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Team:Lyon-INSA/notebook
From 2012.igem.org
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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 | + | <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 made 3 times) | ||
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<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 | + | 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. | ||
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<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 | + | <li>A liquid culture of NM522/pBK1 transformed cells was made so we could extract more plasmid DNA (50 mL LB media + 500 µL Cloramphenicol + 500 µL liquid culture of transformed bacteria );</li> |
<li>We had the 3 genes coding for lysostaphin, dispersin and lacI repressor in pUC57 Ampicillin resistant plasmid delivered;</li> | <li>We had the 3 genes coding for lysostaphin, dispersin and lacI repressor in pUC57 Ampicillin resistant plasmid delivered;</li> | ||
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<li>Transformation of NM522 strain with pUC57-Lyso, pUC57-Disp and pUC57-sfp;</li> | <li>Transformation of NM522 strain with pUC57-Lyso, pUC57-Disp and pUC57-sfp;</li> | ||
| - | <li>200 | + | <li>200 µL 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> | 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> | ||
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<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/ | + | <li>Extraction of the [Promoter+RBS+GFP] in pBK 23 (Tet R) by miniprep and check by electrophoresis after digesting by EcoRI and PstI : gel ok. Congrats (pBK12) -> 122,6 ng/µL.</li> |
<li>Extraction of the pBK6 plasmid (PVeg+RBS+RFP in psB1C3). The electrophoresis confirms the plasmid’s structure.</li> | <li>Extraction of the pBK6 plasmid (PVeg+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> | ||
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<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 electophoresis : lysostaphin clones are okay, promoter clones are not okay.</li> | ||
<li>pUC57 with Dsp put in storage : pBK3.</li> | <li>pUC57 with Dsp 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 | + | <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 µL 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> |
</ul> | </ul> | ||
</description> | </description> | ||
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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 the <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 | + | <li>New Lysostaphin test on LB+Tet plate with a negative control (10 µL of Ampicillin).</li> |
</ul> | </ul> | ||
</description> | </description> | ||
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<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 clones Promoter in Cm iGEM plasmid.</li> | ||
| - | <li>Preliminary test of the liquid lysostaphin (10 | + | <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=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> |
</ul> | </ul> | ||
</description> | </description> | ||
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<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 plasmid pSB1C3 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 | + | <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 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> | ||
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<titre>Stick</titre> | <titre>Stick</titre> | ||
<description> | <description> | ||
| - | Standard ligation between pBK7 (=RBS in pSB1C3) and XylR (obtained by PCR) with | + | 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> | ||
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<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 ( | + | <li>Second standard ligation between pBK7 (=RBS in pSB1C3) and XylR (producted by PCR) with more insert (3µL of insert for 2µL of vector). Transformation into NM522 strains. </li> |
</ul> | </ul> | ||
</description> | </description> | ||
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<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 | + | We made another transformation and we spread the transformed bacteria on GL+Ery plates with different erythromycin concentration (from 5 µg/mL to 10 µg/mL).</li> |
</ul> | </ul> | ||
</description> | </description> | ||
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<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 | + | <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 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 (GL+Ery) plates with different erythromycin concentrations in order to make different tests.</li> | ||
</ul> | </ul> | ||
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<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 | + | <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 the NM522 <i>E. coli</i> strain.</li> |
<li>Another 6 clones with the transformed 3A ligation (done on August 29th) are screened.</li> | <li>Another 6 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> | ||
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<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 | + | <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> | ||
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<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 | + | The bacteria are spread on GL+Ery plates ([ERY]=1 µg/mL and [ERY]=10 µg/mL). |
</description> | </description> | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
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<li>Dispersin in pBK26.</li> | <li>Dispersin in pBK26.</li> | ||
</ul> | </ul> | ||
| - | We spread 200 | + | We spread 200 µL of each transformed cells on GL + Erythromycin ([ERY] = 16 µg/mL) plates. |
</description> | </description> | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
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<li>BS 168 clones transformed by pBK26 and dispersine in pBK26 : nothing on the gel ... </li> | <li>BS 168 clones transformed by pBK26 and dispersine 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 | + | <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 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> | ||
Revision as of 13:54, 23 September 2012
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