Dilution of 100 µL saturated culture in 5 mL LB media.

Incubation time : 2 hours (until O.D =0,3).

• 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.

• 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

• 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

• Lysostaphin in pUC57 Amp resistant (pBK2);
• Dispersin in pUC57 Amp resistant (pBK3);
• Surfactin part 2 (RBS-lacI-terminator) dans pUC57 Amp resistant (pBK4)

• S epi on BL + Tet (BK1)
• Bs abrB on BL + Cm (BK2)
• Bs 168 on BL (BK3)

• 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)

• Extraction of part BBa_K606061 (RBS) Chloramphenicol resistant from transformed NM522 strain;
• Extraction of part BBa_B0010 (terminator) Ampicillin resistant from transformed NM522 strain; (nb : the clones were pink which means that the part contains a RFP gene)
• Extraction of part BBa_K606040 (pLacI) from transformed NM522 strain;
• Extraction of pUC57-Lyso/pUC57-Disp/pUC57-lacI from transformed NM522 strains;
• 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 colour of the colonies is pink). We decided to make another transformation with the same part, only this time the 2011 kit was used.

• PCR product electrophoresis of Promoter PCR : the product is not the good one.
• Reception and storage of the primers (for the amplification of the BBa_K143012 part);
• The transformed NM522 strain with the part BBa_0010 Ampicillin resistant from the 2011 iGEM kit shows the same phenotype as the part found in the 2012 kit;
• Extractions with a miniprep kit are made :
  • 4 clones containing the pLac promotor (1,2,3,4)
  • 4 clones containing the Bacillus subtilis RBS (1,2,3,4)
  • 3 clones containing theTerminator 1,2,3
  • 4 clones containing the gene for Dispersin
  • 4 clones containing the gene for Lysostaphin
  • 4 clones containing the gene for lacI
  Then a digestion is made on a 0.7% agarose gel.

• PCR of the constitutive promotor (part BBa_K143012); → the PCR did not work so we ran a new PCR with a more diluted promotor solution.
• The following strains are put in storage:
  • NM522 containing lacI-pUC57
  • NM522 containing rbs-pUC57
  • NM522 containing dsp-pUC57

• PCR of constitutive promoter → it didn’t work. A new PCR is run with modifications of settings. The annealing temperature was modified from 50°C to 57°C, and 3 solutions with different concentration were tested, however the concentration did not affect the yield.
• Purification of the PCR product.
• Gel electrophoresis of lysostaphin.

• Transformation of B0015 terminator.
• Strains BK13 (Bs arbB), BK10 (Bs 168 GFP) and BK11 (Bs 168 mCherry) are put in storage.