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

Incubation time : 2 hours (until OD₆₀₀ = 0.3).

• For the positive control the pSB1C3 plasmid was used;
• For the transformation, the pBBA_I742123 was used (well A2, iGEM 2012 kit plate 5).

• 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 two other 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 lysostaphin 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 Chloramphenicol + 500 µL liquid culture of transformed bacteria ).
• The 3 genes coding for lysostaphin, dispersin and lacI repressor in pUC57 Ampicillin resistant plasmids were delivered.
• Transformation of NM522 strain with pUC57-Lysostaphin, pUC57-Dispersin and pUC57-sfp.
• 200 µL of each transformed strains were spread on LB + Ampicillin plates.
• Liquid cultures of the following strains were made : Bs 168 in LB, Bs abrB in LB + Chloramphenicol, S. epidermidis in LB + Tetracyclin

• 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) in pUC57 Amp resistant (pBK4).
• and the following strains (in LB broth supplemented with required antibiotic) :
• S. epidermidis = BK1 (Tet^R);
• Bs abrB = BK2 (Cm^R);
• Bs 168 = 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 bands are observed : PROBLEM. Digestion is made again with E, P and E+P → There are 2 PstI 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 promoter (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-Lysostaphin/pUC57-Dispersin/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 color 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 promoter (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 promoter (part BBa_K143012) → the PCR did not work so we ran a new PCR with a more diluted promoter solution.
• The following strains are put in storage:
  • NM522 containing lacI-pUC57;
  • NM522 containing rbs-pUC57;
  • NM522 containing dispersin-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 frenzied psychopath way in order to remember our tasks.
• Ligation of promoter-rbs-GFP in plasmid.
• Genomic DNA extraction of B. subtilis 168: buffers preparation.
• Failure of B0015 transformation.

• Cloning of the constitutive promoter in front of the dispersin part (the gene coding for Dispersin) and RBS/GFP (of E. coli) using a 3A ligation followed by transformation of the ligation in the NM522 strain;
• Extraction of pUC57-lysostaphin. Gel electrophoresis showed a shaded DNA → RNase addition in the extraction kit buffer was forgotten.

• Liquid culture of S. epidermidis in 5mL of TSB (conditions described in protocol “Tests on Staphylococcus aureus biofilms in 24 well plate” ).
• A bacterial suspension of S. epidermidis with OD₆₀₀ close to 0.132 is prepared from a Petri dish containing S. epidermidis. A microtiter plate is inoculated with 2 mL of the suspension diluted 1:100 with TSB supplemented with different concentrations of glucose in order to test the best conditions for biofilm formation. The plate is incubated during 24 hours at 37ºC.

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

• The transformation results of the 3A ligation ([dispersin+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 smear. 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 50µg/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 ([dispersin+RBS/GFP+pSB1T3]) was successful, so 6 clones were tested on a plate containing LB broth supplemented with Tetracyclin.
• The fluorescence test of the transformed bacteria containing [dispersin+RBS/GFP+pSB1T3] confirm that the promoter 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 promoter+pSB1C3 OK → 6 clones are isolated on a LB+Cm plate.
• Quantification and gel electrophoresis of B. subtilis genomic DNA.
• Antibiotic testing of B. thuringensis 407 gfp strain and other strains in LB+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 Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection) and transformation.
• Ligation Lysostaphin in pSB1C3 and transformation.
• Isolation of 6 clones of the Terminator 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 P_veg promoter and RBS from B. subtilis are recognized by E. coli’s ribosome. 4 clones are streaked in LB+Cm.
• LB+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
• Promoter+Dispersin in pIG23 : nothing on the plate;
• Lysostaphin, negative control contains bacteria so the plate is put in junk.
• New digestions, ligations,transformations:
• Lysostaphin+Dispersin in pUC57;
• Dispersin in pSB1C3;
• Lysostaphin in pSB1C3;
• Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection).
• 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 promoter 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.
• Extraction of the pHT315 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.
• S. epidermidis is put in storage in TSB media under the reference BK15.

