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 transformation. For all purposes Tuesday, July 3rd 2012

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

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

Transformation of the NM522 strain (this experiment was repeated 3 times).

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

The transformed bacteria were selected on chloramphenicol (Cm) 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 two other 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 to 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 Phone conference with Romain Briandet.

Terminator was retrieved from 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 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

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) 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).

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-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.

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 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.

Kill Friday, July 13th 2012

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;

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 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.

Kill

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.

Physiological tests : Testing the potential of S. epidermidis to form biofilms

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.

Wednesday, July 18th 2012 For all purposes

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.

Kill

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.

Physiological tests : Testing the potential of S. epidermidis to form biofilms

The microtiter plate prepared the day before is analyzed. Each well is stained with crystal violet and subsequently OD₆₀₀ is measured in order to quantify the adherence of the strain. We can conclude that the concentration of glucose has no impact on the adherence of S. epidermidis.
A microtiter plate is inoculated with 2mL of a suspension obtained from diluting the liquid culture 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.

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 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.

Kill

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.

Physiological tests : Testing the potential of S. epidermidis to form biofilms The microtiter plate prepared the day before is analyzed. Each well is stained with crystal violet and subsequently OD₆₀₀ is measured in order to quantify the adherence of the strain. We can conclude that the concentration of glucose has no impact on the adherence of S. epidermidis and that the fact of cultivating the strain in broth or on solid medium has no impact on the biofilm's quality. Friday, July 20th 2012 For all purposes

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.

Kill

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.

Monday, July 23rd 2012 For all purposes

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.

Kill

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.

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.
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.

Kill

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.

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 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.

Kill

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.

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 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.

Kill

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.

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 had not been mapped. Kill

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)

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)-terminator (pBK4-pBK10) in pSB1C3 = IV.

Stick

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...).

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 (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).

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 diluted but even though, the colonies do not seem to be affected by the presence of Bs 168 pWG100 supernatant → have the Bs 168 pWG100 lost their plasmid? (cultivated without selection pressure, that is without erythromycin). Surfactant

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.

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 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).

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 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.

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+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).

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 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 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.

Kill

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.

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, although they should have been cloned.
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 for each transformation are chosen and spread on LB+Cm and LB+Amp plates.

Surfactant

Transformations 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 containing two relegated plasmids. Sunday, August 5th 2012 For all purposes Liquid culture of 6 NM522 clones with pHT304 S and 6 clones pHT315 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 pHT304 S and NM522 with pHT315 S : Failure ! The SpeI site is still in the plasmids :’(

Kill

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).

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 P_xyl and purification of these PCR products.

Stick

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

Tuesday, August 7th 2012 For all purposes

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.

Kill

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.

Surfactant

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.

Wednesday, August 8th 2012 For all purposes

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.

Kill

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.

Surfactant Results of the transformations :

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...

Miniprep of other clones transformed with the plasmid RBS + xylR : the electrophoresis shows that the ligation still isn’t 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 :

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

Clones are screened on LB + Amp and LB + Cm plates. 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 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!!

Friday, August 10th 2012 For all purposes

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).

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 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.

Surfactant

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.

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 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.

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 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.

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

Surfactant None of the 4 clones tested had the expected fragments, so we screened 6 others. 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 (produced by PCR) with more insert (3µL of insert for 2µL of vector). Transformation into NM522 strains.

Thursday, August 16th 2012 For all purposes

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.

Kill

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.

Surfactant

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.

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. We digested the two plasmids with the XbaI enzyme and this time we incubated the digestion during one hour, instead of 10 minutes. We also tested the enzyme on another plasmid which had a XbaI site. The digestion of the control plasmid was partial. Surfactant Miniprep of 6 clones with the transformed 3A ligation (pBK22, gene abrB and pSB1A3 backbone). The gel verification showed that there were 3 clones having the expected profile. The plasmid was put in storage under the reference pBK29. Stick Plasmid extractions from the 14 clones transformed by [pBK7 + xylR] (with the second ligation). As for the first transformation, the electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they only have the vector without xylR. Monday, August 20th 2012 For all purposes

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.

