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| | </jour> | | </jour> |
| - | ___________________________________________________________________________________
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| - | Wednesday, 15th August:
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| - | For all :
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| - | - The transformation of the pBK20 (pHT315) and pBK19 (pHT304) plasmids was successful. 12 clones per plasmid were tested.
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| - | Kill :
| |
| - | - The lysostaphin test didn’t work : Valérie did the test again by applying first the supernatant on the pastilles before putting them on the plates, and by using TSM media instead of LB media.
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| - | - Transformation of the ligation pSB1C3 + constitutive promoter in NM 522 strain.
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| - | Surfactant :
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| - | - None of the 4 clones tested had the attended fragments, so we screened another 6.
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| - | Stick :
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| - | - Results of the transformation of NM522 by pBK7 and XylR : the negativ control contains nothing, the positiv 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.
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| - | - 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.
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| | | | |
| - | ___________________________________________________________________________________
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| | | | |
| - | Thursday, 16th August:
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| - | For all :
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| - | The electrophoresis after digestion with EcoRI and SpeI showed that the SpeI site was eliminated. 4 clones were chosen for further tests. However, the DNA concentration was to low so the enzymes did not digest the plasmids.
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| - | Kill :
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| - | - Ligation of the Constitutiv promoter (pVeg) produced by PCR in a plasmid IGEM.
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| - | - Result of the lysostaphin tests : didn’t work again. The Bs 168 pWG100 supernatant doesn’t contain any lysostaphine : the lysostaphine is probably degraded in one way or another (sensitivity to the defrosting ?).
| |
| - | - Result of the transformation with pSB1C3 + promoter : a lot of red clones, some white clones (20 - 30). Isolation of 6 white clones and plasmid extractions by miniprep.
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| - | Surfactant :
| |
| - | - Two of the 6 clones sfp+abrB had the expected fragments.
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| - | - Ligation of pXyl produced by PCR in a plasmid IGEM.
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| - | Stick :
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| - | - Ligation of XylR produced by PCR in a plasmid IGEM (pSB1K3).
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| - | - Results of the transformation of NM522 by pBK7 and XylR (for the second ligation) : the plate with bacteria transformed by pBK7 + XylR contains a lot of clones and the controls are standard. 14 clones are selected to do liquid culture and extract their DNA.
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| - | - Plasmid extractions from the 8 clones transformed by PBK7+XylR. The electrophoresis of the digested plasmids showed that the clones have not the right plasmid : they have just the vector without XylR.
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| - | ___________________________________________________________________________________
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| - | Friday, 17th August :
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| - | For all :
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| - | - The plasmids (pBK19 and pBK20) from 2 clones were re-extracted using columns. This time the digestion was successful. The tests showed that there was no SpeI site. However, the pHT315 plasmid also had no XbaI site.
| |
| - | Stick :
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| - | - Plasmid extractions from the 14 clones transformed by PBK7+XylR (with the second ligation). Like for the first transformation, the electrophoresis of the digested plasmids showed that the clones have not the right plasmid : they have just the vector without XylR.
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| - | ___________________________________________________________________________________
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| - | Monday, 20th August:
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| - | For all :
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| - | - The pBK19 with no SpeI site was put in storage under the reference pBK25.
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| - | - The pBK20 with no SpeI site was put in storage under the reference pBK26.
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| - | Kill :
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| - | Launch of 8 Promoter-Dispersin cultures
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| - | - Results of the transformation of NM522 by pSB1T3 and pXyl : the plate with bacteria transformed by psB1T3 + pXyl contains few clones and the controls are standard. 3 clones are selected to do liquid culture and extract their DNA : no plasmid
| |
| - | - Results of the transformation of NM522 by pSB1T3 and rbs-abrB : no clones.
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| - | Stick :
| |
| - | - Results of the transformation of NM522 by pSB1K3 and XylR : the plate with bacteria transformed by psB1K3 + XylR contains a lot of clones and the controls are standard. 3 clones are selected to do liquid culture and extract their DNA: they have just the vector without XylR.
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| - | - A new PCR of xylR is made, in order to increase the stock.
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| - | ___________________________________________________________________________________
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| - | Tuesday, 21st August:
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| - | For all :
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| - | - Meeting at 9 o’clock.
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| - |
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| - | Midiprep of the modified pBK25 and pBK26 shuttle plasmids (without the SpeI site). The extracted DNA was verified by electrophoresis.
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| - | Kill :
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| - | - Standard ligation of pBK23 (=Constitutiv Promoter + RBS + Lysostaphin in pSB1C3) with pBK25 (the shuttle vector E.coli - B.subtilis). Different ligations are made with differents proportions of insert and vector. Transformation in NM522 strain.
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| - | -Digestion of Lysostaphin in pSB1C3 and Dsp in pUC57
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| - | Checking of 8 clones promoter-dispersin, clones are nonstandard
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| - | Surfactant :
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| - |
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| - | Extraction of the plasmid containing the ligation [sfp+abrB+pSB1A3] from a liquid culture of transformed bacteria.
