• Week 6
• Week 7
• Week 8
• Week 9
• Week 10

Transformation protocol :

Sterile conditions needed :

- 2µL of DNA are transferred in eppendorf tube
- Add 100µL of comptentent bacterial cells
- Put on ice for 20 minutes
- Put tubes in thermomixer at 42°C for 45 secondes
- Put on ice for 5 minutes
- Add 900 µL of LB for 100µL of bacterial cells
- Incubate tube for 1 hour at 37°C
- Centrifuge for 5 minutes at 5000 rpm
- Remove 850 µL of supernatant
- Resuspend Pellet in the 150 μL of remaining medium pipet up-down
- Spread total volume on petri dish with the appropriate antibiotic
- Put petri dishes in an incubator overnight at 37˚C

Competent bacterial cells protocol  :

1- Preparation of the Bacterial culture
- Recuperate the overnight bacterial culture
- Determine the OD600 in a 1 ml Tank Spectro, dilute a 100μl of culture in 900μl of distilled LB
- In 1L Erlenmeyer add 200 ml of LB and an appropriate volume of culture to have an OD600 = 0.1
- Put the Erlenmeyer in an incubator for about an hour at 37˚C.
- test the OD600 for the new culture
- the OD600 should be between 0.4 and 0.6
All the handling done in a 15cm radius of an open flame for optimal sterility

2- Preparation of Buffer Tbf1 and Tbf2
Tbf1 Buffer
KAc 1M x 2.4 ml - MnCl2 0.5M x 8 ml - KCl 1M x 8 ml - CaCl2 0.1M x 8 ml - Gly 80% x 15 ml - H2O x 38.6 ml
Tbf2 Buffer
NaMOPS 0.2M x 400 μl - CaCl2 0.1M x 6 ml - Gly 80% x 1.5 ml - KCl 1M x 80 μl - H2O x 500 μl

3- Preparation of Competent Bacterial cells
- Transfer the Bacterial culture in 50 ml Falcon tubes
- Centrifuge for 10 minutes at 3500 rpm in cold
- Remove the supernatant then re-suspend the pellet in 20 ml of Tbf1 for each falcon tube
- Pool in all bacterial culture in two Falcon tubes with 40 ml each
- Centrifuge for 5 minutes at 3500 rpm in cold
- Remove the supernatant then re-suspend the pellet in 4 ml of Tbf2 for each falcon tube
- allocate 200 μl of competent Bacterial cells in each of the eppendorf tubes
- instant freeze the eppendorf tubes in liquid nitrogen
- conserve the tubes at -80˚C

PCR

1- Reaction setup
- Add the following component as listed below

- Assemble all reaction components on ice and quickly transfer the reactions to thermocycler preheated to 94˚C and begin thermocycling
Hint: make a premix of the components mentioned without DNA Template and allocate appropriately 12 μL in each tube.

2- Thermocycling conditions for a routine PCR

Mini prep protocol

1-Material preparation
- Centrifuge Lyse bleu and RNase samples
- Add 20 μL of Lyse to 20 ml of Buffer P1
- Add 200 μL of RNase to previous mix
This mix is considered as buffer P1
2- Culture preparation
- Centrifuge for 2 minutes at 13000 rpm bacterial culture, remove supernatant
- Re-suspend pellet with 250 μL of P1 buffer, pipette up-down
- Add 250 μL of P2 buffer mix thoroughly by inverting the tubes 4-6 times
- Add 350 μL of N3 buffer mix immediately and thoroughly by inverting the tubes 4-6 times
- Centrifuge at 9000 rpm for 10 minutes
3- DNA material separation process
- Transfer 800μL of supernatant to the spin column that should be in a tube
- Centrifuge at 9000 rpm for 45 seconds, discard flow through
- Add 0.5 ml of PB buffer
- Centrifuge at 9000 rpm for 45 seconds ,discard flow through
- Add 0.75 ml of PE buffer
- Centrifuge at 9000 rpm for 45 seconds ,discard flow through
- Centrifuge at 9000 rpm for 1 minute , discard collection tube
- Transfer column to a new eppendorf tube
- Add 50 μL of H2O let stand for 1 minute
- Centrifuge at 9000 rpm for 1 minute
- Keep flow through, discard column
No special condition while preparation. After adding P2, mix N3 in less than 5 minutes

Cloning protocol for IDT sequences :

1- Re-suspending gene fragments
- Centrifuge for 5 seconds at 3000g
- Add 20 μL of TE to the tube for a final concentration of 10ng/μL
- Briefly vortex and centrifuge
- Tube can be stored at -20˚C
2- Digest with the restriction endonucleases
- Add the following components

- Incubate for 45 min at 37˚C
- Incubate for 20 min at 80˚C
Gene Fragment and vector digested separately

3- Ligation
- Add the following components in the order listed below

- Centrifuge for 5 sec at 3000g - Incubate at room temperature for 5 minutes
4- Transformation
- Repeat protocol as mentioned previously

Gel and PCR Clean up

1- Adjust DNA binding condition/Excise DNA fragment
- Mix one volume of sample with 2 volumes of buffer NT1 for PCR sample
- Add 200 μL of NT1 buffer for each 100mg of agarose gel
- Incubate sample for 5-10 minutes at 50˚C. vortex if needed to completely dissolve gel
2- Bind DNA
- Place PCR Clean up column into collection tube 2 mL
- Load up to 700 μl of sample with NT1 buffer
- Centrifuge for 30 seconds at 11 000 g
- Discard flow through and place column back
3- Wash silica membrane
- Add 700 μL of NT3 buffer
- Centrifuge for 30 seconds at 11000 g
- Discard flow through and place column back
4- Dry silica membrane
- Centrifuge for 1 minute at 11 000 g
- Discard flow through
5- Elute DNA
- Place column into new 1.5 mL micro centrifuge tube
- Add 15-30 μL of ddH2O
- Incubate at room temperature for 1 minutes
- Centrifuge for 1 minutes at 11 000 g Note don’t discard flow through
5’- Elute DNA > 1000 bp
- Heat ddH2O to 70˚C
- Incubate ddH2O on column for 5 minutes
- Add 20-30μL of ddH2O
- Centrifuge for 1 minute at 30-50 g
- Centrifuge for 1 minute at 11 000 g
- Repeat elution 2 to 3 times for the best result

 E. coli being aerobic, the solution is stored in LB in a Erlenmeyer flask of 5 × volume, so 500 mL and incubate for 1 h.

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Preparation for transformation : In four steps

For the petri dish LB agar, take a bottle of LB agar ( 400mL for 20 boxes ) and put it in the microwave to liquefy. Heat with microwave 300 watt ( no more ) for 19 minutes with unscrewed plug.

Organizational axis

Subjects organisation

Small theoretical session of Sandra on the manipulations:

1. Transformation

Transformation is a technic used to make the DNA "enter" into the competent cell by destabilizing the membrane:

1. Contact, the cells and plasmids (which are recovered in the iGEM kit)
2. Heat shock, more permeable membrane that will make the DNA enter: 45 seconds at 42 ° C
3. Expression, cells are allowed to express the inserted genes (including antibiotic resistance which serve as a test). There are two types of antibiotics: bacteriostatic (ampicillin, which freezes growth), bactericidal (which kills)
4. Spread on antibiotic (petri dish), that will eliminate the bacteria which have not assimilated the gene

2. Verification of contamination

Negative control: 3 antibiotics, if the bacteria resist: contaminated

3. Cloning

We want to integrate an insert, for this, we need the restriction enzymes

4. Biobrick - iGEM

S and X are compatible

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B. Re-culture

In order to have uncontaminated crops (cultures - -) in case the one made yesterday were. C. Biobrick Putting GFP into solution from the iGEM Kit (Item 5: 16K): 10 μL of H2O miliQ (pure without DNases or RNAses) are deposited and incubated for 10 minutes. The 10 μL of DNA is recovered taking care to take the edge of the cuvée.

Opening of the stopper (in the loom) 19min to 300W MAXIMUM and left at 50 ° C in an incubator.

Recovery of antibiotics and dosages. Here are the different products to respect Ampicillin -> 4 μL / mL Ampi [25mg / mL] -> here 320μL Kanamycin -> 5 μL / mL Kana [10mg / mL] -> here 400 μL Tetracycline -> 1 μL / mL Tetra [15mg / mL] -> here at 80μL Chloramphenicol -> 1.6 μL / mL Chloran [30mg / mL] here 128μL

(Re) preparation of competent bacterial cells: Error of the previous day.

Recovery of precision and OD measurements from diluted sample to tenth: OD TG1 = 5.24 OD DH5α = 4.27

A quantity of bacterium is then taken which has been placed in an LB medium and then incubated for approximately 1 hour at 37 ° C. 1.9 mL of TG1 in 100 mL of LB 2.3 mL of DH5α in 100 mL of LB Then measurement of OD TG1 = 0.474 / DH5a = 0.895 (a little high compared to the desired value).

To make competent cells, it is imperative to work at low temperature (4 ° C) and in sterile medium. The buffers thus prepared were kept and the eppendorf tubes must be cold (cold room and ice cube tray). First, 10 min centrifugation at 3500 rpm at 4 ° C. of the two bacterial strains from the culture (OD 0.4-0.6). The pellet should then be gently resuspended in 80 mL of Tbf1 (20 mL in our case because we do not have 200 mL of culture). 3500 rpm centrifugation with cold pendant 5 mn and resuspension of the new pellet in 2 mL of Tbf2. Incubation of 15mn in ice. Manufacture of 220μL aliquots in annotated eppendorfs STERILE and bath tubes in liquid nitrogen to freeze them quickly. Storage at -80 ° C

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WEEK 2 : 12/06 → 16/06

DAY 6: 13/06/17

BROUILLON

Mini préparation.

Avant de commencer Sur centrifugeuse 20μL de Lyse bleu et 200μL de Rnase dans les tubes initiaux. Sur l'ajoute 20μL de Lyse blue au buffer P1. (Quantité initiale du tampon P1 = 20 ml). Puis sur rajoute 200μL de Rnase 1 au "tampon P1 + Lyse bleu". La solution est conservée dans la glace. L'éthanol (96-100%) "ajouter au tampon PE avant utilisation" était déjà ajouté. Nous avons donc utilisé le buffer PE.