Kill

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.

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 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.

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 cloning : 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 P_xyl clones were tested, but none had integrated the plasmid. Stick

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.

Physiological tests

Liquid culture of B. subtilis 168 in 5mL of LB broth.

Liquid culture of B. subtilis abrB in 5mL of LB broth.

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 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).

Surfactant Transformation of [P_xyl+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.

Physiological tests A microtiter plate is inoculated with B. subtilis 168 and B. subtilis abrB in order to compare the adherence of each strain. (see Protocol 'Tests of Bacillus subtilis adherence in 24 wells plate'). Friday, August 24th 2012 For all purposes The transformation of the cloned shuttle vectors was successful, so 12 clones are put in liquid cultures for further tests. Surfactant 3A ligation of pBK4 (pUC57 containing the lacI gene) and pBK29 (pSB1A3 containing the sfp and abrB genes) in pBK24 (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 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.

Physiological tests The microtiter plate prepared the day before is analyzed. There is a significant difference between the adherence potential of the two strains. B. subtilis abrB strain forms thin layer biofilms while B. subtilis 168 doesn't form any specific kind of biofilms. 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. Stick 6 white clones from the ligation pBK24-xylR are purified and put in liquid cultures. Sunday, August 26th 2012 Kill Extraction from 12 clones of lysostaphin and dispersin in pBK26 (shuttle vector) ligation and gel electrophoresis. The gel didn’t show any good clone. Surfactant Miniprep of 6 clones (3A ligation: pBK24, pBK4, pBK29). The electrophoresis showed that none of the tested clones had integrated the right plasmid. Stick Extraction from the 6 white clones (containing xylR-pBK26). Gel electrophoresis is run, but it didn’t show any good clone. Monday, August 27th 2012 Kill

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.

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(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).

Stick No transformation was done, because we ran out of LB broth. 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 LB+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 cultures and then streaked on LB+Cm plates and LB+Amp plates. Stick Transformation of xylR-pBK10 (blunt end ligation) in NM522. 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 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.

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 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.

Stick

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.

Thursday, August 30th 2012 Kill

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.

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.

Stick

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.

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 digestion was done (EcoRI and SpeI). There was no fragment at 1,000 bp, 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 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.

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. Stick

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
Saturday, September 1st 2012 Kill

Digestion of Lysostaphin in the shuttle vector and dispersin in pUC57 and verification of digestion. Then the vector is treated with CALF protein and a ligation is made using different of insert concentration. Then the ligation is transformed with commercial cells (STLBÉ) according to the manufacturer protocol and another trial is made with higher concentration.

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).

Extraction of 14 clones transformed with Dispersin in pBK25. Gel electrophoresis showed no good clones.

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 E. coli NM522 strain.

6 other 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 overnight. Kill

Shuttle vector containing lysostaphin is treated with an alkaline phosphatase, then a ligation with dispersin is realized, followed by transformation in E. coli NM522 strain.

Dispersin is ligated with pBK26 and then digested by BamHI before transformation. BamHI digestion allows not to transform the circularized vectors which doesn’t contain the insert.

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).

Miniprep of pBK26 shuttle vector is done in order to increase the stock.

Kill

No clones are obtained on the plate lysostaphin+dispersin when we followed the manufacturer protocol even on the positive control. But few colonies appeared on plates done with our own protocol.These clones are put in liquid cultures.

Tests are launched to verify manufacturer cells. And other tests are launched to verify that our plates contained the appropriate antibiotic.

A transformation is made with our cells of their positive control.

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 LB+Ery plates ([Ery]=1 µg/mL and [Ery]=10 µg/mL).
The transformation of NM522 with ligation Dispersin in pBK26 was successful.

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 medium 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

Lots of clones are obtained with our cells.So we have tried a transformation of commercial cells with bigger quantities of DNA.

Miniprep of clones obtained with STLB2 are made. Clones are not okay. A new transformation with the same ligation mix is made but with NM522.

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 :

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

P_xyl (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.

