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.

• Plasmid extraction from Bacillus strain (BT407 GFP). We tested 2 methods in parallel. However, only the one using the extraction kit seems to work.
• Extraction of gfp, cfp, yfp ; test => OK : put in storage.
• YFP transformation was successful.
• Extraction of the pHT 315 GFP plasmid of B. thuringiensis 407 GFP to build a shuttle vector B. subtilis ? E. coli. Transformation in NM522 strain and selection on Ampicillin media : ok.
• B. subtilis 168 is put in storage (BK14) in TSB medium.
• S. epidermidis is put in storage in TSB media under the reference BK15.

• Transformation results:
  • Lyso+Dsp in pUc57 : the plate is covered of clones;
  • Promoter+Dsp : nothing on the plate;
  • Dsp in PSB1C3 : a lot of clones;
  • Lyso in PSB1C3 : a lot of clones.
  Selection of 4 clones of each lyso+igem plasmid and dsp+igem plasmid on Cm plate and in a liquid culture. Isolation of lyso+dsp (because there are too many clones!)
• Extraction of the plasmidic DNA [Promoter in PSB1C3].
• Extraction of pBK6 from the transformed strain.
• Extraction of the [Promoter+RBS+GFP] in pBK 23 (Tet R) by miniprep and check by electrophoresis after digesting by EcoRI and PstI : gel ok. Congrats (pBK12) -> 122,6 ng/µL.
• Extraction of the pBK6 plasmid (PVeg+RBS+RFP in psB1C3). The electrophoresis confirms the plasmid’s structure.
• - Liquid culture of Bs168 pWG100 overnight in order to do the first lysostaphin tests on plates.

• Strains are put in storage :
• S. epidermidis on TSB+Tet (BK17);
• B. thuringensis 407 GFP on GL+Ery (BK16).
• Minipreps to extract the plasmidic DNA of the clones NM522 + pHT 315 GFP and check by electrophoresis : plasmid ok (digested by E, X, S, P and no digested).
• Transformation attempt of Bs 168 by pBK5 with a new procedure : Failure ? the fault of the plasmid which seems not to be the plasmid waited...Try again with the shuttle vector pHT 315 (GFP or not).
• The plasmid extraction protocol from B. subtilis was tested once again, with a few modifications. The results were unsatisfying. After electrophoresis, only genomic DNA was observed.

• Check of the plasmidic DNA extracted of the clones transformed by [Promoter in pSB1C3] : none of the 4 clones is ok.
• Selection of 4 clones transformed by [lyso+dsp] on LB+Amp plates and on liquid cultures.
• Miniprep to extract [Lyso+Cm] and [Disp+Cm] ? Digestion.
• 3A ligation between : lysostaphin in puc57 + Terminator in pSB1K3 + pSB1C3. Check by electrophoresis : Lysostaphin, terminator and vector are ok but not the ligation (no DNA).
• Lysostaphin tests (Bs 168 pWG100 supernatant) on antibiograms (diameter : 6mm) applied on GL+Tet plates having a biofilm already etablished and in development of S. epidermidis.

• Transformation of NM522 strain with pHT304 and pHT315 (2 shuttle vectors for B. subtilis and E. coli).
• gDNA extraction with 2 differents protocols : the first for Bacillus subtilis 168 and the second for Gram- bacteria with some modifications. The first protocol gives a better yield.
• PCR of ARNr 16s on B.s. 168 to test if PCR works in the bacteria without being lysed : it works.
• NM522 + gfp, cfp and yfp in storage.
• pHT 315 GFP put in storage under the reference pBK 18.
• NM522 + pHT 315 GFP strain put in storage under the reference BK 21.

• Other clones transformed with the Constitutive Promoter in pSB1C3 are selected in order to do other check by extracting their plasmidic DNA. ( bur they are not good again).
• Extraction of the plasmidic DNA of the clones transformed with Lyso+Disp in pBK2 and check by electrophoresis gel. The clones are not right.

• Extraction of plasmidic DNA : clones Lysostaphin in PSB1C3, Promoter in pSB1C3.
• Plasmidic DNA check by digestion and electrophoresis : Clones Lysostaphin okay and Promoter clones are not okay.
• New 3A ligation with Lyso/Terminator/pSB1C3 and transformation in NM522 strain.
• Results of the lysostaphin tests on plates : no effect (regular biofilms)

• sfp (surfactin part 1) and abrB put in storage : pBK16 et pBK17.
• Failure of transformation of L1 in NM522.
• 3A assembly of RBS-CFP (pBK7-pBK13) in pSB1C3 = L2. (!! the part2 construction already has a terminator)
• 3A assembly of sfp-part2(lacI)-term (pBK4-pBK10) in pSB1C3 = IV.

