Team:Lyon-INSA/notebook
From 2012.igem.org
(Difference between revisions)
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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/> | 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 to 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 | + | The antibiotic resistance to 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 <i>abrB</i>. |
</description> | </description> | ||
</jour> | </jour> | ||
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<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 168 GFP : no resistance;</li> | ||
- | <li>Bs abrB : Cm resistant;</li> | + | <li>Bs <i>abrB</i> : Cm resistant;</li> |
<li>Bs 168 lysostaphin PWG100 : no resistance;</li> | <li>Bs 168 lysostaphin PWG100 : no resistance;</li> | ||
<li><i>S. epidermidis</i> : Tet resistant.</li> | <li><i>S. epidermidis</i> : Tet resistant.</li> | ||
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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 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 µL Chloramphenicol + 500 µL 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>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-sfp.</li> | + | <li>Transformation of NM522 strain with pUC57-Lysostaphin, pUC57-Dispersin and pUC57-<i>sfp</i>.</li> |
<li>200 µL of each transformed strains were spread on LB + Ampicillin plates.</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>Liquid cultures of the following strains were made : Bs 168 in LB, Bs <i>abrB</i> in LB + Chloramphenicol, <i>S. epidermidis</i> in LB + Tetracyclin</li></ul> |
</description> | </description> | ||
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<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-<i>lacI</i>-terminator) in 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 (in LB broth supplemented with required antibiotic) :</li> | ||
<ul> | <ul> | ||
<li><i>S. epidermidis</i> = BK1 (Tet<sup>R</sup>);</li> | <li><i>S. epidermidis</i> = BK1 (Tet<sup>R</sup>);</li> | ||
- | <li>Bs abrB = BK2 (Cm<sup>R</sup>);</li> | + | <li>Bs <i>abrB</i> = BK2 (Cm<sup>R</sup>);</li> |
<li>Bs 168 = BK3.</li> | <li>Bs 168 = BK3.</li> | ||
</ul> | </ul> | ||
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<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-Lysostaphin/pUC57-Dispersin/pUC57-lacI from transformed NM522 strains.</li> | + | <li>Extraction of pUC57-Lysostaphin/pUC57-Dispersin/pUC57-<i>lacI</i> 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 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> | <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> | ||
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<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 <i>lacI</i>-pUC57;</li> |
- | <li>NM522 containing | + | <li>NM522 containing RBS-pUC57;</li> |
<li>NM522 containing dispersin-pUC57;</li> | <li>NM522 containing dispersin-pUC57;</li> | ||
</ul> | </ul> | ||
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<ul> | <ul> | ||
<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>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- | + | <li>Ligation of promoter-RBS-<i>gfp</i> in plasmid.</li> |
<li>Genomic DNA extraction of <i>B. subtilis</i> 168: 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> | ||
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<description> | <description> | ||
<ul> | <ul> | ||
- | <li>Cloning of the constitutive promoter in front of the dispersin part (the gene coding for Dispersin) and RBS/ | + | <li>Cloning of the constitutive promoter in front of the dispersin part (the gene coding for Dispersin) and RBS/<i>gfp</i> (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 electrophoresis showed a shaded DNA → RNase addition in the extraction kit buffer was forgotten.</li> | <li>Extraction of pUC57-lysostaphin. Gel electrophoresis showed a shaded DNA → RNase addition in the extraction kit buffer was forgotten.</li> | ||
</ul> | </ul> | ||
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<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>Extraction and digestion (E & P) of Bs 168 strain GFP’s plasmid. Gel electrophoresis showed absence of non digested plasmid → extraction failure.</li> | ||
<li>Genomic DNA extraction of <i>B. subtilis</i> 168 (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> <i>abrB</i> failed.</li> |
</ul> | </ul> | ||
</description> | </description> | ||
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<description> | <description> | ||
<ul> | <ul> | ||
- | <li>The transformation results of the 3A ligation ([dispersin+RBS/ | + | <li>The transformation results of the 3A ligation ([dispersin+RBS/<i>gfp</i>+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 smear. 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> | ||