Acrylamide gels and samples are prepared for SDS-PAGE.

Surfactant Gel electrophoresis showed that 5 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. Stick A PCR is run in order to get RBS-xylR. The PCR failed (not enough DNA-polymerase). 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 + crystal violet.

Wednesday, September 5th 2012 Kill Few clones are obtained with commercial cells, there are not very efficient. A new transformation is made with the same ligation mix but in NM522. Surfactant

Standard ligation of pBK22 (sfp gene) and pBK39 (abrB-lacI) and transformation into the E. coli NM522 strain.

A SDS-PAGE gel is run to analyze lysostaphin (in BK32 and BK35 strains) and dispersin (BK29 strain).

Thursday, September 6th 2012 For all purposes Ampicillin resistance tests gave some surprising results : even at 1.3 mg/mL there is bacterial growth. However, the higher Ampicillin concentration is, the lower OD₆₀₀ of the liquid cultures is. 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

Clones obtained on lysostaphin-dispersin plates are put in liquid culture.

Transformation of Bs 168 : new trial ! To improve our protocol, we took periodic OD₆₀₀600 measurements 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 LB + Erythromycin ([Ery] = 16 µg/mL) plates.

Surfactant 12 clones transformed with the ligation pBK22 and pBK39 are screened. Stick 2 new PCR are done to have xylR, but they failed. Friday, September 7th 2012 Kill

A miniprep of potential lysostaphin-dispersin clones is made : no clones are okay.

Result of the transformation of Bs 168 :

All the negative control 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 extract and analyze 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.

A new SDS-PAGE is made using supernatants and pellets as samples. The samples were not concentrated enough, so the gels didn’t show any of the expected bands.

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. Stick A new RBS-xylR PCR is run, now with different concentrations of genomic DNA and a different annealing temperature. The PCR was successful. Physiological tests

Liquid culture of S. epidermidis is incubated without any agitation at 37ºC.

Liquid culture of B. subtilis containing the dispersin gene.

Liquid culture of B. subtilis containing the same plasmid as B. subtilis dispersin but without the dispersin gene.

Liquid culture of B. subtilis containing the lysostaphin gene.

Liquid culture of B. subtilis containing the same plasmid as B. subtilis lysostaphin but without the lysostaphin gene.

Saturday, September 8th 2012 Kill Miniprep of the clones Bs 168 transformed by pBK26, pBK28 : we use the same protocol as DNA extraction for E.coli with addition of Lysozyme to 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. Stick Ligation of P_lac-RBS-xylR and then transformation in NM522. Physiological tests Each of the 24 wells of two microtiter plates are inoculated with 2mL of a suspension obtained from diluting the liquid culture 100 times with TSB enriched with 1% glucose The plate is incubated during 24 hours at 37ºC. 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 dispersin in pBK26 : nothing on the gel...

Transformation of Bs 168 GFP with the five same plasmids as before. The transformed bacteria are spread on LB + 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 + Dispersin 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! Physiological tests The supernatant from the two microtiter plates are decanted. The wells of a third microtiter plate are filled with the supernatant of the liquid culture which is supposed to contain the dispersin. The supernatants are diluted in order to test different concentrations of the supernatants. The wells of a fourth microtiter plate are filled with the supernatant of the liquid culture which is supposed to contain the lysostaphin. Monday, September 10th 2012 For all purposes

4 transformations with the same amount of plasmid are done (pBK19,pBK20, pBK35 and pBK36) in order to characterize the transformation efficiency of the shuttle vectors.

Ampicillin resistance test was repeated, this time with 3 tubes per Ampicillin concentration for each plasmid.

Surfactant We decided to give another try and do the ligation once again, this time using 5 time less vector and a ⅛ ratio vector/insert. Given the fact that we are running out of time, we did two ligations hoping to construct the same biobrick: pBK22+pBK39 and pBK29+pBK37. Both ligations were transformed into the E. coli NM522 strain. Kill

OD₆₀₀ test in tanks to test lysostaphin effect on a S. epidermidis culture. Strains used for the test are E. coli strains which might produce lysostaphin. Supernatant is filtered and applied on the S. epidermidis culture.

