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)