Debut de la mini préparation. Sur rempli nos tubes ependorfs avec les bactéries conservées dans nos entrées. Dans l'ordre -Tube 1: C - -Tube 2: C + -Tube 3: TD1 -Tube 4: TD2 -Tube 5: TD5 -Tube 6: TD6 -Tube 7: RBS -Tube 8: ADN -Tube 9: Eprc -Tube 10: Epr1 -Tube 11: MB -Tube 12: KR -Tube 13: Gfp

Sur centrifugeuse à 13 000 Rpm, puis sur prélève le surnageant et sur le remplissage des tubes et appendorf avec les mêmes compositions. On centrifuge une deuxième fois puis sur prélève à nouveau le surnageant. Une fois le double culot de bactéries débarassé de tout surnageant, sur ajoute 250μL de tampon P1, en haut et en bas pour re-suspendre le culot. Attention sur ne racle pas les bactéries. Ensuite sur ajoute 250μL de Buffer P2. (Pas de up and down pour mélanger ici pour ne pas endommager l'ADN génomique et permettre une bonne extraction de celui-ci) et sur ne dépasse surtout pas les 5min dans ce tampon. (Sur remarque la coloration bleu). Idem pour buffer les 350 μL de Buffer N3 ajouté ensuite. (La coloration bleu disparait et on voit apparaitre un précipité blanc. (Les tubes avec P2 puis N3 sont mélangé au principal en inversant le tube).

WEEK 2

Digestion vector:

We prepared 6 eppendorf tubes, we add in first the buffer ( with the same cone ) and H2O ( with a second cone ). Afterward we add the DNA material for a total mass of 200 ng in each tube. When the initial preparation was done, a short spin was necessary. We extracted the enzyme carefully with a small up action on the edge of the enzymes tubes. Then carefully inject the enzymes in the eppendorf tubes then mix with an up down action. Then we incubate for half an hour at 37° degrees celsius. Afterward, a heat shock is applied at 80°C for 20 minutes to inactivate all enzyme activity.

Tableau / code

Digestion plasmid:

We had 4 tubes of plasmid with each a specific antibiotic resistant gene insert. With a total of volume of 45µL in each. We added 10µL of NE buffer, 41µL of H2O at the end we added our enzymes. ( 2µL for ECORI HF and 2µL for PST1 ). Then we incubate for half an hour at 37° degrees celsius. Afterward, a heat shock is applied at 80°C for 20 minutes to inactivate all enzyme activity.

Ligation:

We prepared 3 tubes for ligation for which we added 2 µL of T4 DNA ligase buffer 10x. Then 11µL of ddH2O, afterward the DNA material ( 2µL upstream 2µL downstream 2µL destination plasmid ). Finally we add T4 DNA ligase and let it incubate at room temperature for 10mn. When the ligation was done, we transformed 5µL of ligation product into 150µL of bacterial cell. ( Previously mentioned ).

Electrophoresis:

As mentioned previously, for each ligation product we added in an eppendorf tube 5µL of DNA 10µL of H2O, 3µL of loading purple dye. In the gel, the order was EPRI cut up, EPRC cut up, EPRC cut down, DNA ladder, RBS cut down, GFP cut down, M13 cut up. We let it migrate for than 30 minutes, at 135 volt which was the limit of the migration, we should have put it for only 25. All fragment under 200 Pb weren't displayed because it had surpassed the migration cap. For EPRI, EPRC cut up and down, we should have seen one bar for the linear plasmid and not for the digested factors.

PHOTO ELECTRO

We also re-did a transformation for RBS which failed previously. This time we used new competent cells and we had a positive result because there were multiple colonies on the gel.

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Préparation of DNA and oligonucleotids:

We recieved 6 tubes of DNA. (P3RsS, p3VcC, p3VcF, p3VcV, p3Xc and p3Xfa). All these tubes are at 1000ng. To be able to prepare them at 50ng / ul we followed the iDT protocol. Centrifuge the tubes 2 min at 3000rpm Addition of 20ul of H20 Vortex Incubation of 20 minutes at 50 ° C Vortex Short spin centrifugation DNA is stored in the freezer -20 ° C

For oligonucleotides: We received the 4 oligonucleotides. To prepare them we followed the recommendations of the producer (Eurogentec). Centrifugation 2 min at 3000rpm Addition of Water to reach a concentration of 100uM Transformation protocol as mentioned before for TD4, Cas9 and dCas9 with the new competent cells prepared on 13/06.

Preparation of competent bacterial cells protocol as mentioned before was done for 100mL of bacterial cells at a OD of 0.478

Resuspension of genomic material from the iGEM plates #4 at the positions as mentioned : 2B, 2D, 2F, 2H, 2G, 2L, 4B, 4D, 4F, 6B, 6D, 6F, 6H, 8B, 8D, 10B, 12B, 12D, 14B This genomic material is a plasmid that codes for a RFP protein with different antibiotic resistances.

A transformation protocol to test competency of bacterial cells was done for the ligation product that was done yesterday. This test was done with the competent bacterial cells from 13/06 and supercompetent cells for a different lab and normal competent cells from another lab. To test the transformation capability we used GFP from our miniprep for a volume of 2 µL.

All transformations were spread out on petri dishes with the specific antibiotic.

We recieved

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Casting of petri dishes (10 amp / 10 kana / 20 chloramphenicol)

PCR checking: Colonies from RBS, TD4, 2B, 2D, 2F, 2H, 2G, 2L, 4B, 4D, 4F, 6B, 6D, 6F, 6H, 8B, 8D, 10B, 12B, 12D and 14B. Box of 15/06 except RBS 14/06. We forgot to put the antibiotics immediately but added them few hours later.

Transformation: Dna from the kit (cas9, 8B and 14B)

Digestion-ligation: The results being negative for the first attempt, we resumed the digestion-ligation of 14/06.

SLIC: We must insert the iDT sequences into the plasmids, which is why it was decided to make a SLIC so that these fragments are inserted into a plasmid with resistance to chloramphenicol (pSB1C3). The DNA selected for SLIC is p3VcV, p3VcF, p3Xc, p3RsS, p3Xfa and pVcC. The ratio used is 3 insert for 1 vector. The number of inserts was calculated using the NEBioCalculator software. The DNA was then transformed into DH5α cells.

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WEEK 3 : 19/06 → 23/06

The PCR done on the 6/16 wasn't used because the plugs were not closed. For that reason, our tubes were dry. Despite a rehydratation Malgré une tentative de réhydratation, nous n'avons pas pu récupérer des résultats satisfaisants lors de notre électrophorèse. Nous avons donc re effectué notre PCR. En plus de cela, nos repiquages sur boîtes n'étaient pas vérifiable pour le 6F ( Kanamycine ). -> Repiquage de 6F en Kana. Ensuite : Incubation 37° overnight.

Infection par phage: -100mL de bactérie Ecoli F+ de la préculture overnight, et ajout de 1µL de phage à 10^5 pfu/µL -Incubation 50mn ( au lieu de 20 ) à room temperature. -Depose sur boite de 100µL sur boîte LB agar sans antibio. Témoin bactérie Ecoli F+ sans phage. -Incubation overnight à 37°

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La PCR réalisé le 6/16/2017 n'a pas pu servir car les bouchons n'avaient pas été fermés dans la machine. Nous avions donc des échantillons sec. Malgré une tentative de réhydratation, nous n'avons pas pu récupérer des résultats satisfaisants lors de notre électrophorèse. Nous avons donc re effectué notre PCR. En plus de cela, nos repiquages sur boîtes n'étaient pas vérifiable pour le 6F ( Kanamycine ). -> Repiquage de 6F en Kana. Ensuite : Incubation 37° overnight.

Infection par phage: -100mL de bactérie Ecoli F+ de la préculture overnight, et ajout de 1µL de phage à 10^5 pfu/µL -Incubation 50mn ( au lieu de 20 ) à room temperature. -Depose sur boite de 100µL sur boîte LB agar sans antibio. Témoin bactérie Ecoli F+ sans phage. -Incubation overnight à 37°

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RBS, TD4, 2B, 2D, 2F, 2H, 2G, 2L, 4B, 4D, 4F, 6B, 6D, 6F, 6H, 8B, 8D, 10B, 12B, 12D and 14B miniprep. Verification PCR to verify the 3 ligations on Kana that did not give colonies, to know if it was ligation or transformation that was not effective. Electrophoresis.

A) Preparation of the DNA: We received 3 tubes. (P3Xfu, MultiTag and Xpr). All these tubes are at 1000ng. To be able to prepare them at 50ng / ul we followed the iDT protocol. The DNA is stored in the freezer -20 ° C.

B) Infection by phage: Since the infection of 17/06 did not work, we start again with a concentration 1000 times higher. 100 mL of Ecoli F + bacteria from the preculture, and addition of 1 μL phage to 10 ^ 8 pfu / μL

C) Transformation of phage DNA Since the infection of 17/06 did not work, we check that it is not DNA. Transformation of 0.5 μl of phage DNA (10 μl pfu / ml) into 50 μL of DH5α.

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Amplification protocol for cas9 dcas9 8B and 14B. We were running out of DNA material for these 4. So an amplification was doe. We added in 4 tubes, 50 µL of reaction compounds, composed of Q5 High fidelity 2X Master mix, forward and reverse primer, template DNA and ddH2O. The cycling routine for PCR was 98°C for initial denaturation, then 30 cycles at 98°C, 60, 72, afterward a final extension for 2mn at 72°C. Then hold at 6°C. We verified our PCR, with an electrophoresis. We added 5µL of DNA template. 10µL of H2O, 3µL of loading purple dye. The electrophoresis was set for 25 minutes at 135 volts. The gel was put in gel red for 10 minutes, then revealed using UV. The result are shown as is:

PHOTO

For cas9 / dcas9 we had a negative result, no bounds at 4000 to 5000 pb. And a positive result for 8B and 14B a distinct bound at a thousand pb. A PCR clean up was then done to recuperate the DNA material. We followed the recommendation of the supplier. To our 45µL of DNA, we added 90µL of NT1 buffer. We placed the solution on the column then added 700µL of NT1 buffer. 30 seconds centri at 11 000g. Then 700µL of NT3, centrifuge then discard. A 1mn centri to dry out the column. To recuperate the DNA, 20µL of ddH2O at 70°C was let to incubate on the column for 5mn. Afterward, 20µL of normal H2O. A centri for 1mn at 30 to 50g then another at 11kg. We repeated two times and kept the flow through. We measured the DNA concentration using nanodrop the values mentioned as is.

Cas9 -> 4,48ng/µL dcas9 -> 4,24ng/µL 8B -> 9,54ng/µL 14B -> 11,88ng/µL

We also did a PCR for Cas9 and dCas9. We have been trying to PCR this two several time in order to be able to use them for mini-prep. ( See PCR protocol ). Once the PCR was done, we injected our samples on the gel and began electrophoresis. Once the electrophoresis was done we putted the gel in red for 15mn and then studied it under UV. Unfortunately no result could be observed on the gel. We decide to stop trying to use Cas9 and dCas9 for the moment.

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Lisa and jeremy made 19 petri dishes with Kanamycin.

Transplantation of 4 plates of phage from 20/01 in a Kanamicin Petri Dish.