• Transformation results:
  • Lysostaphin+Dispersin in pUC57 : the plate is covered of clones;
  • Promoter+Dispersin : nothing on the plate;
  • Dispersin in pSB1C3 : a lot of clones;
  • Lysostaphin in pSB1C3 : a lot of clones.
  Selection of 4 clones of each lysostaphin+iGEM plasmid and dispersin+iGEM plasmid on Cm plate and in a liquid culture. Isolation of lysostaphin+dispersin (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 pBK23 (Tet R) by miniprep and verification by electrophoresis after digestion by EcoRI and PstI : gel ok. Congrats (pBK12) → 122,6 ng/µL.
• Extraction of the pBK6 plasmid (P_veg+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 in TSB+Tet (BK17);
• B. thuringensis 407 GFP in LB+Ery (BK16).
• Minipreps to extract the plasmidic DNA of the clones NM522 + pHT315 GFP and verification by electrophoresis : plasmid ok (digested by E, X, S, P and not digested).
• Transformation attempt of Bs 168 by pBK5 with a new procedure : Failure → blaming the plasmid which seems not to be the expected plasmid... Try again with the shuttle vector pHT315 (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.

• Verification of the extracted plasmidic DNA of the clones transformed by [Promoter in pSB1C3] : none of the 4 clones is ok.
• Selection of 4 clones transformed by [lysostaphin+dispersin] on LB+Amp plates and in liquid cultures.
• Miniprep to extract [Lysostaphin+Cm] and [Dispersin+Cm] → Digestion.
• 3A ligation between : lysostaphin in pUC57 + Terminator in pSB1K3 + pSB1C3. Verification 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 LB+Tet plates having a biofilm already established 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 different 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 rRNA 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 pBK18.
• NM522 + pHT315 GFP strain put in storage under the reference BK21.

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

• Extraction of plasmidic DNA : Lysostaphin in pSB1C3 clones, Promoter in pSB1C3 clones.
• Plasmidic DNA verification by digestion and electrophoresis : Lysostaphin clones okay and Promoter clones are not okay.
• New 3A ligation with Lysostaphin/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)-terminator (pBK4-pBK10) in pSB1C3 = IV.

• PCR of xylR from gDNA extracted yesterday. Test on gel : successful PCR !!
• 3A ligation of RBS (pBK7) and xylR (produced by PCR) in pSB1C3 ( ! the vector plasmid 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 (promoter + lysostaphin) by EcoRI and SpeI, pBK3 (dispersin + terminator) by XbaI and PstI and pSB1C3 by EcoRI and PstI.
• We got 3 clones for the transformation of NM522 bacteria with the ligation’s product Lysostaphin+Terminator-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 (OD₆₀₀ = 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 another 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 LB broth of Lysostaphin+Dispersin are launched.
• Digestion of Promoter clones and lysostaphin clones followed by an electrophoresis : lysostaphin clones are okay, promoter clones are not okay.
• pUC57 with Dispersin put in storage : pBK3.
• Lysostaphin test returns ! This time, we used more S. epidermidis (OD₆₀₀ = 0.75 diluted 1000 times) and a control for the stability of pWG100 plasmid (spreading of 200 µL on LB + 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 broth, so a new transformation was attempted.
• pHT304 and pHT315 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+Dispersin 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 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 NM522 strain (because of the problems on the negative 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 pHT304 and pHT315 plasmids (without SpeI site) on LB+Amp plates → Purification of 12 clones each.

• Velvet replication of Lysostaphin+Dispersin in pSB1C3 transformation plate on LB + Amp.
• Antibiotic resistance checked for 5 Lysostaphin+Dispersin in iGEM plasmid clones (clones 1 to 5).
• BK12 strain verification : spreading on LB + Amp plate.
• Ligation of 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 Promoter in Cm iGEM plasmid clones.
• 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 OD₆₀₀=1.2 (the strains are cultivated overnight at 30°C). This test shows a possible effect of lysostaphin (decrease of OD₆₀₀ 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, although they should have been cloned.
• 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 for each transformation are chosen and spread on LB+Cm and LB+Amp plates.

• Transformations 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 pHT304 S and NM522 with pHT315 S : Failure ! The SpeI site is still in the plasmids :’(

• Selection of clones growing on LB+Cm plates and not on LB+Amp plates :
  • 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 LB+Cm and LB+Amp to test their resistance.
• Transformation of pSB1C3 and pSB1T3 in order to obtain a clone from the PCR matrix (red clone).

• Transformation of the following ligations, in NM522 strain :
• pBK7+pBK13+pSB1K3 (RBS+CFP in Kanamycin resistant backbone);
• pBK7+pBK14+pSB1K3 (RBS+GFP in Kanamycin resistant backbone).
• PCR of RBS-abrB and P_xyl and purification of these PCR products.