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<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 50µg/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 | + | <li>Transformation of <i>yfp</i>, <i>gfp</i> and <i>cfp</i> (from iGEM plates) in NM522;</li> |
<li>B0015 transformation in NM522.</li> | <li>B0015 transformation in NM522.</li> | ||
</ul> | </ul> | ||
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<description> | <description> | ||
<ul> | <ul> | ||
- | <li>The transformation of the 3A ligation ([dispersin+RBS/ | + | <li>The transformation of the 3A ligation ([dispersin+RBS/<i>gfp</i>+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 [dispersin+RBS/ | + | <li>The fluorescence test of the transformed bacteria containing [dispersin+RBS/<i>gfp</i>+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> | ||
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<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>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 <i>B. subtilis</i> genomic DNA.</li> | <li>Quantification and gel electrophoresis of <i>B. subtilis</i> genomic DNA.</li> | ||
- | <li>Antibiotic testing of <i>B. thuringensis</i> 407 gfp strain and other strains in LB+Ery growth medium.</li> | + | <li>Antibiotic testing of <i>B. thuringensis</i> 407 <i>gfp</i> 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> | ||
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<li>Ligation Lysostaphin in pSB1C3 and transformation.</li> | <li>Ligation Lysostaphin in pSB1C3 and transformation.</li> | ||
<li>Isolation of 6 clones of the Terminator 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 <i>yfp</i>. </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> | ||
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<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>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>LB+Ery and TSB+Tet Petri plates are made.</li> | <li>LB+Ery and TSB+Tet Petri plates are made.</li> | ||
- | <li> | + | <li><i>yfp</i> 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> | ||
<li><i>B. subtilis</i> 168 is put in storage (BK14) in TSB medium.</li> | <li><i>B. subtilis</i> 168 is put in storage (BK14) in TSB medium.</li> | ||
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<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 <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 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> | ||
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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+ | + | <li>Extraction of the [Promoter+RBS+<i>gfp</i>] in pBK23 (Tet R) by miniprep and verification by electrophoresis after digestion by <i>Eco</i>RI and <i>Pst</i>I : gel ok. Congrats (pBK12) → 122,6 ng/µL.</li> |
<li>Extraction of the pBK6 plasmid (P<sub>veg</sub>+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> | ||
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<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>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 rRNA 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 + <i>gfp</i>, <i>cfp</i> and <i>yfp</i> in storage.</li> |
- | <li> | + | <li>pHT315 GFP put in storage under the reference pBK18.</li> |
<li>NM522 + pHT315 GFP strain put in storage under the reference BK21.</li> | <li>NM522 + pHT315 GFP strain put in storage under the reference BK21.</li> | ||
</ul> | </ul> | ||
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<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | 3A assembly | + | 3A assembly RBS-<i>gfp</i> (pBK7-pBK14) in pSB1K3 = L1. Transformation of L1 in NM522. |
</description> | </description> | ||
</jour> | </jour> | ||
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<titre>For all purposes</titre> | <titre>For all purposes</titre> | ||
<description> | <description> | ||
- | Extraction of transformed clones (NM522/pHT304 | + | Extraction of transformed clones (NM522/pHT304 and NM522/pHT315). The electrophoresis showed that the plasmids had approximately 7kb and the restriction sites <i>Xba</i>I, <i>Eco</i>RI and <i>Pst</i>I as expected. However, there is a <i>Spe</i>I site which had not been mapped. |
</description> | </description> | ||
<titre>Kill</titre> | <titre>Kill</titre> | ||
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<description> | <description> | ||
<ul> | <ul> | ||
- | <li><i>sfp</i> (surfactin part 1) and <i>abrB</i> put in storage : pBK16 | + | <li><i>sfp</i> (surfactin part 1) and <i>abrB</i> put in storage : pBK16 and pBK17.</li> |
<li>Failure of transformation of L1 in NM522.</li> | <li>Failure of transformation of L1 in NM522.</li> | ||