The competence test reveled that our super competent bacteria are approximately three times more competent than Sandra's one, and approximately 1,5 times more competent than Le test de compétence a révélé que nos bactéries super compétente sont en moyenne 3 fois plus compétentes que les compétentes de Sandra, et environ 1.5 fois plus compétentes que les super compétentes de l’équipe voisine.

Le test de fonctionnement de la ligase montre que la ligase n’est pas efficace, et a perdu son activité. Cela est très probablement dû à une mauvaise conservation de celle-ci et aux nombreux chocs thermique qu’elle a subis.

Then we made new competent bacteria. From the overnight bacteria, we made new competent bacteria. Our first dilution resulted in a OD of 0.54 ( 5.4 because the DO was post dilution ). We then added our culture in 200µL of LB in order to get a OD of 0.1. To do that we had to dilute 1/50: Solution: -200mL of LB -4mL of culture. The OD measured post 1h incubation was 0.7 The rest was the same as in the protocol. Putted in Petri dish overnight.

We had to check RBS because of last night. RBS vérification by PCR. ( RBS used : 681 ng/µL ) Height awaited ~ 3000 pd on mini prep (0.2 µL of DNA ). The results from PCR were putted in an electrophoresis and revealed that we failed. 5 stripes when we awaited only 1 ). We remade digestion because we had suspicious results on our electrophoresis. Remaking digestions M13 Making digestion of “red” vector ( RFP ) in order to get linearized plasmids cutted at EP extremity.

Check digestion in gel of EprC / EprI. Digestion of RFP vectors - Purify on agarose gel - CmR et KmR (respectively C3 and K3 - Digest maximum quantity of vectors ~1000ng with 2x1µL of enzymes dans un volume total de 50µl. Let it migrate a long time (40/45 min) to have a good separation. Une fois que la migration est terminée, on révèle au gel red et aux UV. En respectant les règle de sécurités, nous avons à l’aide d’une lame découpé les bandes gel contenant les plasmides linéaires, que nous avons mis dans des tubes eppendorf. Après avoir pesé nos tubes, pour connaître la masse de gel dans chacun de ceux-ci (K3: 0.4g ; C3: 0.3g), nous avons effectué un gel clean-up en suivant le protocol. A l’issue du gel clean-up, nous avons récupéré 60µl de solution de plasmide linéaire, très peu concentré ( environ 2-5 ng/µl).

We made an amplification of Cas9 dCas9 with Pfu turbo which was long graciously with Eric Durand. This protocol consisted of adding 1mL of our DNA template, to 2.5 mL of primers and 1mL of Dntp 5mL of DMSO and 5mL of PFU mix and to add a sufficient quantity of H2O to complete to 50mL. The program for the PCR was set by Eric: 70°C for lengthening, 68° for denaturation, and 58° for oligomerization. The PCR thermocycling took 4h and 44mn.

We did the digestion of 50 ng of iDT DNA sequences p3 VCV, p3 VCC, p3VcF, p3Xfa ,p3Xfu, p3RsS, p3Xc, MultiTag, Xpr in 15µL. We ligate those fragment in pSB1C3 and we did the transformation in DH5Alpha. For the ligation we put 6µL of insert and 2µL of plasmid with 1µL of T4 DNA ligase Then we made an amplification PCR of idt sequences (same one). We used 10ng of DNA for the amplification.

On the petri dish with GM1 E.coli infected with M13 phage, coming from the 20/06, we seen multiple infection site. Thus we transferred 4 of them to a kanamycin petri dish.

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The M13 infected E.coli Has grown so we pick some colonies in order to make 4 starters. Some of the digestion and ligation of iDT sequence work (p3 VCC, p3 VcV and Xpr) so we make starters. In the temoin plane none colonies were visible.

Week 4

Transplanting GFP, killer red, EprC, EprI, M13. Preparation of starter. Jérémy makes 18 petri dishes Chloram

We received the iDT sequence of p3_Ng and p3_RSM so we eluate them in water (20µL) at the concentration of 5ng/µL.

Camille and Thibault used the starters from MG1 cultures with M13KO7 phages in it. Then, we did mini preps with 4 extracts from different spots on the petri box. GR for “grande”, TP for “tres petit” p1 and p2.

after the minipreps, we measured DNA concentration with nanodrops, our results were : tp :248 ng/µL, GR : 51 ng/µL p1 : 48 ng/µL p2 : 154 ng/µL RBS : 338 ng/µL

In order to verify that we purify M13KO7 genome we digest the phagemid with BamHI and PstI-HF. We digest 100ng of plasmid in 20µL. Thus we observed 2 bands one at 5859 pb and the other at 2830 pb.

With theses concentrations, we made a electrophoresis gel, here are the results :

From left to right : 1 - 2 - 3 - Ladder - Eprc up E/S - M13 up ES - GFP down XP - Epr1 down XP - KillerRed down XP - ladder - 8B - 14B

Digestion:

Vector RFP Ampicillin resistant in order to pick up a linear vector digested on one side by EcorI and on the other side by PftI. The goal is to be able to construct afterward. To do so, we took RFP vector including a gene resistant to ampicillin. We prepared 100µL -> 6µL de plasmide RFP Amp R ( to get approximately 2000 ng ) -> 2µL EcoRI HF -> 2µL Pst I -> 80 H2O -> 10µL of buffer 2.1

30 min 37°C then 20 min 80°C.

We made it migrate on an 1% agarose gel. The gel must be long enough to allow a good separation to see a good split between stripes. Then we revealed it with the usual gel RED+UV protocol. As our stripes were revealed, we cut the corresponding stripes and transfer them in two separate eppendorf tubes. The two tubes weight were 280 and 120 mg respectively. Then we followed the gel clean up protocol : we add 200µL of NT1 to 100 mg of gel, the tube was heated and vortex to dilute the gel in the liquid, after liquification of the gel we poured the solution on a column and let it soak of 5 minute a centrifuge at 10000 g was done to flow through the mix and afterward discarded. 700µL of NT3 was then added, a centrifuge was put in use to pass through NT3 and discarded also. To dry out the column a 1 minute centrifuge was done. To recuperate the DNA we let the column soak for 5 minute with hot water. We eluted the DNA with 30µL of water after centrifuge at 10000g the flow through was kept in a newly added 1.5 mL eppendorf tube. We did the same thing a second time with 20µL of hot water. We measured the DNA concentration using a nanodrop. The concentration were 10 and 12 ng/µL respectively. We finished with an electrophoresis to verify if we actually recuperated the DNA, the extra were kept at -20 degrees. digestion we digested KR, EprC, M13, EprI, and GFP EprC and M13 were digested using the enzymes EcoRi and SpeI KR, EprI and GFP were digested using XbaI and PstI 500 ng of DNA template were digested following the protocol to the letter. to verify an electrophoresis was done, the results are shown and described as follow.

For the binding : Up stream : EprC ; M13 ( EcoRI and Spe I ) Down stream KR ; EprI ; GFP ( XbaI and PstI )

EprC -> 11µL to get 500ng ( 33µL of H2O ) cutsmart M13 -> 7,6µL to get 500ng ( 36,5 of H2O )

KR -> 3,8µL to get 500ng ( 40µL of H2O ) EprI -> 8,5 µL to get 500ng ( 35,5µL of H2O ) GFP -> 5,7 µL to get 500ng ( 38,5 µL of H2O ).

_____________________ WEEK 4 : 26/06 → 30/06

Day 16

Camille and Thibault made minipreps and nanodrops of : Xpr : 4ng/mL -- p3VcV 8ng/mL -- M13 15ng/mL -- EprI 14ng/mL -- GFP 25ng/mL -- KR 57ng/mL -- EprC 18ng/mL.

Jérémy makes 43 competent cell DH5Alpha

Camille did the digestion and ligation of p3Ng and p3RsM in pSB1C3 at a concentration of 10uM. She transform those fragment in DH5a (2µL of dna for 50µL of competent cell). Other ligation where transform by Thibault (6µL of dna for 50µL of competent cell), the fragment were : p3Xc p3Xfu, p3Xfa, MultiT, p3RsS, p3VcF.

Quick changes were made. We wanted to put a mutation on M13KO7 in order to insert a punctual mutation. Thus this mutation will permit the apparition of a restriction site. The Quick Change is a PCR ON. We have two slot : 1 → M13KO7Tp and 2 → M13KO7p2. Firstly, we used the primers Bdw and Bup to insert the BspI.

Quick change : Plasmid : 1µL dNTP(10mM) : 6µL DMSO : 1µL Primer 1 : 2,5 µL Primer 2 : 2,5 µL Tampon pfu turbo : 5 µL pfu Turbo : 1 µL H2O qsp 50 µL

I put primers with a concentration of 100mM. This might be to concentrate….

26/06/17

Camille and Thibault did minipreps and nanodrops of : Xpr : 4ng/mL -- p3VcV 8ng/mL -- M13 15ng/mL -- EprI 14ng/mL -- GFP 25ng/mL -- KR 57ng/mL -- EprC 18ng/mL.

Jérémy makes 43 competent cell DH5Alpha

Camille did the digestion and ligation of p3Ng and p3RsM in pSB1C3 at a concentration of 10uM. She transform those fragment in DH5a (2µL of dna for 50µL of competent cell). Other ligation where transform by Thibault (6µL of dna for 50µL of competent cell), the fragment were : p3Xc p3Xfu, p3Xfa, MultiT, p3RsS, p3VcF.

A quick change were made. We wanted to put a mutation on M13KO7 in order to insert a punctual mutation. Thus this mutation will permit the apparition of a restriction site. The Quick Change is a PCR ON. We have two slot : 1 → M13KO7Tp and 2 → M13KO7p2. Firstly, we used the primers Bdw and Bup to insert the BspI.

Transformation of: -controls : 2uL RFP Amp vector + 100 uL of DH5a 2uL RFP Amp vector cut E/P + 100 uL of DH5a 2uL RFP Amp vector cut E/P and ligate

Quick change : Plasmid : 1µL dNTP(10mM) : 6µL DMSO : 1µL Primer 1 : 2,5 µL Primer 2 : 2,5 µL Tampon pfu turbo : 5 µL pfu Turbo : 1 µL H2O qsp 50 µL

I put primers with a concentration of 100mM. This might be to concentrate….

Quick change Program

98°C 5 min

95°C 1 min 29 cycles 55°C 1 min 68°C 20 min (2 min per kb) 68°C 20 min

16°C hold

Measurements:

Calibrage of TECAN:

First we need to calibrate the TECAN using LUDOX-S40 as a point reference to obtain a ratiometric conversion factor to transform absorbance data into a standard OD600 measurement. We added 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette). Then we added 100 μl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette). We measured absorbance 600 nm of all samples in all standard measurement modes in instrument. We recorded the data into Excel.