• Purification of the xylR gene.
• Miniprep of RBS-xylR and electrophoresis test → the ligation failed again...

• There is a little plasmid pHT304 S and pHT315 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 plates.

• 8 new clones Lysostaphin+Dispersin in pSB1C3 are screened on Ampicillin. Ampicillin sensitive clones are put in liquid culture.
• Verification of plasmids extracted the previous day and electrophoresis : Dispersin in pSB1C3 (clones not okay), Promoter+Dispersin in pSB1C3 (Clones not okay).
• The transformation of pSB1C3 and pSB1T3 didn’t work.
• New Lysostaphin liquid test with 3 measurements and a negative control (Bs 168 supernatant instead of Bs 168 pWG 100). This time, the cultures grew over 36 hours 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 : [P_xyl-RBS-sfp] + [RBS-abrB] + [psB1T3].
• Transformation of the ligation [P_xyl-RBS-sfp] + [RBS-abrB] + pSB1T3 in NM522 strain and spreading LB+Tet plates.
• Results of the transformation by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : Failure! The negative control has a few colonies : the LB+Kan plate was contaminated and the used LB medium too! The transformation is done again...
• Purification of P_xyl produced by PCR.

• Result of the transformation of NM522 by pHT304 S et pHT315 S : 0 clone and 1 clone... this only clone is tested but the test is negative : the SpeI site is always here at 311 bp. The cultures of the transformation are concentrated and spread on LB plate : 0 clone for pHT304 S and 18 for pHT315 S but none of the clones is good. We must do the filling-in again...
• Transformation of the pSB1C3 + RFP and pSB1T3 + RFP plasmids extracts of the iGEM plates to use them as tool of cloning after extraction.

• Plasmidic extraction of Lysostaphin+Dispersin clones, then digestion and electrophoresis : clones not okay.
• New ligations :
  • Promoter+Dispersin in pSB1C3;
  • Lysostaphin+Dispersin in pSB1C3.
  Then transformation in NM522.
• Plasmidic extraction of the BK12 strain : plasmid okay.

• Transformations of NM522 by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : ok.
• Transformation by sfp-abrB-pSB1T3 : the negative control isn’t good, the transformation is done again...

• Extraction of NM522/pHT315S. We expected a plasmid without a SpeI site. However, the selected clone contained a plasmid identical to pHT315 (the digestion by SpeI and EcoRI enzymes gives a fragment at 1000 bp). We decided to screen more clones (8 out of 17).
• The transformation pSB1C3 + RFP and pSB1T3 + RFP is a success ! (red clones)

• Positive control : okay;
• Negative control : okay;
• [Lysostaphin + Dispersin] : 8 clones;
• [Promoter + Dispersin] : more than 30 clones.

• The transformation of NM522 strain by sfp-abrB didn’t work again : although the positive control contains many clones (plasmid A2) and the negative control contains nothing, the real transformation contains nothing either ! We decided to do the sfp-abrB ligation again...
• Plasmid extraction of 4 clones NM522 transformed by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : the electrophoresis of the digested plasmids showed that 3 out of 4 clones had the good fragment.
• PROBLEM : WE RAN OUT OF LIGASE!!

• Deletion of the SpeI site from pHT315 and pHT304 plasmids and filling-in using the Pfu polymerase. This time we used gel electrophoresis to verify the plasmids after each purification.
• Extraction of the plasmid containing the RFP gene in the pSB1C3 iGEM backbone (midiprep).

• New Lysostaphin tests are planned (on plates) : in this purpose, a culture of 60 mL of Bs 168 pWG100 is inoculated and then incubated overnight at 28°C.
• None of the clones Lysostaphin+Dispersin grew in LB + Amp so LB cultures were inoculated.
• Out of 29 clones, 18 Promoter+Dispersin clones are Ampicillin sensitive, LB cultures are launched with these clones.
• Plasmidic extractions of these clones : clones not okay.
• PCR to obtain pSB1C3 : the electrophoresis showed that there was no DNA, so another trial was done, but it was unsuccessful.
• The strain NM522 with Constitutive Promoter + pSB1C3 was put in storage under the reference BK27.

• Ligation of the sfp and abrB parts in an iGEM backbone.
• The NM522 strain with abrB-pSB1K3 was put in storage under the reference BK25.
• The NM522 strain with sfp-pSB1K3 was put in storage under the reference BK26.