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| - | Results of the second transformation of NM522 by pSB1T3 and rbs-abrB : no clones.
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| - | Stick :
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| - | - Two standard ligations are done:
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| - | * pBK7-xylR (xylR cut in X and P sites)
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| - | * pBK24-xylR (xylR cut in E and S sites)
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| - | - Transformation of pBK7-xylR into NM522 strain.
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| - |
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| - | ___________________________________________________________________________________
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| - | Wednesday, 22nd August:
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| - | For all :
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| - |
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| - | Kill :
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| - | - Result of the transformation of NM522 with [Constitutiv Promoter + RBS + Lysostaphin] in pBK25 : all the controls are right, and there are a lot of clones on the different plates with the transformed bacteria. 14 clones are selected to do liquid cultures and extract their DNA.
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| - | - New try of clonage : Digestion, Ligation ,transformation to construct :
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| - | Lyso+Dsp in PSB1C3 and Lyso+Dspin the shuttle vector (vector for E.coli and B.subtilis)
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| - |
| |
| - | Surfactant :
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| - | - 6 clones pXyl were tested, but none had integrated the plasmid
| |
| - | Stick :
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| - | - checking of 6 clones XylR-psB1K3 : They have just the vector.
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| - | - Transformation of pBK24-xylR into NM522 strain.
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| - | - Results of pBK7-xylR transformation: lots of clones and no clones in the negative witness. 12 clones are put in liquid culture for extraction.
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| - |
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| - | ___________________________________________________________________________________
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| - | Thursday, 23rd August:
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| - |
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| - | cloning of the iGEM linker into the shuttle vector (pBK25 and pBK26) and transformation in the NM522 strain;
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| - |
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| - | Kill:
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| - | -Transformations results : All controls are okay
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| - | Too much clones on Lyso+Dsp in shuttle vector transformation plate, so some clones are selected and spread and a new LB plate with the appropriate antibiotic
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| - | 12 liquid culture in LB are launched for the clones Lyso+Dsp in PSB1C3
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| - | - Miniprep of 14 clones transformed with the ligation product [Constitutiv Promoter + RBS + Lysostaphin] in pBK25 and electrophoresis to analyse the extracted DNA : the transformation is succesful for 3 clones =)
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| - | - The first plasmid [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (1).
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| - | - The second plasmid [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (2).
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| - | - The third plasmid [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (3).
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| - | Surfactant :
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| - | - Transformation of pXyl+pSB1T3 in NM522.
| |
| - | Stick :
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| - | - Results of pBK24-xylR transformation: most of the clones are red, however a few of them are white, which is good. 7 white clones are put in liquid culture for extraction.
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| - | - Extraction of pBK7-xylR from 12 clones, and then a gel electrophoresis is run: 2 clones seem interesting. We ran a second gel to verify these 2 clones, but it showed us that they were not good.
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| - | ------------------------------------------------------------------------------------------------------------------
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| - | Friday 24th August
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| - | For all :
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| - |
| |
| - | the transformations of the cloned shuttle vectors was successful, so 12 clones are put in liquid culture for further tests;
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| - | Kill:
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| - | Surfactant :
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| - |
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| - | 3A ligation of pBK4 (pUC57 containing the lacI gene) and pBK29 (pSB1A3 containing the sfp and abrB genes) in pBK24 backbone (pSB1C3 containing a RFP gene). The cloned fragment was transformed in the NM522 strain;
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| - |
| |
| - | Stick :
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| - | - After the bad results with xylR, we decided to cut pBK10 plasmid in Sma1 site, and ligate with xylR, doing a blunt ligation. We also decided to ligate xylR with xylR, creating a big polymer, which will be like a “pre-ligation” molecule.
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| - | - Extraction of pBK24-xylR from 7 clones. Gel electrophoresis is run → ONE CLONE IS GOOD!! meaning that it’s the first time we manage to ligate xylR successfully.
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| - | ------------------------------------------------------------------------------------------------------------------
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| - | Saturday, 25th August
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| - | For all :
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| - |
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| - | miniprep of 12 clones selected from transformed bacteria with the cloned shuttle vectors; we were out of SpeI enzyme, so we could not digest the extracted plasmids.
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| - | Kill:
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| - | Surfactant :
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| - |
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| - | Stick :
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| - | -
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| - | ------------------------------------------------------------------------------------------------------------------
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| - |
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| - | Sunday, 26th August
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| - | For all :
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| - | Kill:
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| - | Surfactant :
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| - |
| |
| - | miniprep of 6 clones (3A ligation: pBK24, pBK4, pBK29). The electrophoresis showed that none of the tested clones had integrated the right plasmid.
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| - | Stick :
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| - | ------------------------------------------------------------------------------------------------------------------
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| - | Monday, 27th August
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| - | For all :
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| - | Kill:
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| - |
| |
| - | - Transformation of BS 168 strain with the pBK28 (1) plasmid which contains the [Constitutive Promoter + RBS + Lysostaphin] in the shuttle vector.
