Team:Lyon-INSA/notebook

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For all purposes Monday, July 2nd 2012 Liquid culture (5 mL LB media) of NM 522 cells and overnight incubation under agitation at 37°C for later transformations. For all purposes Tuesday, July 3rd 2012

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

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

Transformation of the NM522 strain (this experiment was made 3 times)
For the positive control the pSB1C3 plasmid was used ; For the transformation, the pBBA_I742123 was used (well A2, 5th iGEM 2012 kit plate) The transformed bacteria were selected on chloramphenicol plates. For all purposes Wednesday, July 4th 2012 Transformation analysis :

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.

4 liquid cultures (5mL LB medium + 50 µL chloramphenicol) and 4 streaked chloramphenicol plates were made using isolated colonies likely to contain transformed bacteria.

The antibiotic resistance (Ampicillin, Tetracyclin, Chloramphenicol and Kanamycin ) of the following strains was tested: BS 168, BS 168 MCherry, BS 168 GFP, BS 168 Lysostaphin, Staphylococcus epidermidis, BS Abrb. For all purposes Thursday, July 5th 2012 Antibiotics resistance tests :

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

Extraction of 4 clones containing the pBBA_I742123 plasmid. Verification by gel electrophoresis (after EcoRI digestion) For all purposes Friday, July 6th 2012 Telephonic conference with Romain Briandet

Terminator was retrieved from the plate 1 well 13D

Long meeting For all purposes Monday, July 9th 2012

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

For all purposes Tuesday, July 10th 2012 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) dans pUC57 Amp resistant (pBK4)

and the following strains:

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

For all purposes Wednesday, July 11th 2012

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.

For all purposes Thursday, July 12th 2012

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.

Kill Friday, July 13th 2012

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

Monday, July 16th 2012 Kill

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.

For all purposes

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

Tuesday, July 17th 2012 For all purposes

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.

Kill

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.

Wednesday, July 18th 2012 For all purposes

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.

Kill

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.

Thursday, July 19th 2012 For all purposes

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.

Kill

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.

Friday, July 20th 2012 For all purposes

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.

Kill

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.

Monday, July 23rd 2012 For all purposes

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.

Kill

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.

Stick PCR of XylR. Gel electrophoresis: the PCR didn’t work. Tuesday, July 24th 2012 For all purposes

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.

Kill

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.

Stick New PCR of XylR with some modifications in the protocol : the PCR didn’t work. Wednesday, July 25th 2012 For all purposes

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.

Kill

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.

Stick PCR on the B. subtilis 168 genome using universal primers of Phil Oger, and positive control with Bulkhoderia cepacia genome provided by Phil Oger. Goal : determine if the PCR problems come from the primers or the low concentration of genomic DNA of B. subtilis 168. Thursday, July 26th 2012 For all purposes

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.

Kill

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.

Surfactant 3A assembly rbs-gfp (pbk7-pbk14) in psb1K3 = L1. Transformation of L1 in NM522. Friday, July 27th 2012 For all purposes Extraction of transformed clones (NM522/pHT304 et NM522/pHT315). The electrophoresis showed that the plasmids had approximately 7kb and the restriction sites XbaI, EcorI and PstI as expected. However, there is a SpeI site which was not mapped. Kill

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)

Surfactant

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.

Stick

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...).

Monday, July 30th 2012 For all purposes

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...).

Kill

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).

Surfactant

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

Stick Purification of XylR, produced by PCR. Tuesday, July 31st 2012 Kill Results of the lysostaphin tests on plates : negative. There is no biofilm : too diluated but even though, the colonies do not seem to be affected by the presence of Bs 168 pWG100 supernatant ? the Bs 168 pWG100 have lost their plasmide ? (are cultivated without selection pressure, that is whitout erythromycin). Surfactant

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.

Stick Ligation of RBS and XylR and transformation in NM522 strain. For all purposes Wednesday, August 1st 2012

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

Kill

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).

Surfactant

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.

For all purposes Thursday, August 2nd 2012

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.

Kill

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).

Stick

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.

Surfactant The transformation of Sfp and abrB in NM522 strain didn’t work. Friday, August 3rd 2012 For all purposes

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.

Kill

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.
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.

Surfactant

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.

Stick New ligation between RBS and XylR in pSB1A3 and transformation in NM522 strain. Saturday, August 4th 2012 Kill

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.