• We identified the reason why the deletion of the site SpeI from pHT315 and pHT304 plasmids and filling-in was not successful : the Pfu enzyme requires Mg²⁺ and the buffer we used did not contain any, so we decided to give it another try, this time with the proper buffer.
• The extracted plasmids containing the RFP gene in the pSB1C3 backbone were digested and verified by electrophoresis.

• Lysostaphin tests : Flasks of 25mL of Bs 168 pWG 100 filtered supernatant were cultivated on Friday and frozen at -20°C on Sunday (2 flasks of each). The control flask is filled with 25mL of LB broth. These flasks are then frozen at -80°C and freeze-dried overnight.
• The gel electrophoresis of the digested plasmid [Lysostaphin + Dispersin] in pSB1C3 doesn’t correspond to the expected fragments. Similarly, the second electrophoresis shows that the clones with [Constitutive promoter + Dispersin] in pSB1C3 do not have the right plasmid : the digested fragments do not have the right size.

• Ligation of the Constitutive Promoter (extracted by PCR) in the pSB1C3 plasmid containing the RFP gene (purified by midiprep) in order to obtain the Constitutive Promoter in the pSB1C3.
• Lysostaphin tests on plates return again ! The freeze-dried products are put in five times less water than at first. Volumes up to 100 µL of concentrated supernatant are applied on antibiograms which are used for tests on the S. epidermidis biofilm.

• The lysostaphin test didn’t work : Valérie did the test again by applying first the supernatant on the paper disks before putting them on the plates, and by using TSM medium instead of LB broth.
• Transformation of the ligation [pSB1C3 + constitutive promoter] in NM 522 strain.

• Results of the transformation of NM522 by pBK7 and xylR : the negative control contains nothing, the positive control (transformed with pBK5) is full of bacteria and the plate with NM522 transformed by pBK7 + xylR contains a lot of clones too. 8 clones are selected to do liquid culture and extract their DNA.
• Second standard ligation between pBK7 (=RBS in pSB1C3) and xylR (produced by PCR) with more insert (3µL of insert for 2µL of vector). Transformation into NM522 strains.

• Miniprep of the clones having integrated the modified shuttle vector (after filling-in). 4 clones seem to have the right plasmid.
• The electrophoresis after digestion with EcoRI and SpeI showed that the SpeI site was eliminated. 4 clones were chosen for further tests. However, the DNA concentration was too low so the enzymes did not digest the plasmids.

• Ligation of the Constitutive promoter (P_veg) produced by PCR in an iGEM plasmid.
• Result of the lysostaphin tests : didn’t work again. The Bs 168 pWG100 supernatant doesn’t contain any lysostaphin : the lysostaphin is probably degraded in one way or another (sensitivity to defrosting ?).
• Result of the transformation with [pSB1C3 + promoter] : a lot of red clones, some white clones (20 - 30). Isolation of 6 white clones and plasmid extractions by miniprep.

• Miniprep of 4 clones with the transformed 3A ligation (pBK22, gene abrB and pSB1A3 backbone). The electrophoresis did not turn out as expected for any of the 4 clones, so we decided to screen 6 others.
• Ligation of P_xyl produced by PCR in an iGEM plasmid.

• Ligation of xylR produced by PCR in an iGEM plasmid (pSB1K3).
• Results of the transformation of NM522 by [pBK7 + xylR] (for the second ligation) : the plate with bacteria transformed by [pBK7 + xylR] contains a lot of clones and the controls are standard. 14 clones are selected to do liquid culture and extract their DNA.
• Plasmid extractions from the 8 clones transformed by [pBK7 + xylR]. The electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they have just the vector without xylR.

• pBK19 with no SpeI site was put in storage under the reference pBK25.
• pBK20 with no SpeI site was put in storage under the reference pBK26.

• Launch of 8 [Promoter+Dispersin] cultures.
• Results of the transformation of NM522 by [pSB1T3 + P_xyl] : the plate with bacteria transformed by [pSB1T3 + P_xyl] contains few clones and the controls are standard. 3 clones are selected to do liquid cultures and extract their DNA : no plasmid.
• Results of the transformation of NM522 by [pSB1T3 + rbs-abrB] : no clones.

• Results of the transformation of NM522 by [pSB1K3 + xylR] : the plate with bacteria transformed by [pSB1K3 + xylR] contains a lot of clones and the controls are standard. 3 clones are selected to do liquid culture and extract their DNA : they have just the vector without xylR.A new PCR of xylR is made, in order to increase the stock.

