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.

• Long morning meeting to discuss results and to write some posters in a frenetic psychopath way in order to remember our tasks.
• Ligation of promoter-rbs-GFP in plasmid.
• Bs’ genomic DNA extraction: buffers preparation.
• Failure of B0015 transformation.

• Cloning of the constitutive promoter in front of the disp part (the gene coding for Dispersin) and RBS/GFP (of Ecoli) using a 3A ligation followed by transformation of the ligation in the NM522 strain;
• Extraction of pUC57-lysostaphin. Gel electrohporesis showed a shaded DNA → forgetting of RNase during the extraction.

• Extraction and digestion (E & P) of strain Bs 168 GFP’s plasmid. Gel electrohporesis showed absence of non digested plasmid → extraction failure.
• Bs 168’s genomic DNA extraction (Kit Genomic DNA from tissue) .
• Transformation of pBK5 in B. subtilis abrB failed.

• The transformation results of the 3A ligation ([disp+RBS/GFP+pSB1T3]) was unsuccessful, so it was repeated, this time with both a positive control (strain 39) and a negative one with NM522 culture ;
• Addition of RNase to the miniprep from pUC57-lysostaphin clones. Gel electrophoresis: there is still a layered cut. However, the bands are as expected.

• TSS tests are also made to be sure that all TSS solutions that we have are ok because some transformations did not work;
• A 50ug/mL erythromycin solution is made in order to test the strains’ resistance;
• Transformation of yfp, gfp et cfp (from iGEM plates) in NM522;
• B0015 transformation in NM522.

• The transformation of the 3A ligation ([disp+RBS/GFP+pSB1T3]) was successful, so 6 clones were tested on a plate containing BL media supplemented with Tetracyclin;
• The fluorescence test of the transformed bacteria containing [disp+RBS/GFP+pSB1T3] confirm that the promotor is functional
• Ligation of the promoter in a Cm resistant iGEM plasmid was made in order to send it to the registry.

• Transformation results : all TSS work (except maybe 1 and 2 with a smaller yield) and prom+pSB1C3 OK → 6 clones are isolated on a GL+Cm plate.
• Quantification and gel electrophoresis of B.subtilis’ genomic DNA.
• Antibiotic testing of B. thuringensis 407 gfp strain and other strains in BL+Ery growth medium.
• Failure of transforming pBK5 in B. subtilis 168 with the same protocol as previously. New task: find a new protocol.

• Transformation of NM522 strain with the part BBa_K606030 (pBK6) and promoter+dispersin;
• Ligation Prom+Dsp in P23 and transformation;
• Ligation Lysostaphin in pSB1C3 and transformation;
• Isolation of 6 clones of the Term transformation;
• Transformation results of fluorescent genes: ok, except yfp
• NM522 strain containing pUC57-lysostaphin is put in storage under the name of BK12.

• Selected clones (NM522+pBK6) are red, meaning that pVeg promoter and RBS from subtilis are recognized by E. coli’s ribosome. 4 clones are streaked in LB+Cm.
• GL+Ery and TSB+Tet Petri plates are made.
• YFP transformation was successful.
• GFP and CFP plasmids’ extraction showed a failure in ligation.
• B. subtilis 168 is put in storage (BK14) in TSB medium.
• Extraction of B0015 terminator. Gel electrophoresis showed 4 good clones.

• Transformation results
• Prom+Dsp in p23 : nothing on the plate;
• Lysostaphin, negative witness contains bacteria so the plate is put in junk.
• New digestions, ligations,transformations:
• Lyso+Dsp in pUc57;
• Dsp in PSB1C3;
• Lyso in PSB1C3;
• Prom+Dsp in p23.
• streaking of 4 clones of the transformed strain NM522/pBK6 on a Cm + LB plate. All of them formed red colonies which shows that the constitutive promotor and the RBS specific to the Bacillus subtilis strain are recognized by the RNA polymerase of E coli.