                        LUDOX 100%    H2O	

Replicate 1 0.1 0.0896 Replicate 2 0.099 0.0895 Replicate 3 0.1007 0.0915 Replicate 4 0.1028 0.0933

With C+, C-, TD1, TD2, TD3, TD4, TD5, TD6, we made starters over night.

� 27/06/17

Quick change M13KO7

Since the PCR is over we have two slot : 1 → M13KO7Tp and 2 → M13KO7p2. In order to remove methylated DNA we put in each 1µL of DpnI enzyme and we incubate 4h at 37°C. After that, we transform 5µL of the mix in 50µL of super competent DH5a.

A miniprep of VcV, VcC and Xpr has been done. The protocol has been followed but the centrifugation has been done at 9000 rpm and not 13 000. In the end, the nanodrop results were : VcV : 27,23 ng/µL, Vcc : 52,80 ng/µL, Xpr : 23,63 ng/µL

D/L of Deps (10uM), p3SS (5uM) and p3Ec (10uM) with EcoRI and PstI in 2.1 tampon. Ligation with pSBIC3 plasmid.

Transformation : In DH5a competent cell with 5µL of digestion with 50µL of cell.

Hussein and Thibault did : Digestion - Electrophoresis - Nanodrop - Gel extract from the 2kb strip - PCR cleanup

Digested with EP → nanodrop results from minipreps : psb1c3 : 249,7 ng/µL psb3c5 135,2 ng/µL psb4c5 : 585,2 ng/µL

After PCR cleanup :

psb3c5 : 8,03 ng/µL psb1c3 : 12ng/µL psb4c5 : 15,66 ng/µL

Soraya and Jérémy did digestion/ligation and transformation :

- 3 controls : Control AmpR, check plasmids and transformation => cellular colony Control Plasmid AmpR cut, check digestion => no cellular colony Control Plasmid AmpR cut/lig => cellular colony

- 3 ligations : Lig EprC-KR => no cellular colony Lig M13-EprI => no cellular colony Lig EprC-GFP => no cellular colony

Measurements: Fluorescein fluorescence standard curve We prepared a dilution series of fluorescein in 4 replicates and measure the fluorescence in a 96 well plate in a plate reader in order to generate a standard curve of fluorescence for fluorescein concentration.

We prepared 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. Then, we diluted the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM (500μL of 2x fluorescein in 500 μL 1x PBS will make 1 mL of 50 μM (1x) fluorescein solution.)

28/06/17

Preparing ~20 pétri dish of ampicillin

Vérification PCR from RBS and VcF starters

Jérémy did the transformation of M13 with TG1 competent cells Jérémy did the transformation of of RPF puc19 with TG1 competent cells Thibault did verification PCR and minipreps from starters of RBS and VcF. Here are the results : from left to right : RBS / empty / ladder / empty / VcF RBS was expected around 300 kb and VcF 1098 kb That’s what we have (+300 for VcF) For the minipreps, the nanodrops results are : RBS : 256 ng/µL and 25 ng/µL for VcF

6 starters where done of p3_VcV, Xpr, p3_VcF two of each. We’ve got colonies from the d/l of 27/06 Deps1 Deps2, p3_Ng, p3SS, p3_Xc, p3Ec et p3MT.

Camille did the transformation, of p3_Xfa, p3_Xfu, p3_RsS et p3_RsM with 5uL of ligation and 50uL DH5a.

M13-EprC not good > 1000pb to electro PCR M13-EprI seems to be good find the miniprep

29/06/17 we did the mini prep for M13 ( 9,35 ),KR (169 ), RBS (291) and GFP (71), P3VCF (117), EprC (55), Xpr (72) P3Xc (62), Ligation M13-EprC (19), EprI (65), P3VcV (328) (ng/microlitre)

Thibault repicked the petri dishes of : psb1c3 / psb1k3 / psb3c5 / psb3t5 / psb4c5 / psb2k3 / psb3k3 / AC3 / psb4k5 Repicked it on pétri dishes and made starters that will incubate ON for futures minipreps

PCR of verification from iDT sequence ligation onto pSB1C3 and repiquage of them. Séquences vérifiées: _p3Ng : 3 colonies _Deps: 2 colonies

_p3VcC: 3 colonies _p3Ec :3 colonies

_p3Xc: 3colonies _p3VcF: colonies

_Xpr: 2 colonies _p3Ss: 1 colonies

_p3VcV: 3 colonies

The electrophorèse shows that p3Ec 1 and 2, and p3Xc1, p3Xc2 and p3Xc3, might be good.

We’ve got p3Pa from iDT, and we add water until we reach the concentration of 50ng/µl.

Digestion of p3_Pa with SpeI and PstI. 3uL p3_Pa (150ng) 5uL 2.1 Buffer 0,5uL PstI 0,5uL SpeI H2O qsp 150uL 30 min at 37°C then 20 min at 80°C This is a error from us because we should cut p3_pa w/ EcoRI rather than SpeI.

p3Xfu, p3Xfa,p3RsS, p3RsM and p3Pa were ligate onto pSB1C3 (6uL of digestion and 2uL of linearised and cut plasmid).

Deps, p3VcF, Xpr, p3Ss, p3VcC p3Xfu, p3Xfa,p3RsS and p3RsM were transformed.

5 starter where done: p3Ec (1); p3Ec (2); p3Xc (1); p3Xc(2); p3Xc (3).

Repiquage and PCR of p3_VcV. The electrophoresis shows us that those colonies didn’t have the right fragment.

� 30/06/17

PCR Amplification of all expected M13 on the gel Verification PCR of M13 we used yesterday

And miniprep of p3E1, p3E2, p3Xc1, p3Xc2, p3Xc3.

Results from nanodrop : psb2k3 : 31 ng/µL psb3k3 : 71: ng/µL psb4k5 : 72 ng/µL psb3c5 : 107 ng/µL psb1k3 : 96 ng/µL psb3T5 : 63 ng/µL psb4c5 : 47 ng/µL AC3 : 62 ng/µL psb1c3 : 250 ng/µL p3Ec1 : 41 ng/µL p3Ec2 : 48 ng/µL p3Xc1 : 16 ng/µL p3Xc2 : 49 ng/µL p3Xc2 : 28 ng/µL

The concentration of p3Xc1 et p3Xc2 is too low for the sequencing, thus we make 2 staters of each ON.

Transformation of M13, GFP, KR and RBS.

Transformation des biobricks measurements TD1, TD2, TD3, TD4, TD5, TD6, C+, C-, et de 2B. PCR d’amplification des p3.

Miniprep of M13, concentration 42,47 ng/uL.

Repiquage et mise en culture de 8 starters: 6 M13KO7 mutB Tp et 2 M13KO7 mutB p2.

PCR d’amplification of sequence IDT p3Vcc, p3 vcf, Mtag, p3 Pa, p3 Rsm, Xpr, p3Xfu, p3 Xca, p3 Ng, p3 Vcv, p3 Ec, p3SS, p3 Rss. Ensuite, nous avons réalisé une électrophorèse pour vérifier les fragments.

Température d’hybridation 55°C (30secondes) Durée d’extention 45 secondes 30 cycles Température de dénaturation 98°C Q5 master mix x2

Digestion of p3_Pa with EcoRI and PstI. 3uL p3_Pa (150ng) 5uL 2.1 Buffer 0,5uL EcoRI 0,5uL SpeI H2O qsp 150uL 30 min at 37°C then 20min at 80°C

Ligation of p3_Pa and p3_VcF with pSB1C3 For p3_Pa 2uL of digestion and for p3_VcF 6uL of DNA. For both 2uL of Buffer and 1uL of T4 DNA ligase with H20 qsp 50uL.

Transformation of p3_Pa and p3_VcF in DH5a (5uL of DNA w/ 50 of cells).

PCR amplification and clean up of all p3 iDT sequence amplified with Q5 enzymes. p3Ec = 54 ng/uL, Xpr = 28 ng/uL, p3Ng = 89 ng/uL, p3Pa = 72 ng/uL, p3VcC = 93 ng/uL, p3Xc = 103 ng/uL, p3RsM = 134 ng/uL, MT = 33 ng/uL, p3SS = 29ng/uL, p3_Xfa = 86ng/uL, p3RsS = 98ng/uL, p3Xfu = 98 ng/uL, p3 VcF = 96ng/uL et p3VcV = 64ng/uL.

Minipreps of all the psb1A10/psb1AX3/psb4A5/psb1A3/psb1AX3/psb6A1/psb1A3 And miniprep of p3Xc1, p3Xc3.

Results from nanodrop : psb1A10 : 157 ng/µL psb1AX3 : 85 ng/µL psb4A5 : 89 ng/µL psb1A3 : 48 ng/µL psb1AX3 : 68 ng/µL psb6A1 : 277 ng/µL psb1A3 : 308 ng/µL p3Xc1 : 133 ng/µL and p3Xc2 : 108 ng/µL.520

Digestion and gel extract of GFP, KR and M13ori. Result from nanodrop : pSB1C3 (1) E/S = 7ng/uL, pSB1C3 (2) E/S = 8ng/uL, pSB1C3 (1) X/P = 10ng/uL, pSB1C3 (2) X/P = 13ng/uL, pSB1C3 (3) X/P = 9ng/uL, pSB1C3 (4) X/P = 9ng/uL, M13ori (1) E/S = 4ng/uL, M13ori (2) E/S = 5ng/uL, KR(1) X/P = 5ng/uL, KR(2) X/P = 13ng/uL, GFP(1) X/P = 7ng/uL and GFP(2) X/P = 7ng/uL

Transformation de la biobrick Contrôle positif. Gel interpretation for M13 : 800~1000 pb, M13 is 529 + 300 = 529 ( the results are ok ) Soraya manips : Digestion-ligation of EprC, EprI and RBS are little sequences, respectively 55, 61 and 15 Bp. This explains why it is so hard to bind them with other vectors. ( They are lost in the huge mix of other strands ). Ori M13 -EprC / EprC-Kr | EprC-GFP Ori M13 -EprI / EprI-RBS / RBS-KR EprI-RBS / RBS-GFP

Thibault : Elaboration of the “Phages In Soil protocol”

_____________________________________________________

WEEK EDIT

30/06/17 ~Again and again and agaaaiiiinn!~ EprC, EprI and RBS are short sequences (respectively 55pb, 61pb and 15pb), which can explain why it is so hard to bind them with other strand into a vector. That is why we chose to try another protocol, that consist in cut the original vector of those short sequences and directly insert in the second sequence that is longer. -For the upstream part : (Ori M13, 529pb) We want to cut about 1500ng of OriM13 plasmid, we have the miniprep from the 13/06/17, at 66ng/µL. So we have to use 22,7 µL of this miniprep by EcoRI and SpeI. -For the downstream parts : (Killer Red(KR), 723pb; GFP, 711pb) In the same way as before we want to cut about 1500ng of each plasmid: -For KR, we have the miniprep from the 13/06/17 at 130ng/µL, so we have to use 11,5µL of this miniprep. - For GFP, we have the miniprep from the 13/06/17 at 88ng/µL, so we have to use 17µL of this miniprep. Each one will be cut by XbaI and PstI. Restriction : (Total volume = 100 µL)

Gel extract : Cut the gel as closely as possible to each band, make a Gel-Clean up (see protocols). Restriction and gel extract of GFP, KR and M13ori. Result from nanodrop :

==> 3h at room temperature.

