> biofilm inhibits the stick of other bacteria !
</description>
</description>
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Liquid cultures were inoculated with the strains BK49 and BK52 and then incubated at 37C for 48 hours.
Liquid cultures were inoculated with the strains BK49 and BK52 and then incubated at 37C for 48 hours.
</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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<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>
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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>
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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>
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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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