• Meeting at 9 o’clock.
• Midiprep of the modified pBK25 and pBK26 shuttle plasmids (without the SpeI site). The extracted DNA was verified by electrophoresis.

• Standard ligation of pBK23 (= Constitutive Promoter + RBS + Lysostaphin in pSB1C3) with pBK25 (the shuttle vector E. coli - B. subtilis). Different ligations are made with different proportions of insert and vector. Transformation in NM522 strain.
• Digestion of Lysostaphin in pSB1C3 and Dispersin in pUC57.
• Verification of 8 [promoter-dispersin] clones : clones happen to be nonstandard.

• Extraction of the plasmid containing the ligation [sfp+abrB+pSB1A3] from a liquid culture of transformed bacteria.
• Results of the second transformation of NM522 by pSB1T3 and rbs-abrB : no clones.

• Two standard ligations are done :
• pBK7-xylR (xylR cut in X and P sites);
• pBK24-xylR (xylR cut in E and S sites).
• Transformation of pBK7-xylR into NM522 strain.

• Result of the transformation of NM522 with [Constitutive Promoter + RBS + Lysostaphin] in pBK25 : all the controls are right, and there are a lot of clones on the different plates with the transformed bacteria. 14 clones are selected to do liquid cultures and extract their DNA.New trial of cloning : Digestion, Ligation, transformation to construct :
• [Lysostaphin + Dispersin] in pSB1C3;
• [Lysostaphin + Dispersin] in the shuttle vector (vector for E. coli and B. subtilis,).

• Verification of 6 [xylR-pSB1K3] clones : they only have the vector.
• Transformation of [pBK24-xylR] into NM522 strain.
• Results of [pBK7-xylR] transformation : lots of clones and no clones in the negative control. 12 clones are put in liquid culture for extraction.

• Transformations results : All controls are okay.
• Too many clones on [Lysostaphin + Dispersin] in shuttle vector transformation plate, so some clones are selected and spread and a new LB plate with the appropriate antibiotic.
• 12 liquid culture in LB are launched for the clones [Lysostaphin + Dispersin] in pSB1C3.
• Miniprep of 14 clones transformed with the ligation product [Constitutive Promoter + RBS + Lysostaphin] in pBK25 and electrophoresis to analyze the extracted DNA : the transformation is successful for 3 clones =)
• The first clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (1);
• The second clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (2);
• The third clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (3).

• Results of [pBK24-xylR] transformation : most of the clones are red, however a few of them are white, which is good. 7 white clones are put in liquid culture for extraction.
• Extraction of pBK7-xylR from 12 clones, and then a gel electrophoresis is run: 2 clones seem interesting. We ran a second gel to verify these 2 clones, but it showed us that they were not good.

• After the bad results with xylR, we decided to cut pBK10 plasmid in SmaI site, and ligate with xylR, doing a blunt ligation. We also decided to ligate xylR with xylR, creating a big polymer, which will be like a “pre-ligation” molecule.
• Extraction of pBK24-xylR from 7 clones. Gel electrophoresis is run → ONE CLONE IS GOOD!! Meaning that it’s the first time we manage to ligate xylR successfully.

• Transformation of Bs 168 strain with pBK28 (1) plasmid which contains the [Constitutive Promoter + RBS + Lysostaphin] in the shuttle vector.
• Midiprep : Extraction of P_veg-dispersin-pSB1C3 of clone 22.
• BK33 strain was put in storage.

• Given the fact that the digested plasmids extracted from transformed NM522 E. coli with the 3A ligation (pBK24, pBK4, pBK29) had unexpected fragments (and impossible to explain), the ligation was done again, this time changing the vector/insert ratio (1/1 and 1/3, as opposed to the first trial with a ⅙ ratio ).
• Liquid cultures of two potentially good clones of the NM522 strain transformed with a 3A ligation (pBK22(promoter P_xyl, RBS and sfp gene), abrB gene and pSB1A3 backbone) were made for miniprep (the two plasmids were extracted using the classic ABC protocol, with extraction solutions that we made, so the yield and the purity of the extractions were poor; that’s why we decided to extract the plasmids again, this time using an extraction kit).

• Ligation of [Constitutive promoter + Dispersin] in the shuttle vector pBK25.
• Results of the transformation of BS 168 strain with the pBK28 (1) in the shuttle vector : the negative control is full of bacteria → Is the Bs 168 strain resistant to erythromycin ? Or is the erythromycin concentration too low ?
  We made another transformation and we spread the transformed bacteria on LB+Ery plates with different erythromycin concentration (from 5 µg/mL to 10 µg/mL).