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| - | - Midiprep : Extraction of pveg-disp-pSB1C3 of clone 22
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| - | - BK33 was put in storage
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| - | Surfactant :
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| - |
| |
| - | given the fact that the digested plasmids extracted from transformed NM522 E. coli with the 3A ligation (pBK24, pBK4, pBK29) had unexpected fragments (and impossible to explain), the ligation was done again, this time changing the vector/insert ratio (1/1 and 1/3,as opposed to the first essay with a ⅙ ratio );
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| - | liquid cultures of two potentially good clones of the NM522 strain transformed with a 3A ligation (pBK22(promotor Pxyl, RBS and sfp gene), abrB gene and pSB1A3 backbone ) were made for miniprep (the two plasmids were extracted using the classical protocol, with extraction solutions that we made, so the yield and the purity of the extractions were poor; that’s why we decided to extract the plasmids again, this time using an extraction kit);
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| - | Stick :
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| - | - no transformation is done, because we ran out of LB growth medium.
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| - |
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| - | ------------------------------------------------------------------------------------------------------------------
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| - | Tuesday, 28th August
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| - | For all :
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| - | - Meeting at 1:30 pm
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| - | Kill:
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| - | - Ligation of Constitutive promoter-disp in the shuttle vector pBK25.
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| - | - 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 ?
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| - | We make an other transformation and we spread the transformed bacteria on GL+Ery plates with different erythromycin concentration (from 5 µg/mL to 10 µg/mL).
| |
| - | Surfactant :
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| - |
| |
| - | the transformations with the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios ) was successful. 12 clones per transformation are put in liquid culture and then streaked on LB+Cm plates and LB+Amp plates.
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| - |
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| - | Stick :
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| - |
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| - | ------------------------------------------------------------------------------------------------------------------
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| - | Wednesday, 29th August
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| - | For all :
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| - | Kill:
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| - | - Transformation of the previous ligation promoter-disp-pBK25 in E. Coli NM522.
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| - | - Results of the second transformation of BS 168 strain with the pBK28 (1) in the shuttle vector : the negative control is always full of bacteria whereas the erythromycin concentration was higher than the previous try (0,5 µg/mL the first time, 5 and 10 µg/mL the second time).
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| - | We made (GL+Ery) plates with different erythromycin concentrations in order to make different tests.
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| - |
| |
| - | Surfactant :
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| - |
| |
| - | only one of the 24 screened colonies concerning the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios ) did not turn up to be red (LB+Cm plate) and did not grow on a LB+Amp plate. The clone was used to inoculate a liquid culture for an extraction.
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| - |
| |
| - | miniprep of the two clones containing the genes sfp and abrB. 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;
| |
| - | given the fact we were not very optimistic concerning the 3A ligation of pBK24, pBK4 and pBK29, we decided to do two more essays, this time using two ligated genes (sfp and abrB) coming from different 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 classical protocol because we were running out of time and we were behind the schedule.
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| - | in parellel, 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.
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| - |
| |
| - | Stick :
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| - |
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| - | ------------------------------------------------------------------------------------------------------------------
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| - | Thursday, 30th August
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| - | For all :
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| - | Kill:
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| - | Transformation results, too much clones on the control digest vector not liguate so the transformation can’t be used for the transformation Lyso+Dsp in PSB1C3
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| - | 12 clones put in culture from the transformation plate Lyso+Dsp in the shuttle navette treated by BamHI
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| - | Preparation of tubes for sequencing, launch of miniprep and midiprep
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| - | - Standard ligation between the shuttle vector pBK26 (PHT315 modified) and the pBK23 plasmid (= Lysostaphine in pSB1C3). Transformation of the ligation product in NM522 strain.
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| - | Surfactant :
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| - |
| |
| - | all 5 transformations were successful. White colonies are selected (pBK24 is a plasmid containing RFP in the pSB1C3 backbone, so we can exclude the colonies having integrated the religated backbone) and tested on LB+Cm and LB+Amp plates.
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| - | the miniprep of the one clone that had the expected phenotype (obtained after the transformation of the 3A ligation of pBK24, pBK4, pBK29) was digested, but the gel electrophoresis din not turn out as expected.
| |
| - | Stick :
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| - |
| |
| - | ------------------------------------------------------------------------------------------------------------------
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| - | Friday, 31th August
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| - | For all :
| |
| - | Kill:
| |
| - | Check of clones Lysostaphin-Dispersine in the shuttle vector (pBK26): clones are not good
| |
| - | Check of the miniprep for sequencing (Prom+Dsp in PSB1C3): DNA is okay
| |
| - | - Result of the transformation of NM 522 strain with the Lysostaphin inPBK26 :
| |
| - | - the negativ control is ok.
| |
| - | - the positiv control is full of colonies.
| |
| - | - the transformation is full of colonies : 24 clones are selected (on plates and in liquid culture) in order to extract their DNA and check it.
| |
| - | Surfactant :
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| - |
| |
| - | miniprep of the saturated cultures with transformed bacteria containing the ligation of the sfp and abrB genes. The electrophoresis confirmed that this time the bacteria contained the rigth plasmids.
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| - |
| |
| - | Stick :
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| | | | |
| | </month> | | </month> |
"