• PCR of xylR from gDNA extracted yesterday. Test on gel : PCR successful !!
• 3A ligation of RBS (pBK7) and XylR (produced by PCR) in pSB1C3 ( ! the vector plamid has the same resistance as the plasmid containing the RBS...).

• Meeting at 9 o’clock.
• It was also a fire day : a man’s hair and a lab bench on fire !!!! As the saying goes, ”everything comes in threes, if it's happened twice, it'll happen a third time ” (we’re still waiting for the third fire...).

• Digestion test and gel of pBK2 (prom + lyso) by EcoRI and SpeI, pBK3 (disp + term) by XbaI and PstI and pSB1C3 by EcoRI and Pst1.
• We got 3 clones for the transformation of NM522 bacteria with the ligation’s product Lyso+Terminater-pSB1C3 : the plasmidic DNA of these 3 clones is extracted and tested by electrophoresis gel ? the 3 clones aren’t right.
• Lysostaphin test on plate : this time, by trying with biofilm in formation less dense (OD = 0,5, dilution the culture x100, 1000 and 10 000).

• Transformation of L1, L2 et IV in NM522.
• Transformation of sfp and abrB in NM522.

• Given the fact that the previous transformation didn’t work, we made an other transformation of NM522 strain with the abrB and sfp parts.
• Results of the transformations of L1, L2 and IV : ok. Selection of some clones in order to do minipreps.

The transformation protocol for Bacillus was tested using the pHT315 GFP plasmid extracted from the B. thuringiensis BT407GFP strain.

• Transformation results :
  • Lysostaphin+terminator : nothing;
  • Lysostaphin+dispersin : a lot of clones.
  Cultures in liquid LB of Lyso+Dsp are launched.
• Digestion of Promoter clones and lysostaphin clones followed by an electophoresis : lysostaphin clones are okay, promoter clones are not okay.
• pUC57 with Dsp put in storage : pBK3.
• Lysostaphin test : the come back ! This time, we used more S. epidermidis (DO= 0.75 diluted 1000 times) and a control for the stability of the pWG100 plasmid (spreading of 200 µL on GL + Ery plates), after the liquid culture without selective pressure. Spreading on LB plates without Tetracycline (but addition of Tetracycline into the Bs 168 pWG100 supernatant).

• The strain NM522/pBK6 was put in storage under the reference BK22.
• Transformations of the NM522 strain with the ordered constructions (sfp and abrB in pUC57 AmpR). The transformation was unsuccessful, so we did it again.
• Quantification of Sfp and abrB constructions provided by Genecust using the Nanodrop.
• Miniprep of the L1, L2 and IV ligations : it didn’t work.

• The transformation of the Bacillus strain failed because of contaminated LB media, so a new transformation was attempted.
• pHT 304 and pHT 315 plasmids supernumerary site SpeI deletion attempt by digestion, filling-in, ligation and transformation in NM522.

• Extraction of plasmidic DNA from the Lysostaphin + Dispersin clones in Cm iGEM plasmid (pSB1C3) and from the BK12 strain clones. Plasmidic DNA is checked by digestions.
  Lysostaphin+Dsp clones digestion : clones not okay.
• Ligations of :
  • Constitutive promoter in Cm iGEM plasmid;
  • Dispersin in Cm iGEM plasmid.
  Ligation were verified by electrophoresis.
• Results of the Lysostaphin tests : some Bs 168 pWG100 contaminated the plate. However, we noticed a halo of 2-3 mm around the Bacillus colonies where the S. epidermidis didn’t grow due to lysostaphin activity.
• New Lysostaphin test on LB+Tet plate with a negative control (10 µL of Ampicillin).

• Miniprep of 5 clones of the transformed NM522 strain with RBS-xylR and 2 clones of the NM522 transformed with the abrB construction. Given the fact that the NM522 strain used for the transformations was contaminated, the electrophoresis showed unexpected results.
• New ligation of RBS and XylR in pSB1A3 and transformation in NM522 strain.

• Meeting at 9 o’clock.
• Purification of the NM 522 strain (because of the problems on the negativ control during the transformation) and resistance tests on the purified clones.
• The results of Bacillus transformation are inconclusive : there are only a few clones on the plate. However, the negative control has a few colonies too. Despite this result, we decided to verify six clones and extract their DNA.
• A lot of clones for the transformation of the pHT 304 and pHT 315 plasmids (without SpeI site) on LB+Amp plates ? Purification of 12 clones of each.