- | <li>3A assembly of RBS- | + | <li>3A assembly of RBS-<i>cfp</i> (pBK7-pBK13) in pSB1C3 = L2. (!! the part2 construction already has a terminator)</li> |
- | <li>3A assembly of sfp-part2(lacI)-terminator (pBK4-pBK10) in pSB1C3 = IV.</li> | + | <li>3A assembly of <i>sfp</i>-part2(<i>lacI</i>)-terminator (pBK4-pBK10) in pSB1C3 = IV.</li> |
</ul> | </ul> | ||
</description> | </description> | ||
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<description> | <description> | ||
<ul> | <ul> | ||
- | <li>Transformation of L1, L2 | + | <li>Transformation of L1, L2 and IV in NM522.</li> |
- | <li>Transformation of sfp and abrB in NM522.</li> | + | <li>Transformation of <i>sfp</i> and <i>abrB</i> in NM522.</li> |
</ul> | </ul> | ||
</description> | </description> | ||
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<description> | <description> | ||
<ul> | <ul> | ||
- | <li>Given the fact that the previous transformation didn’t work, we made another transformation of NM522 strain with the <i>abrB</i> and <i>sfp</i> parts.</li> | + | <li>Given the fact that the previous transformation didn’t work, we made another transformation of NM522 strain with the <i>abrB</i> and <i><i>sfp</i></i> 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> | ||
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<ul> | <ul> | ||
<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 (<i>sfp</i> and <i>abrB</i> 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 <i>sfp</i> and <i>abrB</i> 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> | ||
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<description> | <description> | ||
<ul> | <ul> | ||
- | <li>Ligation (3A protocol) of pBK7, pBK13 and iGEM backbone pSB1K3 (=RBS+ | + | <li>Ligation (3A protocol) of pBK7, pBK13 and iGEM backbone pSB1K3 (=RBS+<i>cfp</i>+pSB1K3).</li> |
- | <li>Ligation (3A protocol) of pBK7, pBK14 and iGEM backbone pSB1K3 (=RBS+ | + | <li>Ligation (3A protocol) of pBK7, pBK14 and iGEM backbone pSB1K3 (=RBS+<i>gfp</i>+pSB1K3).</li> |
<li>Because of the difficulties to transform the bacteria with the plasmids containing <i>sfp</i> and <i>abrB</i>, we decided to do an electrophoresis directly on the constructions sent by Genecust. Result : the genes were not inserted in a pUC57 plasmid, although they should have been cloned.</li> | <li>Because of the difficulties to transform the bacteria with the plasmids containing <i>sfp</i> and <i>abrB</i>, we decided to do an electrophoresis directly on the constructions sent by Genecust. Result : the genes were not inserted in a pUC57 plasmid, although they should have been cloned.</li> | ||
<li>Ligation of <i>abrB</i> and <i>sfp</i> in pSB1K3 and transformation in NM522.</li> | <li>Ligation of <i>abrB</i> and <i>sfp</i> in pSB1K3 and transformation in NM522.</li> | ||
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<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | Plasmid extractions from 3 clones NM522/abrB and 5 clones NM522/sfp. The electrophoresis of the digested plasmids with EcoRI and PstI confirmed the presence of <i>sfp</i> construction (at 800 bp) and <i>abrB</i> construction (at 500 bp). | + | Plasmid extractions from 3 clones NM522/<i>abrB</i> and 5 clones NM522/sfp. The electrophoresis of the digested plasmids with EcoRI and PstI confirmed the presence of <i>sfp</i> construction (at 800 bp) and <i>abrB</i> construction (at 500 bp). |
</description> | </description> | ||
<titre>Stick</titre> | <titre>Stick</titre> | ||
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<li>Transformation of the following ligations, in NM522 strain :</li> | <li>Transformation of the following ligations, in NM522 strain :</li> | ||
<ul> | <ul> | ||
- | <li>pBK7+pBK13+pSB1K3 (RBS+ | + | <li>pBK7+pBK13+pSB1K3 (RBS+<i>cfp</i> in Kanamycin resistant backbone);</li> |
- | <li>pBK7+pBK14+pSB1K3 (RBS+ | + | <li>pBK7+pBK14+pSB1K3 (RBS+<i>gfp</i> in Kanamycin resistant backbone).</li> |
</ul> | </ul> | ||
- | <li>PCR of RBS-abrB and P<sub>xyl</sub> and purification of these PCR products.</li> | + | <li>PCR of RBS-<i>abrB</i> and P<sub>xyl</sub> and purification of these PCR products.</li> |
</ul> | </ul> | ||
</description> | </description> | ||
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<description> | <description> | ||
<ul> | <ul> | ||
- | <li>3A ligation : [P<sub>xyl</sub>-RBS-sfp] + [RBS-abrB] + [psB1T3].</li> | + | <li>3A ligation : [P<sub>xyl</sub>-RBS-<i>sfp</i>] + [RBS-<i>abrB</i>] + [psB1T3].</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>Transformation of the ligation [P<sub>xyl</sub>-RBS-<i>sfp</i>] + [RBS-<i>abrB</i>] + 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 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>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 P<sub>xyl</sub> produced by PCR.</li> | <li>Purification of P<sub>xyl</sub> produced by PCR.</li> | ||