Surfactant

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

Stick Sorting of the RBS + xylR clones to eliminate the clones having two plasmids religated. Sunday, August 5th 2012 For all purposes Liquid culture of 6 NM522 clones with pHT 304 S and 6 clones pHT 315 S. Surfactant Plasmid extractions from 3 clones NM522/abrB and 5 clones NM522/sfp. The electrophoresis of the digested plasmids with EcoRI and PstI confirmed the presence of sfp construction (at 800 bp) and abrB construction (at 500 bp). Stick Liquid cultures of RBS+XylR clones are launched to do plasmid extractions. Monday, August 6th 2012 For all purposes

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 :’(

Kill

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).

Surfactant 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 pXyl and purification of these PCR products.

Stick

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

Tuesday, August 7th 2012 For all purposes

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.

Kill

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.

Surfactant

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.

Wednesday, August 8th 2012 For all purposes

Result of the transformation of NM522 by pHT 304 S et pHT 315 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 GL plate : 0 clone for pHT 304 S and 18 for PHT 315 S but none of the clones is good. We must do the filling-in again...
Transformation of the pSB1C3 + RFP and T3 + RFP plasmids extracts of the iGEM plates to use them as tool of cloning after extraction.

Kill

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

Surfactant Results of the transformations :

Transformations of NM522 by BK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : ok.
Transformation by Sfp-abrB-pSB1T3 : the negative control isn’t good, the transformation is done again...
Miniprep of other clones transformed with the plasmid RBS + XylR : the electrophoresis shows that the ligation isn’t still good…

Thursday, August 9th 2012 For all purposes

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)

Kill Transformation results :

Witness + : okay;
Witness - : okay;
Lyso+Dsp : 8 clones;
Prom+Dsp : more than 30.

Clones are screened on Ampicillin and Chloramphenicol. Surfactant

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 true transformation contains nothing too ! We decided to do the ligation Sfp-abrB 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.

Friday, August 10th 2012 For all purposes

Deletion of the site SpeI 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).

Kill

New Lysostaphin tests are planned (on plates) : in this purpose, a culture of 60 mL of Bs 168 pWG100 is inoculated and then incubated over night at 28°C.
None of the clones Lyso+Dsp grew on Ampicillin so LB cultures were inoculated.
Out of 29 clones, 18 clones Prom+Dsp 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 essay was done, but it was unsuccessful.
The strain NM522 with Constitutive Promotor + pSB1C3 was put in storage under the reference BK27.

Surfactant

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

Saturday, August 11th 2012 For all purposes Gel electrophoresis showed that the elimination of the SpeI site was not successful. Sunday, August 12th 2012 Kill Culture of 60 mL of Bs 168 pWG 100 at 28°C for the lysostaphin tests on plates. Monday, August 13th 2012 For all purposes

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.

Kill

Lysostaphin tests : Flasks of 25 mL of Bs 168 pWG 100 filtered supernatant were cultivated Friday and frozen at -20°C Sunday (2 flasks of each). The control flask is filled with 25 mL of LB media. 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 promotor + Dispersin] in pSB1C3 do not have the right plasmid : the digested fragments do not have the right size.

Surfactant Transformation of the ligation [sfp + abrB + pSB1A3] in the NM522 strain. Tuesday, August 14th 2012 For all purposes The gel electrophoresis of the digested plasmids pHT315 and pHT304 after the final purification shows promising results, so the two plasmids were transformed in the NM522 strain. Kill

Ligation of the Constitutive Promoter (extracted by PCR) in the plasmid pSB1C3 containing the RFP gene (purified by midiprep) in order to obtain the Constitutive Promoter in the pSB1C3.
Lysostaphin tests on plates : the come back ! The freeze-dries 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.

Surfactant The transformation of the ligation [sfp+abrB+pSB1A3] was successful, so 4 clones were put in liquid culture in order to be tested. Stick Standard ligation between pBK7 (=RBS in pSB1C3) and XylR (obtained by PCR) with 1µL of insert for 5µL of vector. The ligation was transformed into the NM522 strain. Wednesday, August 15th 2012 For all purposes The transformation of the pBK20 (pHT315) and pBK19 (pHT304) plasmids was successful. 12 clones per plasmid were tested. Kill

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 media instead of LB media.
Transformation of the ligation [pSB1C3 + constitutive promoter] in NM 522 strain.