S. epidermidis cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic.

According to the results there is an effect of lysostaphin but we observed an effect due to the initial OD₆₀₀ of cultures that were different, so we don’t know if the results can be used. A new protocol is tried out, culture are diluted to have the same OD₆₀₀.

New SDS-PAGE 12% gel is done with just the supernatants. The samples preparation didn’t go well and they were not enough concentrated. Therefore the gel didn’t show anything.

Plac and Pxyl kinetics

Measurement of the NM522 + P_lac kinetics (with Cm) during 12h in a 96 well plate with and without different IPTG concentration addition. (1 mM; 0.5mM and 0.1mM)

In the same 96 well plate NM522 + P_xyl kinetics is also measured during 12h with and without different xylose concentration. (2%; 0.5% and 0.2%)

Results : kinetics decreases so there is a problem. But it can be explained by the fact that the promoters need to be activated by a sigma factor which is active in the exponential phase and 12h is not enough. So this experiment has to be done during 24h.

Physiological tests The microtiter plates prepared the day before are analyzed. Each well is stained with crystal violet and subsequently OD₆₀₀ is measured in order to quantify the adherence of the strain. We can see that the biofilm is not adherent enough and it is destroyed no matter what kind of supernatant is tested. Unfortunately, we cannot say what the effect of the dispersin or lysostaphin is using this kind of test. Tuesday, September 11th 2012 Experiments realized in Massy

Seeding a 96 well plate with S. aureus to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+disp in pBK26 and Romain Briandet’s strains (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)

Seeding a 96 well plate with S. aureus to produce a biofilm (plate 2).

For all purposes

Design of the primers required to sequence the modified sequences of the shuttle vectors (after elimination of SpeI site by filling-in and cloning of the iGEM linker).

Ampicillin resistance test was a complete failure : the negative control (NM522 strain grown in LB broth supplemented with Ampicillin at the usual selective concentration), so the Ampicillin that was used lost it’s biological activity.

Surfactant 12 clones per transformation are screened. Kill

OD₆₀₀ test in tanks to test lysostaphin effect on a S. epidermidis culture. Strains used for the test are E. coli strains which might produce lysostaphin. Supernatant is filtered and applied on the S. epidermidis culture. S. epidermidis cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic. According to the results there is an effect of lysostaphin but we observed an effect of dilution due to the protocol, so we don’t know if the results can be used. So a new protocol is worked out.

SDS-PAGE is done using pellets, to find a good concentration.

Physiological tests Liquid culture of S. epidermidis is incubated without any agitation at 37ºC. Wednesday, September 12th 2012 Experiments realized in Massy

We remove the supernatant in the plate 2 made on Tuesday and added Bs168+dispersin in pBK26 and Bs168+pBK26 with different dilutions. Observation of the plate at the confocal laser scanning microscope after 5 hours of incubation. Exploitation of the pictures with Imaris software. Dispersin seems to have an effect on the biofilm.

Reading of the plate 1 with a confocal laser scanning microscope (after an overnight incubation). Exploitation of the pictures with Imaris software.

For all purposes The transformed plates with the modified and unmodified shuttle vectors do not show any significant difference, so we decided to make another essay, this time with 3 transformation for each plasmid. Surfactant Unfortunately, the screened clones did not integrate the expected plasmid. Kill

OD₆₀₀ test in tanks to test lysostaphin effect on a S. epidermidis culture with the new protocol. Strains used for the test are Bacillus subtilis strains which may produce lysostaphin. Supernatant is filtered and applied on the S. epidermidis culture. There is a problem with the results : the control is not good.

Miniprep of one clone containing dispersin in pBK26 is done. Gel electrophoresis showed that the clone was good. The plasmid is then put in storage (pBK41).

Physiological tests

5 test tubes containing a lamella are inoculated with the culture prepared the day (S. epidermidis) before diluted 1:100 with TSB supplemented with 1% glucose.

Liquid culture of B. subtilis containing the dispersin gene.