Line 1,092: | Line 1,092: | ||
<description> | <description> | ||
<ul> | <ul> | ||
- | <li>Result of the transformation of NM522 by pHT304 S | + | <li>Result of the transformation of NM522 by pHT304 S and pHT315 S : 0 clone and 1 clone... this only clone is tested but the test is negative : the <i>Spe</i>I 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 pSB1T3 + 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> | ||
Line 1,114: | Line 1,114: | ||
<ul> | <ul> | ||
<li>Transformations of NM522 by pBK7+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 <i>sfp</i>-<i>abrB</i>-pSB1T3 : the negative control isn’t good, the transformation is done again...</li> |
</ul> | </ul> | ||
<li>Miniprep of other clones transformed with the plasmid RBS + xylR : the electrophoresis shows that the ligation still isn’t good…</li> | <li>Miniprep of other clones transformed with the plasmid RBS + xylR : the electrophoresis shows that the ligation still isn’t good…</li> | ||
Line 1,144: | Line 1,144: | ||
<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 real transformation contains nothing either ! We decided to do the sfp-abrB ligation again...</li> | + | <li>The transformation of NM522 strain by <i>sfp</i>-<i>abrB</i> 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 <i>sfp</i>-<i>abrB</i> 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> | <li><strong>PROBLEM : WE RAN OUT OF LIGASE!!</strong> | ||
Line 1,175: | Line 1,175: | ||
<ul> | <ul> | ||
<li>Ligation of the <i>sfp</i> and <i>abrB</i> parts in an iGEM backbone.</li> | <li>Ligation of the <i>sfp</i> and <i>abrB</i> parts in an iGEM backbone.</li> | ||
- | <li>The NM522 strain with abrB-pSB1K3 was put in storage under the reference BK25.</li> | + | <li>The NM522 strain with <i>abrB</i>-pSB1K3 was put in storage under the reference BK25.</li> |
- | <li>The NM522 strain with sfp-pSB1K3 was put in storage under the reference BK26.</li> | + | <li>The NM522 strain with <i>sfp</i>-pSB1K3 was put in storage under the reference BK26.</li> |
</ul> | </ul> | ||
</description> | </description> | ||
Line 1,215: | Line 1,215: | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | Transformation of the ligation [sfp + abrB + pSB1A3] in the NM522 strain. | + | Transformation of the ligation [<i>sfp</i> + <i>abrB</i> + pSB1A3] in the NM522 strain. |
</description> | </description> | ||
</jour> | </jour> | ||
Line 1,234: | Line 1,234: | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | The transformation of the ligation [sfp+abrB+pSB1A3] was successful, so 4 clones were put in liquid culture in order to be tested. | + | The transformation of the ligation [<i>sfp</i>+<i>abrB</i>+pSB1A3] was successful, so 4 clones were put in liquid culture in order to be tested. |
</description> | </description> | ||
<titre>Stick</titre> | <titre>Stick</titre> | ||
Line 1,288: | Line 1,288: | ||
<description> | <description> | ||
<ul> | <ul> | ||
- | <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>Miniprep of 4 clones with the transformed 3A ligation (pBK22, gene <i>abrB</i> 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 P<sub>xyl</sub> 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> | ||
Line 1,332: | Line 1,332: | ||
<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 + 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 + 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 + | + | <li>Results of the transformation of NM522 by [pSB1T3 + RBS-<i>abrB</i>] : no clones.</li> |
</ul> | </ul> | ||
</description> | </description> | ||
Line 1,365: | Line 1,365: | ||
<ul> | <ul> | ||
<li>Extraction of the plasmid containing the ligation [<i>sfp</i>+<i>abrB</i>+pSB1A3] from a liquid culture of transformed bacteria.</li> | <li>Extraction of the plasmid containing the ligation [<i>sfp</i>+<i>abrB</i>+pSB1A3] from a liquid culture of transformed bacteria.</li> | ||
- | <li>Results of the second transformation of NM522 by pSB1T3 and | + | <li>Results of the second transformation of NM522 by pSB1T3 and RBS-<i>abrB</i> : no clones.</li> |
</ul> | </ul> | ||
</description> | </description> | ||
Line 1,410: | Line 1,410: | ||
<ul> | <ul> | ||
<li>Liquid culture of <i>B. subtilis</i> 168 in 5mL of LB broth.</li> | <li>Liquid culture of <i>B. subtilis</i> 168 in 5mL of LB broth.</li> | ||
- | <li>Liquid culture of <i>B. subtilis</i> abrB in 5mL of LB broth.</li> | + | <li>Liquid culture of <i>B. subtilis</i> <i>abrB</i> in 5mL of LB broth.</li> |
</ul> | </ul> | ||
</description> | </description> | ||
Line 1,448: | Line 1,448: | ||