• Replication with velvet of the Lysostaphin+Dispersin in pSB1C3 transformation plate on LB + Amp.
• Antibiotic resistance checked for 5 clones Lysostaphin+Dispersin in iGEM plasmid (clones 1 to 5).
• BK12 strain check : spread on LB ampicillin.
• Ligation Promoter+Dispersin in Cm iGEM plasmid followed by transformation of the 3 ligations.
Ligation were verified by electrophoresis.
• Transformation of Promoter+Dispersin ligation.
• PCR to check the presence of the promoter in the clones Promoter in Cm iGEM plasmid.
• Preliminary test of the liquid lysostaphin (10 µL of tetracycline to kill Bacillus, 500 µL of BS 168 pWG100 supernatant, 500 µL of S. epidermidis with OD=1,2 (the strains are cultivated overnight at 30°C). This test shows a possible effect of lysostaphin (decrease of the OD with the time, during 3h30). To be repeated with a negative control without Bs 168 pWG100.

• Ligation (3A protocol) of pBK7, pBK13 and iGEM backbone pSB1K3 (=RBS+CFP+pSB1K3).
• Ligation (3A protocol) of pBK7, pBK14 and iGEM backbone pSB1K3 (=RBS+GFP+pSB1K3).
• Because of the difficulties to transform the bacteria with the plasmids containing Sfp and abrB, we decided to do an electrophoresis directly on the constructions sent by Genecust. Result : the genes were not inserted in a pUC57 plasmid.
• Ligation of abrB and sfp in pSB1K3 and transformation in NM522.

• Transformation results :
  • The negative control is ok;
  • There are clones on every transformation plates (Promoter in pSB1C3, Dispersin in pSB1C3, Promoter+Dispersin in pSB1C3).
  8 clones per type of transformation are chosen and spread on LB+Cm and LB+Amp plates.

• Transormations of abrB and sfp are a success !! :D
• Liquid cultures of abrB and sfp clones are launched to do plasmid extractions.

• Plasmid extraction from 6 clones BS 168 transformed by pH315 GFP : the electrophoresis of the digested plasmids showed that the clones had the right plasmid. The Bacillus transformation also worked, but the yield must be improved.
• Miniprep and digestions by SpeI of the plasmid extracted from the 6 clones of NM522 with pHT 304 S and NM 522 with pHT 315 S : Failure ! The SpeI site is still on the plasmids :’(

• Selection of clones growing on LB Cm and not on LB Amp:
  • 4 clones Dispersin in pSB1C3;
  • 7 clones Promoter pSB1C3;
  • 5 clones Promoter+Dispersin in pSB1C3.
  Liquid cultures in LB+Cm are made with these clones. Then extraction of plasmidic DNA.
• 8 clones Lysostaphin + Dispersin in pSB1C3 are spread on GL+Cm and GL+Amp to test their resistance.
• Transformation of pSB1C3 and pSB1T3 in order to obtain a clone from the PCR matrix (red clone).

• pBK7+pBK13+pSB1K3 (RBS+CFP in Kanamycin resistant backbone);
• pBK7+pBK14+pSB1K3 (RBS+GFP in Kanamycin resistant backbone);
• PCR of RBS-abrB and pXyl and purification of these PCR products.

• Purification of the xylR gene.
• Miniprep of RBS-xylR and electrophoresis test ⇒ the ligation didn’t work again...

• There is a little plasmid pHT 304 S and pHT 315 S post filling-in, new digestion by Spel enzyme to eliminate the plasmids which are not full on the SpeI site in order to increase the amount of plasmids without SpeI site.
• Transformation in NM522 and spreading on LB Amp.

• 8 new clones Lysostaphin+Dsp in PSB1C3 are screen on Ampicilline. Ampicilline sensitive clones are put in liquid culture.
• Checking of plasmids extract the previous day and electrophoresis : Dsp in pSB1C3 (clones not okay), Promoter+Dsp in pSB1C3 (Clones not okay).
• The transformation of pSB1C3 and pSB1T3 didn’t work.
• New Lysostaphin liquid test with 3 measures and a negativ control (Bs 168 supernatant instead of Bs 168 pWG 100). This time, the cultures grew up one and a half day at 37°C (efficiency of the lysostaphin in stationary phase ?) ⇒ the test is positive, lysostaphin has a right effect on the S. epidermidis cultures.

• 3A ligation : [pXyl-RBS-Sfp] + [RBS-abrB] + [psB1T3].
• Transformation of the ligation [pXyl-RBS-Sfp] + [RBS-abrB] + pSB1T3 in NM522 strain and LB+Tet medium.
• Results of the transformation by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : Failure! The negativ control has few colonies : the LB+Kan plate was contaminated and the used LB medium too! The transformation is done again...
• Purification of pXyl produced by PCR.