Surfactant None of the 4 clones tested had the attended fragments, so we screened another 6. Stick

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 (producted by PCR) with more insert (3µL of insert for 2µL of vector). Transformation into NM522 strains.

Thursday, August 16th 2012 For all purposes 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. Kill

Ligation of the Constitutive promoter (pVeg) 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 the 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.

Surfactant

Two of the 6 [sfp+abrB] clones had the expected fragments.
Ligation of pXyl produced by PCR in an iGEM plasmid.

Stick

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.

Friday, August 17th 2012 For all purposes The plasmids (pBK19 and pBK20) from 2 clones were re-extracted using columns. This time the digestion was successful. The tests showed that there was no SpeI site. However, the pHT315 plasmid also had no XbaI site. Stick Plasmid extractions from the 14 clones transformed by [pBK7 + XylR] (with the second ligation). Like for the first transformation, the electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they have just the vector without XylR. Monday, August 20th 2012 For all purposes

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

Kill

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

Stick

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.

Tuesday, August 21st 2012 For all purposes

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.

Kill

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

Surfactant

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.

Stick

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.

Wednesday, August 22nd 2012 Kill

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 clonage : Digestion, Ligation, transformation to construct :

[Lysostaphin + Dispersin] in pSB1C3;
[Lysostaphin + Dispersin] in the shuttle vector (vector for E. coli and B. subtilis,/i>).

Surfactant 6 pXyl clones were tested, but none had integrated the plasmid. Stick

Check 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 witness. 12 clones are put in liquid culture for extraction.

Thursday, August 23rd 2012 For all purposes Cloning of the iGEM linker into the shuttle vector (pBK25 and pBK26) and transformation in the NM522 strain. Kill

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 analyse the extracted DNA : the transformation is succesful 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).

Surfactant Transformation of [pXyl+pSB1T3] in NM522. Stick

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.

Friday, August 24th 2012 For all purposes The transformation of the cloned shuttle vectors was successful, so 12 clones are put in liquid culture for further tests. Surfactant 3A ligation of pBK4 (pUC57 containing the lacI gene) and pBK29 (pSB1A3 containing the sfp and abrB genes) in pBK24 backbone (pSB1C3 containing a RFP gene). The cloned fragment was transformed in the NM522 strain. Stick

After the bad results with xylR, we decided to cut pBK10 plasmid in Sma1 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.

Saturday, August 25th 2012 For all purposes Miniprep of 12 clones selected from transformed bacteria with the cloned shuttle vectors; we ran out of SpeI enzyme, so we could not digest the extracted plasmids. Sunday, August 26th 2012 Surfactant Miniprep of 6 clones (3A ligation: pBK24, pBK4, pBK29). The electrophoresis showed that none of the tested clones had integrated the right plasmid. Monday, August 27th 2012 Kill

Transformation of BS 168 strain with the pBK28 (1) plasmid which contains the [Constitutive Promoter + RBS + Lysostaphin] in the shuttle vector.

Midiprep : Extraction of pveg-disp-pSB1C3 of clone 22.

BK33 strain was put in storage.

Surfactant

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(promotor pXyl, RBS and sfp gene), abrB gene and pSB1A3 backbone) were made for miniprep (the two plasmids were extracted using the classical 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).

Stick No transformation was done, because we ran out of LB growth medium. Tuesday, August 28th 2012 For all purposes Meeting at 1:30 pm. Kill

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 GL+Ery plates with different erythromycin concentration (from 5 µg/mL to 10 µg/mL).

Surfactant The transformations with the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios ) was successful. 12 clones per transformation are put in liquid culture and then streaked on LB+Cm plates and LB+Amp plates. Wednesday, August 29th 2012 Kill

Transformation of the previous ligation [promoter-dispersin-pBK25] in E. coli NM522.

Results of the second transformation of BS 168 strain with the 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 (GL+Ery) plates with different erythromycin concentrations in order to make different tests.

Surfactant

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 genes sfp and abrB. 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 different 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 classical protocol because we were running out of time and we were behind the schedule.

In parellel, 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.

Thursday, August 30th 2012 Kill

Transformation results : too many clones on the control digestion vector not ligated so the transformation can’t be used for the transformation [Lysostaphin+Dispersin] in pSB1C3.

12 clones put in culture from the transformation plate [Lysostaphin + Dispersin] in the shuttle navette 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.

Surfactant

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.