<titre>Physiological tests</titre> | <titre>Physiological tests</titre> | ||
<description> | <description> | ||
- | A microtiter plate is inoculated with <i>B. subtilis</i> 168 and <i>B. subtilis</i> abrB in order to compare the adherence of each strain. (see Protocol 'Tests of Bacillus subtilis adherence in 24 wells plate'). | + | A microtiter plate is inoculated with <i>B. subtilis</i> 168 and <i>B. subtilis</i> <i>abrB</i> in order to compare the adherence of each strain. (see Protocol 'Tests of Bacillus subtilis adherence in 24 wells plate'). |
</description> | </description> | ||
</jour> | </jour> | ||
Line 1,460: | Line 1,460: | ||
<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 (pSB1C3 containing a RFP gene). The cloned fragment was transformed in the NM522 strain. | + | 3A ligation of pBK4 (pUC57 containing the <i>lacI</i> gene) and pBK29 (pSB1A3 containing the <i>sfp</i> and <i>abrB</i> 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 1,471: | Line 1,471: | ||
<titre>Physiological tests</titre> | <titre>Physiological tests</titre> | ||
<description> | <description> | ||
- | The microtiter plate prepared the day before is analyzed. There is a significant difference between the adherence potential of the two strains. <i>B. subtilis</i> abrB strain forms thin layer biofilms while <i>B. subtilis</i> 168 doesn't form any specific kind of biofilms. | + | The microtiter plate prepared the day before is analyzed. There is a significant difference between the adherence potential of the two strains. <i>B. subtilis</i> <i>abrB</i> strain forms thin layer biofilms while <i>B. subtilis</i> 168 doesn't form any specific kind of biofilms. |
</description> | </description> | ||
</jour> | </jour> | ||
Line 1,566: | Line 1,566: | ||
<li>Miniprep of the two clones containing the <i>sfp</i> and <i>abrB</i> 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>Miniprep of the two clones containing the <i>sfp</i> and <i>abrB</i> 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 (<i>sfp</i> and <i>abrB</i>) 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>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 (<i>sfp</i> and <i>abrB</i>) 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 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> | + | <li>In parallel, we decided to do a standard ligation in order to transfer the <i>lacI</i> 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> | </ul> | ||
</description> | </description> | ||
Line 1,656: | Line 1,656: | ||
<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>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>6 other 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 <i>abrB</i> gene and the plasmid containing <i>lacI</i> in pSB1C3 backbone (pBK37) and transformation. In parallel, another ligation is attempted : [pBK37 + pBK29].</li> |
</ul> | </ul> | ||
</description> | </description> | ||
Line 1,712: | Line 1,712: | ||
<li><i>abrB</i> gene obtained by PCR was cloned in pSB1T3 backbone.</li> | <li><i>abrB</i> gene obtained by PCR was cloned in pSB1T3 backbone.</li> | ||
<li>6 clones transformed with the mixture of two plasmids are screened.</li> | <li>6 clones transformed with the mixture of two plasmids are screened.</li> | ||
- | <li>Miniprep of 12 clones per transformed ligation : only one had the expected fragments (ligation of the lacI and abrB genes). The clone is put in storage under the reference pBK39.</li> | + | <li>Miniprep of 12 clones per transformed ligation : only one had the expected fragments (ligation of the <i>lacI</i> and <i>abrB</i> genes). The clone is put in storage under the reference pBK39.</li> |
</ul> | </ul> | ||
</description> | </description> | ||
Line 1,731: | Line 1,731: | ||
<li>Standard ligations between : | <li>Standard ligations between : | ||
<ul> | <ul> | ||
- | <li>P<sub>lac</sub> in pSB1C3 (pBK9) digested by <i>Spe</i>I and <i>Pst</i>I and [RBS- | + | <li>P<sub>lac</sub> in pSB1C3 (pBK9) digested by <i>Spe</i>I and <i>Pst</i>I and [RBS-<i>gfp</i>] in pSB1T3 digested by <i>Xba</i>I and <i>Pst</i>I;</li> |
- | <li>P<sub>xyl</sub> (amplified by PCR) digested by <i>Spe</i>I and <i>Eco</i>RI and [RBS- | + | <li>P<sub>xyl</sub> (amplified by PCR) digested by <i>Spe</i>I and <i>Eco</i>RI and [RBS-<i>gfp</i>] in pSB1T3 digested by <i>Xba</i>I and <i>Eco</i>RI.</li> |
</ul> | </ul> | ||
These two ligation products are transformed in NM522 strain.</li> | These two ligation products are transformed in NM522 strain.</li> | ||
Line 1,765: | Line 1,765: | ||
<description> | <description> | ||
<ul> | <ul> | ||
