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Revision as of 10:48, 29 August 2012
For all purposes
Monday, July 2nd 2012
Liquid culture (5 mL LB media) of NM 522 cells and overnight incubation under agitation at 37°C for later transformations.
For all purposes
Tuesday, July 3rd 2012
Dilution of 100 µL saturated culture in 5 mL LB media.
Incubation time : 2 hours (until O.D =0,3).
Transformation of the NM 522 strain (this experiment was made 3 times)
For the positive control the pSB1C3 plasmid was used ;
For the transformation, the pBBA_I742123 was used (well A2, 5th iGEM kit plate)
The transformed bacteria were selected on chloramphenicol plates.
For all purposes
Wednesday, July 4th 2012
Transformation analysis:
- Positive control: lots of colonies
- Negative control: one of the three negative controls was contaminated (the correspondent transformed colonies were not used). No colonies were observed on the other two negative control plates.
- Test plate: between 1 and 8 were observed.
4 liquid cultures (5mL LB media + 50 µL chloramphenicol) and 4 streaked chloramphenicol plates were made using isolated colonies likely to contain transformed bacteria.
The antibiotic resistance (Ampicillin, Tetracyclin, Chloramphenicol and Kanamycin ) of the following strains was tested: BS 168, BS 168 MCherry, BS 168 GFP, BS 168 Lysostaphine, Staphylococcus epidermidis, BS Abrb.
For all purposes
Thursday, July 5th 2012
Antibiotics resistance tests :
- Bs 168 : no resistance
- Bs 168 M cherry : no resistance
- Bs 168 GFP : no resistance
- Bs abrB : Cm resistant
- Bs 168 lysostaphine PWG100 : no resistance
- S. Epidermidis : Tet resistant
Extraction of 4 clones containing the pBBA_I742123 plasmid. Verification by gel electrophoresis (after EcoR1 digestion)
For all purposes
Friday, July 6th 2012
Telephonic conference with Romain Briandet
Terminator was retrieved from the plate 1 well 13D
Long meeting
For all purposes
Monday, July 9th 2012
- pBBa_I742123 was put in storage (under the reference pBK1);
- 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 );
- we had the 3 genes coding for lysostaphin, dispersin and lacI repressor in pUC57 Ampicillin resistant plasmid delivered;
- transformation of NM522 strain with pUC57-Lyso, pUC57-Disp and pUC57-sfp;
- 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
For all purposes
Tuesday, July 10th 2012
the following parts were put in storage:
- Lysostaphin in pUC57 Amp resistant (pBK2);
- Dispersin in pUC57 Amp resistant (pBK3);
- Surfactin part 2 (RBS-lacI-terminator) dans pUC57 Amp resistant (pBK4)
and the following strains:
- S epi on BL + Tet (BK1)
- Bs abrB on BL + Cm (BK2)
- Bs 168 on BL (BK3)
ligation of Dispersin and Lysostaphin biobricks:
- digestion of pBK2 with the restriction enzymes EcoRI and SpeI;
- digestion of pBK3 with the restriction enzymes PstI and XbaI;
- digestion of pBK4 with the restriction enzymes EcoRI and PstI;
- 3A ligation of the digested parts;
- electrophoresis control showed the expected fragment for lysostaphin and dispersin (2,1 kb). However, the digested backbone plasmid had 3 fragments instead of 2.
Plasmid A2 extraction with a midiprep (pBK5) and digestion → 3 stripes are observed : PROBLEM. Digestion is made again with E, P and E+P → There are 2 Pst1 sites !!! WRONG PLASMID
- transformation of NM522 strain with the part BBa_K606061 Chloramphenicol resistant;
- transformation of NM522 strain with the part BBa_B0010 Ampicillin resistant;
- transformation of NM522 strain with the part BBa_K606040 Chloramphenicol resistant;
- design and order of the primers for the constitutive promotor (part BBa_K143012)
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