04/07/17:

- Transformation of the cloning of the day before, into competent DH5α ( 3 µL of ligation mix with 50 µL of bacteria, on chloramphénicol) . (Help Camille to make SLIC during the rest of the day) -Preparation of more linear Psb1C3, using Psb1C3 vector containing RFP (transplanting of the 29/06/17). Launch a starter.

05/07/17:

Colonies on Petri dishes are too small to transplant them and make a PCR, so we just made the transplant and let them grow up. (1,2 and 3 are EprC-GFP colonies 1,2 and 3; 11 to the end are EprC-KR).

Digestion and clean-up: In order to obtain more linear Psb1C3, we previously launch two big starters of transformed bacteria with RFP into Psb1C3 vector. Then we made minipreps which had very low concentrations. So we used a device which centrifuge at hot temperature to have better concentrations to simplify the digestion and gel extract.

06/07/17:

-PCR of verification on colonies, for the ligations EprC-GFP and EprC-KR.

PHOTO

Only the assembly EprC-KR succeed. ==> launch a starter. RBS failed again.

Digestion of 2000ng of Psb1C3 containing RFP. We never succeeded cloning EprI and RBS so we decided to use LacI-RBS instead of these biobricks.

07/07/17:

PHOTO Electrophoresis of what supposed to be Psb1C3-RFP cut E/P. (The size are wrong, this is not Psb1C3) We remade it from the new miniprep.

-Miniprep of the assembly EprC-KR. -Cloning: We tried to obtain EprC-GFP, OriM13-EprC-KR, LacI-RBS-KR and LacI-RBS-GFP.

digestion: - EprC-KR : 500ng=> 14.5µL of miniprep 07/07/17 , H2O=29.5 µL (see protocol) - LacI-RBS: 500ng=> 18.5 µL of miniprep 07/07/17 , H2O=25.5 µL (see protocol)

1h at 37°C, then inactivate 20min at 80°C.

TABLEAU

3h at room temperature.

Transformation : 3 µL of ligation mix with 50 µL of bacteria (be careful, EprC-GFP on chloramphenicol, the others on ampicillin).

10/07/17:

- No colony on Amp Petri dishes ... But there is one on Petri dish Chloram ! (EprC-GFP) And when we put it in the MAGIC BOX, we could saw its green light ! (Happy!!!). ==> PCR control and starter.

-Digestion (restriction): 100ng of each.

TABLEAU

1h at 37°C, then inactivate 20min at 80°C.

Ligation:

TABLEAU

3h at room temperature.

Transformation : 3 µL of ligation mix with 50 µL of bacteria, on chloramphenicol.

PHOTO

RBS are not good again... 1: miniprep 23/06 2: miniprep 28/06

But the results of LacI-RBS are right. 3: miniprep 07/07 4: miniprep 09/07 6: directly from the kit.

TABLEAU

==> 1h at 37°C. ==>electrophoresis to control these enzyme (first time I use them).

We can't see anything, probably not enough DNA.

PHOTO

12/07/17:

11/07/17: -We didn't see colonies so we transformed the ligation once more time. -PCR control of the different RBS, and LacI-RBS.

WEEK 5 : 03/07 → 07/07 03/07/17

PCR of verification of the clone coming from the ligation between iDT sequence and pSBIC3. For each ligation we had multiple colonies that we tested. Finally it seems that only a few got the right length : p3Pa1, p3Pa2 p3Pa4, p3Pa6, p3Xfa4, p3RSM2, p3RSM3, p3RSM4, p3RSM6, p3RSM7 and p3RSM8.

As DH5alpha infected with M13KO7mutB are really slow to grow, we transformed 5uL M13KO7mutB with 50uL of TG1 E. coli bactgerium.

03/07/2017 Thibault : Large quantities of helper phage  : 200µL of TG1 cells with a 0,4 DO, added 10µL of M13KO7 helper φ (10^9 pfu/mL). After 30min 37°C growing, spread on pétri dish without antibiotics

Morning after : I made a 5mL starter of TG1 and added a small plaque of the yesterday culture. After 2 hours of shaking at 37°C, I transfered that in a 2L flask with 500mL 2YT and 50µg/mL kanamycin.

The morning after, I spinned for 15min at 10800g, kept the supernatant and added 100mL of PEG/NaCl and made another spin to be able to resuspend the pellet in PBS.

04/07/17

We did the miniprep of all the good ligation between iDT and pSBIC3. p3RsM7 = 116ng/uL, p3Xfa = 92ng/uL, p3RsM2 = 67ng/uL, p3Pa4 = 76ng/uL, p3RsM3 = 75ng/uL, p3RsM4 = 80ng/uL, p3RsM8 = 102ng/uL, p3Pa1 = 104ng/uL, p3Pa2 = 85ng/uL, p3RsM6 = 67ng/uL and p3Pa6 = 103ng/uL

We prepared the petri dishes: 16 of Chloramphenicol and 19 of Kanamycin.

We did a competency test for the DH5α that we prepared earlier.

We did a transformation for strong promoter medium RBS, strong promoter weak RBS, medium promoter and medium RBS. We did a transformation for strong promoter medium RBS RFP, strong promoter weak RBS RFP, medium promoter and medium RBS RFP. We did a transformation for strong promoter medium RBS GFP, strong promoter weak RBS GFP, medium promoter and medium RBS GFP, HisTag double stop, HisTag RFP.

05/07/17

Amplification with PCR of the smallest iDT sequences = MT, Xpr and p3SS. As the elongation times are different between each fragment we choose to amplify only the smallest (between 120 and 150bp) in order to improve the success of the amplification. In each tube we put = 25uL Q5 master Mix 2,5uL of OGC36 2,5uL of OGC37 1uL of DNA 19uL H2O (qsp 50uL)

98°C 30 sec

95°C 15 sec 29 cycles 55°C 30 sec 72°C 10 sec (30 sec per kb) 72°C 2 min

16°C hold

We prepared the petri dishes: 20 of Chloramphenicol.

We did a competency test for the TG1 that we prepared earlier. And we realised a transformation for RBS and Kanamycin resistance.

Sequençing p3Pa1, p3Xfa4, p3RsM3.

PCR clean up of Xpr = 60ng/uL, p3SS = 53ng/uL and MT = 60 ng/uL

06/07/2017 We did a transformation for MT, Deps, Xpr, p3SS, p3Ng, p3Xfu, p3VcC, p3VcV, p3RsS, p3VcF in DH5α with Chloramphenicol antibiotics petri dishes. We put 5μL of DNA for 50μL of bacterial cells.

We digest RFP pSB1C3 in E/P to do a gel extract.

the IDT kit for DEPS present some mutation this was previously observed after transferring the sequence into a plasmid and realising a pcr verification on colony.

to countermeasure this problem a pcr amplification is done with the setting as mentioned in the protocols with the single different where the oligomerization duration was set to a minute and 15 seconds. an electrophoresis verified that the desired band between 1500 and 2000 was actually present to clean the solution from any and all contaminant we did a pcr clean up afterward the concentration was measured with a nanodrop a concentration of 140 ng/μL was retrieved. later this day a SLIC was attempted using a circular plasmid digested with EcoRI-HF and PstI. Deps was digested with the same enzymes as well. a small difference to the original protocol was done when digesting an appropriate quantity of DNA for the vector and insert was present but they weren’t digested separately. this small change gave us a negative result the next day after the transformation with competent cells.

07/07/2017 The IDT kit for DEPS present some mutation this was previously observed after transferring the sequence into a plasmid and realising a PCR verification on colony.

to countermeasure this problem a pcr amplification is done with the setting as mentioned in the protocols with the single different where the oligomerization duration was set to a minute and 15 seconds. an electrophoresis verified that the desired band between 1500 and 2000 was actually present to clean the solution from any and all contaminant we did a pcr clean up afterward the concentration was measured with a nanodrop a concentration of 140 ng/μL was retrieved. later this day a SLIC was attempted using a circular plasmid digested with EcoRI-HF and PstI. Deps was digested with the same enzymes as well. a small difference to the original protocol was done when digesting an appropriate quantity of DNA for the vector and insert was present but they weren’t digested separately. this small change gave us a negative result the next day after the transformation with competent cells.

We digest psb1 rfp vector and then made a gel extract to isolate empty vector rfp.

We made TG1 + phage pétri dish + htop agar to be able to get colonies of infected TG1 ( on kanamycin pétri dishies)

___________________________________

WEEK 5 : 10/07 → 14/07

08/07/2017 with the negative results of the transformation another attempt was launched this time by following the protocol to the letter so we used linearized plasmid and digested DEPS in a separate tube. the construction were as followed 3 slic with deps in three different plasmid psb1C3, psb1A3 and psb1K3. and an standard digestion ligation was done also with the three different plasmid this was done just for fun and no practical outcome was intended but maybe this was done to increase our chances to succeed. the different preparation are as mentioned.

Digestion of deps for ligation 500 ng of Deps 0.5 μL of EcoRI-HF 0.5 μL of PstI 5 μL of buffer 2.1 39 μL of H2O

ligation of Deps AmpR ligation of Deps KanaR ligation of Deps ChloranR 150 ng of digested Deps 150 ng of digested Deps 150 ng of digested Deps 2μL of linearized psb1A3 1μL of linearized psb1K3 2μL of linearized psb1C3 2μL of T4 buffer 2μL of T4 buffer 2μL of T4 buffer 1μL of T4 ligase 1μL of T4 ligase 1μL of T4 ligase 10 μL of H2O 10 μL of H2O 10 μL of H2O

Deps digestion for Slic X2 500 ng of Deps 0.5μL of EcoRI-HF 0.5μL of PstI 5μLof buffer 2.1 9μL of H2O

Slic Deps with AmpR Slic Deps with KanaR Slic Deps with ChloranR 3μL of linearized psb1A3 2μL of linearized psb1k3 3μL of linearized psb1C3 250 ng of Deps 250 ng of Deps 250 ng of Deps 2μL of buffer 2.1 2μL of buffer 2.1 2μL of buffer 2.1 0.5μL of T4 polymerase 0.5μL of T4 polymerase 0.5μL of T4 polymerase 4.5μL of H2O 5.5μL of H2O 4.5μL of H2O

when all is done a transformation was performed.