Friday, August 31st 2012 For all purposes We have finally received the enzymes, so we tested the minipreps containing the modified shuttle vectors (containing the iGEM linker). The results are promising : 4 plasmids are digested by SpeI, so the ligation was successful. The four plasmids are further tested for the other 3 iGem sites. To make sure that the site SpeI is not the initial site that we eliminated by filling-in and that the plasmid used for transformation and ligation was not contaminated with the original plasmid, a double digested was done (EcoRI and SpeI). There was no fragment at 1000 pb, so the ligation was successful. Kill

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 NM 522 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 their DNA and check it.

Surfactant Miniprep of the saturated cultures with transformed bacteria containing the ligation of the sfp and abrB genes. The electrophoresis confirmed that this time the bacteria contained the right plasmids. Saturday, September 1st 2012 For all purposes Kill

Digestion of Lysostaphin in the shuttle vector and dispersine in pUC57 and verification of digestion.

Verification of midiprep : Midiprep of Lysostaphin in pSB1C3 is okay but not the one of Dispersin in pUC57.

Miniprep of 14 clones NM522 transformed with Lysostaphin in pBK26. The digestions and electrophoresis show that 7 clones are ok ! They are put in storage under the references pBK38 (1) to pBK38 (6).

Surfactant

One of the 12 screened clones transformed with the ligation of pBK22 and pBK39 has the expected fragments. Unfortunately, the plasmid is mixed with another one. To separate the two plasmids we decided to transform 1 µL of the miniprep into the NM522 E. coli strain.

Another 6 clones with the transformed 3A ligation (done on August 29th) are screened.

Standard ligation of the abrB gene and the plasmid containing lacI in pSB1C3 backbone (pBK37) and transformation. In parallel, another ligation is attempted : [pBK37 + pBK29].

Sunday, September 2nd 2012 For all purposes Liquid cultures containing the modified shuttle vectors with the iGEM linker are inoculated and incubated over night. Kill Shuttle vector containing the lysostaphin is treated with an alcaline phosphatase, then a ligation with the dispersin is realised followed by transformation in NM522 E. coli strain. Surfactant

Plasmids from the 6 clones containing the 3A ligation are extracted and digested. Gel electrophoresis showed that one of the clones has the expected fragments. However, it was mixed with another plasmid. In order to separate them, we decided to transform the NM522 strain with 1µL of the miniprep.

12 clones for each ligation (done the day before) are screened.

Monday, September 3rd 2012 For all purposes Midiprep of the cloned shuttle vectors (containing the iGEM linker). Kill Transformations of BS 168 :

Trial n°1 : negative control with H₂O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK25 (=pBK28).

Trial n°2 : negative control with H₂O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK26 (=pBK38).

The bacteria are spread on GL+Ery plates ([ERY]=1 µg/mL and [ERY]=10 µg/mL). Surfactant

abrB gene obtained by PCR was cloned in pSB1T3 backbone.

6 clones transformed with the mixture of two plasmids are screened.

Miniprep of 12 clones per transformed ligation : only one had the expected fragments (ligation of the lacI and abrB genes). The clone is put in storage under the reference pBK39.

Tuesday, September 4th 2012 For all purposes Multiple tubes containing LB media and Ampicillin at different concentrations ranging from 0 to 1,3 mg/mL are inoculated with bacteria containing the shuttle vectors (pBK35 and pBK36). Kill

Results of the transformations of BS 168 : not coherent. However, the problem seems to be identified : it is the erythromycin solution. Indeed, the erythromycin must be diluted in ethanol and not in water and must be kept refrigerated at -20°C.

Standard ligations between :

pLac in pSB1C3 (pBK9) digested by SpeI and PstI and [RBS-GFP] in pSB1T3 digested by XbaI and PstI;

pXyl (amplified by PCR) digested by SpeI and EcoRI and [RBS-GFP] in pSB1T3 digested by XbaI and EcoRI.

These two ligation products are transformed in NM522 strain.

Surfactant Gel electrophoresis showed that 5 clones out of 6 clones screened after the transformation with the miniprep containing two plasmids had the expected fragments. However, we had doubts and we decided to search for restriction sites in the sequences that the plasmid was supposed to contain to make sure we had the right construction. We found out that a digestion with EcoRV would give 5 fragments of different sizes, enough to confirm the presence of the expected genes. Unfortunately, the electrophoresis was disappointing. Biofilm

One plate with S. epidermidis to evaluate the adhesion.