• Transformation of the previous ligation [promoter-dispersin-pBK25] in E. coli NM522.
• Results of the second transformation of Bs 168 strain with pBK28 (1) in the shuttle vector : the negative control is still full of bacteria even though the erythromycin concentration was higher than the previous trial (0,5 µg/mL the first time, 5 and 10 µg/mL the second time).
• We made (LB+Ery) plates with different erythromycin concentrations in order to make different tests.

• Only one of the 24 screened colonies concerning the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios) did not turn up to be red (LB+Cm plate) and did not grow on a LB+Amp plate. The clone was used to inoculate a liquid culture for an extraction.
• Miniprep of the two clones containing the sfp and abrB genes. The gel electrophoresis did not turn out as expected, probably because the bacteria used for inoculating the liquid cultures were contaminated or did not come from the same colony. We decided to transform the low purity plasmids into the NM522 strain.
• Given the fact that we were not very optimistic concerning the 3A ligation of pBK24, pBK4 and pBK29, we decided to do two more trials, this time using two ligated genes (sfp and abrB) coming from clones other than the one containing pBK22. However, the miniprep of the bacteria which were supposed to contain the two plasmids was unsatisfying, so we decided to do the 3A ligation with the low purity plasmids, isolated with the classic protocol because we were running out of time and we were behind the schedule.
• In parallel, we decided to do a standard ligation in order to transfer the lacI gene into a plasmid having an antibiotic resistance other than Ampicillin. This way, in case the 3A ligations fails, we would not be forced to do again a 3A ligation because the plasmids would have different antibiotic resistances.

• Transformation of xylR-pBK24 in NM522. The transformation of xylR-pBK24 is done in order to see if xylR was successfully ligated or not. If the bacteria are red, then xylR was not ligated.
• 24 clones containing pBK10-xylR are patched in LB+Amp growth medium, and then incubated.

• Transformation results : too many clones on the control digestion not ligated vector so the transformation can’t be used for the transformation [Lysostaphin+Dispersin] in pSB1C3.
• 12 clones are put in culture from the transformation of [Lysostaphin + Dispersin] in the shuttle vector treated by BamHI.
• Preparation of tubes for sequencing, launch of miniprep and midiprep.
• Standard ligation between the shuttle vector pBK26 (pHT315 modified) and the pBK23 plasmid (= Lysostaphin in pSB1C3). Transformation of the ligation product in NM522 strain.

• All 5 transformations were successful. White colonies are selected (pBK24 is a plasmid containing RFP in the pSB1C3 backbone, so we can exclude the colonies having integrated the religated backbone) and tested on LB+Cm and LB+Amp plates.
• The miniprep of the one clone that had the expected phenotype (obtained after the transformation of the 3A ligation of pBK24, pBK4, pBK29) was digested, but the gel electrophoresis did not turn out as expected.
• The miniprep confirmed that the ligation containing lacI was successful.

• The patched plate is used as a master for replica-printing. The replica plate contains LB+Kan growth medium. Results : only one clone didn’t grow in the replica plate, meaning that it might be a clone containing xylR-pBK10 ligation. The clone is put in liquid culture overnight.
• Another test is run to confirm that pBK34 plasmid (xylR-pBK24) doesn’t contain xylR : we digested the plasmid using NdeI restriction enzyme. Since xylR contains a NdeI restriction site (pBK24 doesn’t contain it), if pBK34 is opened by the enzyme, it means that it contains xylR. Result : pBK34 doesn’t contain xylem.

• Verification of [Lysostaphin-Dispersin] in the shuttle vector (pBK26) clones : clones are not good.
• Verification of the miniprep for sequencing ([Promoter + Dispersin] in pSB1C3) : DNA is okay.
• Result of the transformation of NM522 strain with the Lysostaphin in pBK26 :
• The negative control is ok;
• The positive control is full of colonies;
• The transformation is full of colonies : 24 clones are selected (on plates and in liquid culture) in order to extract and check their DNA.

• Extraction of the good clone from the replica-printing. Then a electrophoresis gel is run : the blunt end ligation was successful.
• The plasmid is digested by EcoRI, XbaI, SpeI and PstI to find out if the problem comes from a bad site sequence. Result : the EcoRI restriction site is not good, therefore is not recognized by Eco