We made a starter of fresh TG1 that we repicked with the infected TG1. We put the mix in 500mL of 2YT + 2,5 ml of kanamycin and let it ON at 30°C with shaking.

09/07/2017

After a night of wait the petri dishes presented a number of colonies but no fireworks wet a PCR verification on colony was necessary so we followed the protocol and began some starters for tomorrow in the process. the results of the electrophoresis of the PCR were very promising we had the suitable bands between 1500 and 2000. but we can’t be 100% sure if the sequence is intact only after sequencing.

10/07/2017

We made a repiquage of TG1 M13KO7mutB (33 colony).

With the ON flasque at 30°C with shaking we made a 6000 rpm spin for 30min Then we put it on ice for an hour and added 100 ml of PEG/Nacl (22g of PEB 6000 + 16,1g Nacl) Another 6000g spin for an hour.

Added 8 mL PBS and 2 ml PEG and spinned

�11/07/2017

We made starters of TG1 and GM1 for future tests with phages.

We did a transformation of M13KO7mutB in TG1 in a petri dish with Kanamicin antibiotic as well for p3RSS, p3SS, p3VcC, p3Xc, P3Ng, p3Xfa, MT, p3Xpr, p3Xfu, p3Pa, p3VcF in DH5α in petri dishes with Chloramphenicol antibiotics. We put all of these in a 37°C incubator. We also did the transformation of M13KO7mutB two more times and put one in a 30°C incubator and the other one in a 42°C incubator.

12/07/2017

Today we have done a digestion of: PSB1C3, P3xfa, Deps, P3VcF, Xpr, P3RSS, P3xc, P3vcc, P3xfu, P3Ng, P3ss, P3vcv, MT. We have used buffer 2.1, PST1 et ecor1 for enzymes. We wanted a final concentration of 10 ng/µL. For all the vectors we have used 0,5 µL of eco1 and pst1 and 5 µL of 2.1 PSB1C3: Vector 2uL (500ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 50uL

P3xfa: Insert 2uL (200ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

P3vcf: Insert 2uL (200ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

P3rss: Insert 2uL (200ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

Xpr: Insert 6uL (200ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

P3Xc: Insert 2uL (200ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

P3vcc: Insert 3uL (200ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

MT: Insert 2uL (200ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

P3vcv: Insert 3uL (200ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

P3Ng:

Insert 2uL (200ng)

EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

Deps: Insert 2uL (200ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

P3xfu: Insert 2uL (200ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

P3ss : Insert 6uL (200ng) EcoRI 0,5 uL PstI 0,5 uL Buffer 2.1 5uL H2O qsp 20uL

After it we have incubated all of this tubes at 37°C for 2 hour and 20 minutes at 80°C

We did a mini prep of p3RSM which was in PSB1C3. We obtained a concentration of 132.66ng/µL and 127.35ng/µL.

We did a transformation of Deps in 100µL of DH5α. Ligation of iDT sequence w/ pSBIC3. We choose a ratio of 1 vector of 2 insert. P3xfa: Insert 2,5uL Vector 5uL (50ng) T4 DNA ligase 0,1 uL Buffer 1 uL H2O qsp 10uL

P3vcf: Insert 3uL Vector 5uL (50ng) T4 DNA ligase 1 uL Buffer 1 uL H2O qsp 10uL

P3rss: Insert 2uL Vector 5uL (50ng) T4 DNA ligase 1 uL Buffer 1 uL H2O qsp 10uL

Xpr: Insert 0,5uL Vector 5uL (50ng) T4 DNA ligase 1 uL Buffer 1 uL H2O qsp 10uL

P3Xc: Insert 1,5uL Vector 5uL (50ng) T4 DNA ligase 0,1 uL Buffer 1 uL H2O qsp 10uL

P3vcc: Insert 2uL Vector 5uL (50ng) T4 DNA ligase 1 uL Buffer 1 uL H2O qsp 10uL

MT: Insert 0,5uL Vector 5uL (50ng) T4 DNA ligase 1 uL Buffer 1 uL H2O qsp 10uL

P3vcv: Insert 3uL Vector 5uL (50ng) T4 DNA ligase 1 uL Buffer 1 uL H2O qsp 10uL

P3Ng: Insert 3uL Vector 5uL (50ng) T4 DNA ligase 1 uL Buffer 1 uL H2O qsp 10uL

P3xfu: Insert 1,5uL Vector 5uL (50ng) T4 DNA ligase 1 uL Buffer 1 uL H2O qsp 10uL

P3ss: Insert 0,5uL Vector 5uL (50ng) T4 DNA ligase 1 uL Buffer 1 uL H2O qsp 10uL

Ligation 1H and transformation in DH5alpha (5uL of DNA and 50uL of cells), incubation at 37°C ON. We make a negative control (bacteria only) and a positive controle (pB1C3 rfp).

Quick Change: 2 tubes in order to have 2 different test 1uL pfu turbo 6uL dNTP (10uM) 2,5uL PrimerAup 2,5uL PrimerAup 5uL pfu buffer 1uL DMSO 32uL H2O (qsp 50uL)

95°C 2 min

95°C 30 sec 29 cycles 55°C or 58°C 30 sec 68°C 20 min(2 min per kb) 68°C 2 min

16°C hold

Transformation of SuperNova (Bba).

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13/07/17

Phage crossing over test : Yesterday we transformed TG1 with oriM13 and put two petri dishes incubation. We used the culture to make new starters we will use to mix up with phages later.

Quick change : We’ve got the result of the quick change of yesterday (M13KO7mutA1 & 2). We put 1µL of DpnI in each tube and we did a 4h incubation at 37°C. The gel electrophoresis shows us no amplification but Valerie told me to transform anyway. With the advise of Gauthier I put 50µL of the PCR mix w/ 100µL of TG1 cells. 1H of incubation on ice then 45sec in 42°C then 1h at 37°C. Then we put on 4 kanamycine petri dish ON at 37°C for each.

Starters of p3EC and p3RsM for glycerolization

Colonies PCR of SuperNova.

We did a repiquage of New Lig p3Pa4-GFP DH5alpha.

14/07/17

Result of the Quick change : none of the colonies coming from M13KO7mutA2 grown, but we’ve got 13 colonies for M13KO7mutA1. We decided to grow 2 starters for 3 first colonies : M13KO7mutA1.1, M13KO7mutA1.2 and M13KO7mutA1.3.

PCR on colonies (vérification of the ligation between pSB1C3 and iDT sequences). We choose to only verify 4 colonies per petri dish.

p3VcC1, p3VcC2, p3VcC3, p3VcC4, p3RsS1, p3RsS2, p3RsS3, p3RsS4, p3Xc1, p3Xc2, p3Xc3, p3Xc4, Xpr1, Xpr2 , Xpr3, Xpr4, p3SS1, p3SS2, p3SS3, p3SS4, p3VcF1, p3VcF2, p3VcF3, p3VcF4, p3Xfu1, p3Xfu2, p3Xfu3, p3Xfu4, p3Ng1, p3Ng2, p3Ng3, p3Ng4, p3Xfa1, p3Xfa2, p3Xfa3, p3Xfa4, MT1, MT2, MT3, MT4, p3VcV1, p3VcV2, p3VcV3, p3VcV4, Deps1, Deps2, Deps3, Deps4.

But none of the colonies were good.

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WEEK 7 : 17/07 → 21/07

17/07/17

Cultures from infection grew, results : TG1+ phages : lysis + irregular growth (I’ll make another one and a strike control to make sure it’s not a TG1 normal growth. Negative control and positive are ok GM1 + phages grew correctly, less than the GM1 (due to the phage infection).

PCR Clean up pa0744 / pa0745 / thioesterase (quorum sensing)

Marie: I did a mini prep of M13KO7mutA1.1, M13KO7mutA1.2 et M13KO7mutA1.3. I obtained 210ng/µL, 277ng/µL and 236ng/µL.

Myriam/Husein: The program for the day was to try to insert the different IDT sequences that did work in a plasmid. The sequences were Mt, P3Xfu, Deps, p3Xc, P3Ng, Pa0744, Xpr, P3Pa4, Pa0745, P3SS, P3Xfa, thioesterase, P3VCV, p3vcc, P3RSS, P3VCF.

Firstly we did a digestion of Psb1C3 (460ng/µL and 2070 bp) we wanted 2000 ng in 100 µL so we have taken 4 µL of Psb1C3, 0,5µL of ecoRI, 0,5µL of Xba, 10 µL of buffer 2.1, 0,5 µL PST1 and 84,5 µL of H20 qq.

After it, we done a ligation of these sequences with the plasmid Psb1C3 (3µL per ligation)

Plasmid lenght (ng) quantity for ligation (µL) H20qq (µL) MT 7,5 1 13 P3SS 6,04 1 13 Xpr 7 1 13 Xc 31,74 3 11 Xfu 27,10 3 11 Ng 53,48 5 9 Vcc 39,86 4 10 Xfa 47,25 5 9 Vcv 54,06 5 9 Rss 45,51 5 9 Vcf 53,04 5 9 Deps 2.0 76,81 3 11 PA0744 55,31 6 8 PA0745 42,66 4 10 Thiostérase 40,77 4 10

After it we did a transformation of it and P3PA4 in DH5 alpha with 3 µL of plasmid and 50 µL of bacteria.

18/07/17 We did a verification PCR of New Lig p3Pa4-GFP DH5alpha and an electrophoresis which showed that the ligation wasn't successful.

Marie/Camille: I did a digestion of M13KO7mutA1.1, M13KO7mutA1.2 and M13KO7mutA1.3 (miniprep from yesterday) with 3 couples of restriction enzymes: pst1/BamH1 , pst1/Avr2 , pst1/BspE1. I put, in each tube, around 100ng of plasmid (0,5µL), 2µL of buffer (2.1 and 3.1), 0,5µL per enzyme, and 16,5µL of water. As a result none of them got the the restriction site of AvrII which is bad, and none of them got the restriction site BspEI which is good. Everyone was cut by PstI and BamHI which is good. Quick Change : As none of the three colonies were good we decided to retry a quickchange based on M13KO7p2. 1µL pfu turbo 6µL dNTP (10uM) 2,5µL PrimerAup 2,5µL PrimerAdw 5µL pfu buffer 32µL H2O (qsp 50µL) But we forgot to put the hybridization time back to 55°C.