- | <li>Standard ligation of pBK22 (sfp gene) and pBK39 (abrB-lacI) and transformation into the <i>E. coli</i> NM522 strain.</li> | + | <li>Standard ligation of pBK22 (<i>sfp</i> gene) and pBK39 (<i>abrB</i>-<i>lacI</i>) 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> | <li>A SDS-PAGE gel is run to analyze lysostaphin (in BK32 and BK35 strains) and dispersin (BK29 strain).</li> | ||
</ul> | </ul> | ||
Line 1,783: | Line 1,783: | ||
<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 : | <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 | + | <li>pBK 25 (= modified shuttle vector pHT304);</li> |
- | <li>pBK 26 (= modified shuttle vector | + | <li>pBK 26 (= modified shuttle vector pHT315);</li> |
- | <li>pBK 28 (= Lysostaphin in the modified shuttle vector | + | <li>pBK 28 (= Lysostaphin in the modified shuttle vector pHT304);</li> |
- | <li>pBK 38 (= Lysostaphin in the modified shuttle vector | + | <li>pBK 38 (= Lysostaphin in the modified shuttle vector pHT315);</li> |
<li>Dispersin in pBK26.</li> | <li>Dispersin in pBK26.</li> | ||
</ul> | </ul> | ||
Line 2,048: | Line 2,048: | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <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. | + | Gel electrophoresis of the extracted plasmids shows that there are 5 possible plasmids containing the ligated insert (P<sub>xyl</sub>-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>-terminator). The five plasmids are digested by EcoRV and the electrophoresis confirms that, this time, the plasmids have the expected fragments. |
</description> | </description> | ||
<titre>Physiological tests</titre> | <titre>Physiological tests</titre> | ||
Line 2,076: | Line 2,076: | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | Transformation of the ligations between the plasmid pBK42 containing the construction [ | + | Transformation of the ligations between the plasmid pBK42 containing the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] and the shuttle vectors pBK35 and pBK36 into the NM522 strain . |
</description> | </description> | ||
Line 2,092: | Line 2,092: | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | Screening of 15 clones transformed with the | + | Screening of 15 clones transformed with the ligation pBK42+pBK35 and pBK42+pBK36 |
</description> | </description> | ||
Line 2,113: | Line 2,113: | ||
<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 [ | + | <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> |
</description> | </description> | ||
Line 2,129: | Line 2,129: | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | <li>30 clones containing the construction [ | + | <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 |
</li> | </li> | ||
</description> | </description> | ||
Line 2,147: | Line 2,147: | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | <li> Miniprep of the transformed clones with the construction [ | + | <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. |
</li> | </li> | ||
</description> | </description> | ||
Line 2,168: | Line 2,168: | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | <li> Transformation of <i>B. subtilis | + | <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. |
</li> | </li> | ||
</description> | </description> | ||
Line 2,208: | Line 2,208: | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | <li> 24 clones transformed with the construction [pXyl-sfp-abrB-lacI] are screened; | + | <li> 24 clones transformed with the construction [pXyl-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>] are screened; |
</li> | </li> | ||
</description> | </description> | ||
Line 2,226: | Line 2,226: | ||
<titre>Kill</titre> | <titre>Kill</titre> | ||
<description> | <description> | ||
- | A last SDS-PAGE is run. This time the problems were | + | A last SDS-PAGE is run. This time the problems were successfully solved. But the gels were not conclusive due to a low dispersin concentration. |
</description> | </description> | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | <li> 6 of the 24 clones are put in liquid culture for further testing in LB | + | <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%; |
</li> | </li> | ||
</description> | </description> | ||
Line 2,247: | Line 2,247: | ||
<titre>Surfactant</titre> | <titre>Surfactant</titre> | ||
<description> | <description> | ||
- | <li> In order to characterize the construction [ | + | <li> In order to characterize the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] two tests are made. To confirm the presence of <i>sfp</i> gene, we made emulsions with the filtered supernatant of the transformed strains and sunflower oil. Concerning <i>abrB</i> gene, a biofilm formation test was made in a 24-well microplate in order to compare the transformed strain to the wild-type strain. |
</li> | </li> | ||
</description> | </description> |
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