One plate with S. epidermidis + supernatant of different cultures (lysostaphin, dispersin...).

Same plate as the one above one + crystal violet.

Wednesday, September 5th 2012 Surfactant Standard ligation of pBK22 (sfp gene) and pBK39 (abrB-lacI) and transformation into the NM522 E. coli strain. Thursday, September 6th 2012 For all purposes Ampicillin resistances tests had some surprising results : even at 1.3 mg/mL there is bacterial growth. However, the higher Ampicillin concentration is, the lower is the OD of the liquid cultures. Ampicillin resistance test of the two shuttle vectors did not show any significant difference between the two strains (BK37 and BK38). The result is not surprising, given the fact that both plasmids (pBK36 and pBK35) derive from the shuttle vectors pHT315 and pHT304 which have the same origin replication as pUC19. However, the test must be done again, this time with at least two samples for each Ampicillin concentration in order to be able to estimate an error. Kill Transformation of BS 168 : new try ! To improve our protocol, we took periodic OD600 readings in order to stop the cell growth at the best stage (just between the exponential and the stationary phase). The BS 168 strain is transformed by :

pBK 25 (= modified shuttle vector pHT 304);

pBK 26 (= modified shuttle vector pHT 315);

pBK 28 (= Lysostaphin in the modified shuttle vector pHT 304);

pBK 38 (= Lysostaphin in the modified shuttle vector pHT 315);

Dispersin in pBK26.

We spread 200 µL of each transformed cells on GL + Erythromycin ([ERY] = 16 µg/mL) plates. Surfactant 12 clones transformed with the ligation pBK22 and pBK39 are screened. Friday, September 7th 2012 Kill

Result of the transformation of BS 168 :

All the negative controls plates are clear : the erythromycin concentration is high enough to do a selection;

Some plates with the transformed bacteria are empty but some have from 1 to 4 clones : these clones are put in liquid cultures in order to make miniprep and analyse their DNA;

Transformation of BS 168 : new trial by electroporation. As the last transformation, the BS 168 strain is transformed by pBK25, pBK26, pBK28, pBK38 and dispersin in pBK26.

Surfactant All the screened clones had the religated vector, so we decided to use commercial competent cells from STLB2 strain to transform the ligation of pBK39 and pBK22 in order to diminish the number of clones so that the chances of finding the recombinant plasmid containing the 3 genes (sfp, abrB and lacI) would be higher. Saturday, September 8th 2012 Kill Miniprep of the clones BS 168 transformed by pBK26, pBK28 : we use the same protocol as DNA extraction of E.coli with addition of Lysosyme the buffer A1. Electrophoresis to analyze extracted DNA : there is nothing on the gel => Maybe the DNA isn’t concentrated enough ?
New clones are put in liquid cultures in order to make other minipreps. Surfactant 16 clones containing the ligation of pBK22 and pBK39 transformed into STLB2 commercial competent cells are screened. Sunday, September 9th 2012 Kill

Miniprep of the clones BS 168 transformed by pBK25, pBK26, pBK28, and Dispersin in pBK26. Electrophoresis to analyze extracted DNA :

BS 168 clone transformed by pBK25 has the right plasmid !! =)

BS 168 clone transformed by pBK28 has the right plasmid !! BACILLUS LYSOSTAPHIN IS HERE !! :D :D :D

BS 168 clones transformed by pBK26 and dispersine in pBK26 : nothing on the gel ...

Transformation of BS 168 GFP with the same five plasmids as before. The transformed bacteria are spread on GL + ERY plates, with two different concentrations : [ERY]=10 µg/mL and [ERY]=15 µg/mL.

Given the fact that there was nothing on the miniprep for pBK26 and pBK28, we made a new electrophoresis after using the seeDNA method to concentrate the DNA of the miniprep. We see very light stripes on the gel, but the results for pBK26 and dispersin in pBK26 have to be confirm again...

Thanks to the good results, we put in storage the transformed Bacillus under the reference :

BK41 : BS 168 + pBK25

BK42 : BS 168 + pBK28

BK43 : BS 168 + pBK26 (to confirm)

BK44 : BS 168 + Disp in pBK26 (to confirm)

Surfactant The electrophoresis of the 16 digested plasmids extracted did not turn out as expected : all clones are in fact the initial plasmid, pBK39!

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