95°C 2 min

95°C 30 sec 29 cycles 58°C 30 sec 68°C 20 min(2 min per kb) 68°C 2 min

16°C hold

Myriam/Hussein: The result of these transformation were mostly positive some petri-dishes, had more than one colony and thioesterase and pa0745 had many colonies. (result of 17/07 transformation) We started early in the morning to test one colony in each petri-dish ( deps, xc,Ng,Pa0744,Xpr,p3pa4, pa0745,p3ss,xfa, thioesterase, Histag, 5p, Vcv) Therefore, an order of picking the colony then smear the toothpick in a PCR tube, and petri-dishe is scratch to keep a stock of the colonies, the toothpick was then put in a starter for an overnight culture, and adequate volume of antibiotic was added to each tube. The PCR verification of colony had positive results sequence Desired length (pb) Actual length(pb) Deps 1890 Null Xc 957 200-400 Ng 1407 1000-1500 Pa0744 1445 1000-1500 Xpr 344 1000-1500 P3Pa4 1346 1000-1500 Pa0745 1183 Null P3ss 425 Null Xfa 1278 1000-1500 thioesterase 1144 1000-1500 vcv 1125 400-600

The sequences, which was not tested, did not have any colonies like MT, p3xfu, p3vcc, P3vcf.

We did a transformation of Super Nova (SN) without promoter, we have taken it from the plate 5 igem range 1L (we pierced the well and added 10 µL H20q and after 3 min taken it to an ependorff) For the transformation we have taken 3µL of SN without promoter and 50µL bacteria DH5 alpha. The rest of SN without promoter have been put into the -20°C.

19/07/17 Marie/Camille: We’ve got the result of the quick change of yesterday M13KO7mutA3. We put 1µL of DpnI in each tube and we did a 4h incubation at 37°C. For the transformation we put 50µL of the PCR mix w/ 100µL of TG1 cells. 1h of incubation on ice then 1min in 42°C then 1h at 37°C. Then we put on 4 kanamycine petri dish ON at 37°C for each. We also decided to make 8 starters from M13KO7mutA1 of the last time 2 for each colonies (4, 5, 9 and 8) in order to test other colonies.

Myriam/Hussein: Verifying the transplanting we found out that some colonies were red and these where the one that had the appropriate length from the results from the PCR verification on colony for the exception of P3Pa4. The afternoon we redid the transformation of the Monday 17/07 and we did an electrophoresis of the digested part of the pre-mentioned plasmid, all of it was at the good lenght. (Xc, Ng, PA0744, thioestérase, P3ss, Xpr, Xfu, Xfa, PA0745, MT, Vcv, Rss,Vcc, Vcf)

We have glyceroled Supernova Lac I, 5p and P3PA4 (800µL glycerol and 800µL of each) and put it in -80°C

we have done the mini prep of P3PA4, SN lac I and Histag. P3PA4: 167,34 ng/µL SN Lac I: 363,05 ng/µL Histag: 136,13 ng/µL

20/07/17 Marie: I did the mini prep of the starters of the colonies 4,5,8 and 9 from M13KO7mutA1. I obtained 165ng/µL, 199ng/µL and 202ng/µL. The starter 9 didn’t grow so I didn’t get any DNA. I digest those 3 mini preps with 3 couples of restriction enzymes: pst1/BamH1 , pst1/Avr2 , pst1/BspE1. I put, in each tube, around 100ng of plasmid (0,5µL), 2µL of buffer (2.1 and 3.1), 0,5µL per enzyme, and 16,5µL of water. The results showed that the colony 5 doesn’t have the mutation A. And maybe the colonies 4 and 8 do. The positive and negative control look good. It looks like the digestion isn’t complete. I need to do the digestion again and make the gel migrate longer to be sure. As none of the three colonies were good we decided (with Camille) to retry a quickchange based on M13KO7p2 for the mutation A. 1µL pfu turbo 6µL dNTP (10uM) 2,5µL PrimerAup 2,5µL PrimerAdw 5µL pfu buffer 32µL H2O (qsp 50µL)

95°C 2 min

95°C 30 sec 29 cycles 55°C 30 sec 68°C 20 min(2 min per kb) 68°C 2 min

16°C hold

Myriam: We have done a verification PCR under colonie (colonie taken from a transformation done with a miniprep except super nova taken from a transformation done with a lgem plate) We have done the PCR for: Super Nova without promoter (2 colonies tested) (SN 1 and SN2), Killer Red, GFP, Eprc, LacI-RBS, OriM13 and negative control. The electrophoresis revealed that all was good except Eprc and LacI-Rbs and the negative control (no glove) For Eprc and LacI-RBS we decided to do a verification PCR under miniprep for to check if we have the same result.

We have also done a verification PCR from miniprep for the assemblages : Eprc-GFP (3 tubes: 90,90 ng/µL, 20 ng/µL, 29 ng/µL), Eprc-KR (4 tubes: 20 ng/µL, 34 ng/µL, 32,16ng/µL, 33,15 ng/µL), LacI-RBS-KR (4 tubes: 330 ng/µL, 222 ng/µL, 105 ng/µL, 227 ng/µL) , OriM13-Eprc-KR (4 tubes: • 6R: 148ng/µL, • 20: 170 ng/µL, • 8R: 167 ng/µL, • 26: 193 ng/µL) The electrophoresis for all these assemblages was good even the negative control (glove: yeah!)

Eprc-Kr: 32,16 ng/µL, LacI-RBS-Kr: 222ng/µL, Eprc-GFP: 90,90 ng/µL, OriM13-Eprc-Kr 6R: 148 ng/µL were sent to the sequencing the same day.

We also did a transplanting of Super nova without promoter 1 and SN 2 (without promoter), Kr, GFP, OriM13 and starter to do a mini prep the next day.

21/07/17 Marie: I launched the digestion of the mini prep of the colonies 4 and 8 of M13KO7p2 again. This time I put 1µL of plasmid in each tube to prevent the incomplete digestion.So I put, in each tube, around 200ng of plasmid (1µL), 2µL of buffer (2.1 and 3.1), 0,5µL per enzyme, and 16,5µL of water. I let it for 2h30. The gel red was not good so the results were difficult to analyse. Follow-up of the quick-change from yesterday: This morning, I had 1µL of DpnI in the tube and I put it in the incubator 37°C for 4 hours. For the transformation, I put 50µL of the PCR mix w/ 100µL of TG1 cells. 1h of incubation on ice then 45s in 42°C then 1h at 37°C. Then I put on 4 kanamycine petri dish at 37°C for each.

Myriam: We did a mini prep with the starter done the 20/07: SN1, SN2, Kr, GFP, OriM13 (50 µL in the final mini prep): SN1: 118 ng/µL; SN2: 128 ng/µL; Kr: 105,5 ng/µL, OriM13: 76 ng/µL, GFP: 92 ng/µL

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WEEK 8 : 24/07 → 28/07

24/07/17 Marie: I did a mini-prep of p3VcF-C3 that was verified previously by sequençage and I obtained 88ng/µL and 111ng/µL. I did the digestion of the colony 8 of M13KO7mutA again to have a good gel this time. I put, in each tube, around 200ng of plasmid (1µL), 2µL of buffer (2.1 and 3.1), 0,5µL per enzyme, and 16µL of water. I let it for 3h. Then I did a gel and the results showed that AvrII did digest the plasmid so there is the A mutation. But there is a contamination and I don’t know where it comes from. It can be from the dye or the plasmid can be degraded or the enzyme can be contaminated. Since there is the A mutation on the plasmid M13KO7, I can try to add the B mutation. To do so, I did a quick-change (I just noticed that we forgot to put the DMSO in the two last quick-change at least): 1µL pfu turbo 6µL dNTP (10uM) 2,5µL PrimerBup 2,5µL PrimerBdw 5µL pfu buffer 1µL DMSO 32µL H2O (qsp 50µL)

95°C 2 min

95°C 30 sec 29 cycles 55°C 30 sec 68°C 20 min(2 min per kb) 68°C 2 min

16°C hold

The plan for today is to test the different colonies found on the petri dishes which were prepared the previous week The test was on p3xfu colony 4 to 10, p3RSS colony 4 to 10, P3VCV colony 4 to 10.the migration profile of the PCR product were all wrong the problem was then narrowed down to the fact that when the plasmid was digested xbaI was used as a third enzyme to completely eliminate The possibility to re-circularize the plasmid but doing so we added a supplementary fragment to the ligation mix that may have aided the circulation of the plasmid. Because in all the colonies tested we saw two distinct bands one between 200 and 400 and another band between 400 and 600. One band may be the empty plasmid that recirculated and the other band may have the fragment between xbaI and EcorI but the fragment is too small to be seen on the gel so the hypothesis can be neglected. Neither the digest of the IDT sequence nor the digest had any weird band on the ge. The negative and positive were verified and no indication of the problem were observed. So the only reason for this problem is a contamination of the colonies or mutation of the bacteria. So a new set of 3 sequences will be amplified digested and transplanted in a verified plasmid.

Myriam: We recieved the IDT sequence: SgE, SgP, SgR, SgX. We prepared them with 20 µL of H2Oq, there concentration are 12,5 ng/µL. We also did an amplification PCR of these sequences with 50µL for total volume and an electrophoresis after it. All was fine.

25/07/17 We maked a ligation of P3 proteins : P3 VcV, P3 Ng, P3 Xfu, into Psb1C3 plasmid. In a final volume of 20 µL, we add 5µL of P3VcV, 10µL of plasmid, 2µL of buffer ligase, 2µL of H20, 1µL of T4 ligase. We ligated P3 Ng with the same volumes. We ligated P3 Xfu with 2.5 µL of DNA, 10µL of plasmid, 2 µL of buffer ligase, 4.5µL of H20, 1µL of T4 ligase. We let the ligation all night at 16°C.

Marie: For the follow up of the quick change, I added 1μL of DpnI and put the tube at 37℃ for 4h. Then I did the transformation of the new plasmid in TG1 cells in 4 Petri dishes with kanamycin antibiotics. I also did the transformation of the mini prep of M13KO7mutA.8 because there is almost nothing left. I did one Petri dish with kanamycin antibiotics. I did the digestion of M13KO7mutA.8 and M13KO7pt with Pst1 only to check if the enzyme was contaminated. I put, in each tube, around 200ng of plasmid (1µL), 2µL of buffer (3.1), 0,5µL of enzyme, and 17.5µL of water. The results showed that it is M13KO7mutA.8 which is contaminated.

Myriam: We did a Clean-up PCR of SgE, SgX, SgP, SgR: SgP: 112 ng/µL SgX: 52 ng/µL SgR: 120 ng/µL SgE: 82 ng/µL We did a starter of a colony from a transformation (from the miniprep) of PSB1C3 (248 ng/µL from 24/07/17).

26/07/17 Yesterday we set an overnight ligation of p3ng, p3xfu, and P3VCV. The ligation is transformed in a DH5alpha.