Liquid culture of B. subtilis containing the same plasmid as B. subtilis dispersin but without the dispersin gene.

Liquid culture of B. subtilis containing the lysostaphin gene.

Liquid culture of B. subtilis containing the same plasmid as B. subtilis lysostaphin but without the lysostaphin gene.

Thursday, September 13th 2012 Experiments realized in Massy

Seeding of 2 96 well plate with S. aureus to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+dispersin in pBK26 and Romain Briandet's strain (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)

OD₆₀₀ measurements with filtered supernatants.

Mobility test of Bs168 on agar 0.25% plate.

For all purposes

The transformed plates with the modified and unmodified shuttle vectors do not show any undeniable difference, so we decided to make another essay, this time with 3 transformation for each plasmid.

Ampicillin resistance test is repeated (3 samples for each Ampicillin concentration).

Surfactant Another 12 clones per transformation were screened (from the transformation made on 10th September). Kill OD₆₀₀ test in tanks to test dispersin effect on a S. epidermidis culture. Strains used for the test are Bacillus subtilis strains which may produce dispersin. Supernatant is filtered and applied on the S. epidermidis culture. Dispersin has no effect on S. epidermidis culture according to the results. Physiological tests

One of the tubes and lamella prepared the day before containing S. epidermidis cultures is quantified using crystal violet staining and OD₆₀₀ is measured. S. epidermidis cells are quite adherent on glass surfaces.

The supernatant of the four other tubes are decanted and tubes are filled with the supernatants produced by the four liquid cultures of B. subtilis prepared the day before in order to test the effect of lysostaphin and dispersin.

Friday, September 14th 2012 For all purposes

Transformation efficiency tests confirm our supposition : there is no significant difference between the transformation yields of the 4 plasmids (unmodified shuttle vectors, pBK19 and pBK20 and modified shuttle vectors, pBK35 and pBK36).
Moreover, OD₆₀₀ of the bacterial cultures in LB medium at various Ampicillin concentrations shows no significant difference between the BK37 and BK38 strains.

TG1 (E. coli) strain containing pJIM2241 shuttle vector is put in storage (BK47).

Kill

OD_600s test in tanks to test lysostaphin effect on a S. epidermidis culture. The strains used for the test are Bacillus subtilis strains which may produce lysostaphin. Supernatant is filtered and applied on the S. epidermidis culture.

SDS-PAGE is done with some changes in the protocol. The gels showed a band that matches with the expected lysostaphin molecular weight.

Surfactant Gel electrophoresis of the extracted plasmids shows that there are 5 possible plasmids containing the ligated insert (P_xyl-sfp-abrB-lacI-terminator). The five plasmids are digested by EcoRV and the electrophoresis confirms that, this time, the plasmids have the expected fragments. Physiological tests The four tubes and lamella prepared the day before are quantified using crystal violet staining and OD₆₀₀is measured. The same result is obtained : S. epidermidis biofilms are not adherent enough and biofilms are destroyed when the growth medium is changed. Saturday, September 15th 2012 Kill OD₆₀₀ test in 96 well plate to test impact of lysostaphin and dispersin on a culture of S. epidermidis (test during 12 hours). Physiological tests : Mobility of B. subtilis LB plates containing a 0.3% agarose gel are inoculated with 5 μL of a 5 hour liquid culture of B. subtilis containing the plasmids used in transformations. Sunday, September 16th 2012 Physiological tests : Mobility of B. subtilis The diameter of the disks formed by B. subtilis is measured. The fact of introducing the plasmids did not impact the swarming mobility of our bacteria. Friday, September 21st 2012 Plac and Pxyl kinetics A 7-hour-long kinetics is made with P_lac and P_xyl to be sure to have measurement in the exponential phase. Only one IPTG and xylose concentration is used. P_lac = IPTG 0.5% and P_xyl = xylose 0.5%.
Moreover, a 24-hour-long kinetics is also made in a 96 well plate. The conditions are the same as the one done for 12h except that bacteria are inoculated in a 1/100 dilution (instead of 1/50).

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