Marie: The transformation of M13KO7mutA.8 succeeded so I did a repiquage of 8 colonies and I prepared 1 starter for each colony.

Transformation of the TG1 cellules with the ori M13 (miniprep) for further crossing over experiments

Phages in soil : positive control ( GM1 + soil water : nature dish) ,negative : GM1 + soil extract on kanamycin ; GM1 + soil extract + phages  : all did well , all T+ grew and T- didn’t

Myriam: We did a miniprep of the PSB1C3 starter done the 25/07. The obtained concentration was 185,5 ng/µL for a total volume 50 µL We also did a digestion of this PSB1C3 miniprep: 2000 ng for 100 µL: we took 11 µL of the plasmid (PSB1C3), 10 µL of cutsmart buffer, 1µL of ecoR1HF and SPE1HF (enzyme), 77 µL H2Oq. We also did a digestion of SgX, SgE, SgP and SgR: 500 ng for 40µL SgX: 9,4µL of this insert, 0,5µL of the two enzymes (same of PSB1C3), 4µL of cutsmart buffer and 25,6 µL of H2Oq SgP: 4,5 µL of this insert, 0,5µL of the two enzymes (same of PSB1C3), 4µL of cutsmart buffer and 30,5 µL of H2Oq SgR: 4µL of this insert, 0,5µL of the two enzymes (same of PSB1C3), 4µL of cutsmart buffer and 31 µL of H2Oq SgE: 6µL of this insert, 0,5µL of the two enzymes (same of PSB1C3), 4µL of cutsmart buffer and 29 µL of H2Oq We put them 2 hour and half at 37°C and then 20 minutes at 80°C.

We did a gel extract of PSB1C3, we put 50 µL of PSB1C3 in each well (2 well), the lenght was good so we did a gel clean-up of each strip. One of the strip was big so for the clean up we did 2 tubes, so we have gotten 3 tubes at the end: 1, 1’ and 2. PSB1C3 1: 5ng/µL PSB1C3 1’: 5ng/µL PSB1C3 2: 10ng/µL After it, we did a ligation of the various inserts digested previously with the PSB1C3 2 plasmid. We have taken 50 ng of plasmid so we have put 5µL into each ligation. For SgX, SgP and SgE we put 1,3 µL of them in there ligation (because they are all at 219 bp and have the same concentration: 12,5 ng/µL), we also put 2,2 µL of H2Oq, 1µL of T4 ligase buffer and 1µL of T4 ligase (diluted to 1/10). For SgR we put 1,5 µL of it for the ligation, 2µL of H2Oq, 1µL of T4 ligase and T4 ligase buffer. We put these ligation at the 16 °C incubator for an overnight incubation.

27/07/17 Marie: I did the mini prep of the starters prepared yesterday of the transformation of M13KO7mutA.8 in TG1. I obtained 78ng/µL, 68ng/µL, 69ng/µL, 50ng/µL, 78ng/µL, 74ng/µL, 87ng/µL and 107ng/µL. Then I did the digestion of the first 3 colonies with PstI/BamHI, PstI/AvrII and PstI/BspEI for each. I put, in each tube, around 150ng of plasmid (2µL), 2µL of buffer (2.1 and 3.1), 0,5µL per enzyme, and 15µL of water. Then I remembered that I forgot to put the antibiotics in the starters, so I did them again with Kanamycin.

The sequences p3xc, multitag, p3ss, p3xfr. Which were done by our benefactress Prima Valérie. These sequences are verified and a mini-prep was done. To keep a stock of these sequences a transformation using the mini-prep is done. The sequences p3vcv, p3ng, p3xfu that failed the previous week were restarted from scratch as we saw this past week these sequences are being verified with a pcr on colony. The result are revealed with an electrophoresis. we had positive results for p3vcv and p3xfu but no colonies for p3ng.

Myriam: I did a transformation of the ligation done the day before: 10,5 µL of the ligation for 100µL of DH5alpha bacteria.

28/07/17 Marie: I did the mini-prep of the starters I prepared yesterday. I obtained 61ng/µL, 66ng/µL, 66ng/µL, 88.7ng/µL, 46.5ng/µL, 58ng/µL, 77.5ng/µL and 65ng/µL. Then I did the digestion of the first 3 colonies with PstI/BamHI, PstI/AvrII and PstI/BspEI for each. I put, in each tube, around 130ng of plasmid (2µL), 2µL of buffer (2.1 and 3.1), 0,5µL per enzyme, and 15µL of water.

Myriam: Unfortunately the transformation done the 27/07 didn’t work out. I did a miniprep of LacI-RBS (cm): 133 ng/µL Pa0744-C3: 150 ng/µL Pa0745-C3: 107ng/µL Thioestérase-C3: 94 ng/µL P3RSS-C3: 204ng/µL ______________________________________________

WEEK 9 : 31/07 → 04/08 31/07/17 Hussein: a set of starter for p3RSS, thioesterase, PA0744, PA0745, P3VCV, P3xfu, P3VCF, P3RSS was done. we created a petri dish which we transplanted all of the verified IDT sequences to keep in stock, this dish was kept in a cold room all sequences were marked on the dish. a PCR-amplification of the sequences P3Xfa, P3VCC and D3 was done for the purpose to insert these sequences in a plasmid.

Marie:

01/08/17 Marie: The transformation of yesterday succeded. I prepared 2 starters of the same colony with kanamycin antibiotics. There was 2 types of colonies (big and small), I choose a small isolated colony. Follow-up of the quick-change: I added 1µL of DpnI and put in the incubator 37°C for 4h. Then I did a transformation with TG1 cells in 4 petri dishes with kanamycin antibiotics. I did the transformation of SGE-C3, SGP-C3, SGR-C3, SGX-C3 with DH5α cells in 4 petri dishes with chloramphenicol antibiotics.

Hussein: a mini-prep of the starters launched the previous day was done. a pcr clean up also was done for p3xfa, p3vcc, D3. these sequences were digested with EcorI-Hf and SpeI-HF.

02/08/17 Marie: I did the mini-prep of the 2 starters I prepared yesterday. I obtained 69.75ng/µL, 74.25ng/µL. I launched a Phusion cycle to remove the p3 part in the M13KO7p2 plasmid: 1µL of DNA 1µL of Betaïne 5µL of dNTP (1mM) 2.5µL M13p3VF (10pM) 2.5µL M13p3VR (10pM) 5µL Tampon Phusion 32.7µL of H2O 0.3µL of Phusion 98°C 30 sec

98°C 10 sec 30 cycles 70°C 30 sec 72°C 5 min 72°C 10 min

16°C hold

Then I did a gel and after that, I did a PCR clean-up.

hussein: 1000ng of PSB1C3 RFP with an initial concentration of 100ng/ul was digested with EcorI-HF and Spe-HF in a final volume of 20 ul this cut backbone was used to ligate sge, sgx, sgp, sgr, p3vcc, p3xfa, p3ng, D3. an old digest of Deps which was done before was used to be inserted in a plasmid psb1c3-rfp cut EcorI-HF and PstI. these ligation were transformed with 100ul of Dh5alpha bacteria. 2 ul of each ligation was used for this purpose.

03/08/17 Marie: I put 1µL of DpnI in the PCR clean-up I did yesterday, and let it incubate at 37°C for 2h. Then I put it at 80°C for 20mn. And then I started a transformation with 100µL of TG1 cells. I put the mixture in ice for 1h, then at 42°C for 2mn, then 5mn in ice. I added 900µL of LB and let it incubate at 37°C for 1h. Then I put 250µL per petri dish in 4 petri dishes with kanamycin antibiotics. After that, I started a PCR overnight (Turbo cycle) with M13KO7p2: 1µL of DNA 1µL of Betaïne 5µL of dNTP (1mM) 2.5µL M13p3VF (10pM) 2.5µL M13p3VR (10pM) 5µL Tampon Turbo Pfu 32.7µL of H2O 0.3µL of Turbo Pfu 95°C 2 min

95°C 30 sec 30 cycles 55°C 30 sec 72°C 20 min 72°C 10 min

16°C hold

hussein: the transformation for D3, P3Ng, P3vcv, p3xfa, sgp, sgr, sgx, sge was done but we doubled the quantity of DH5 alpha and ligation

04/08/17 Marie: In the morning, I did the PCR clean-up of the PCR overnight, I obtained 59.8ng/µL in around 15µL. I put 2.3µL of the PCR clean-up in a PCR tube and gave it to Gauthier so he can do a SLIC. I did a digestion with the remaining liquid. I put the 12µL left of M13KO7Δp3, 3µL of CutSmart, 1µL of DpnI and qsp 30µL of water. I put the mixture in the incubator 37°C for 2h. After that, I did a transformation in TG1 cells.

Hussein: the transformation done on the thursday were checked with the magic box to eliminate any rfp colonies and marke the colonies that are only white. these colonies were transplanted on a box to be tested next week.

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WEEK 10 : 07/08 → 11/08

07/08/17 Marie: The transformation of Friday didn’t succeed, but Gauthier’s SLIC did. So I prepared 4 starters (1 per colony with one colony per petri dish). I picked 12 colonies per petri dish and I did a verification of all 48 colonies. It showed that the colonies 27 and 48 were maybe good. So I prepared one starter for each colony with kanamycin antibiotics.

Camille: The transplantation done by Hussein the 04/08 still had too many rfp clones. I restart the processus from 3 insertion of iDT sequence : p3-D3, p3Xfa and Depart. First I digest pSB1C3 (from 25/06/2016) with EcoRI and SpeI in CutSmart Buffer. I digest 2000ng of vector in 150uL. CutSmart 15uL pSB1C3 15uL EcoRI 2uL SpeI 2uL H2O 116uL Digestion 3 hours at 37°C, then 20min at 80°C.

Then digestion of insert : p3D3 from pcr cleanup (01/08) 128ng/uL Deps directly from iDT sequence 50ng/uL p3Xfa from pcr cleanup (01/08) 160 ng/uL I put 250 ng of DNA for 20uL (12,5ng/uL)

p3D3 : CutSmart 2uL p3D3 2uL EcoRI 0,2uL SpeI 0,2uL H2O 13,6uL Deps : CutSmart 2uL Deps 5uL EcoRI 0,2uL SpeI 0,2uL H2O 8,6uL p3Xfa : CutSmart 2uL p3Xfa 1,5uL EcoRI 0,2uL SpeI 0,2uL H2O 14,1uL 3h of digestion at 37°C and 20 min at 80°C.

For the ligation part I used NEBcalculator with a ration of 3:1 and for 50ng of vector.

p3D3 (30,8ng) : Buffer T4 2uL p3D3 2uL pSBIC3 5uL T4 ligase 0,5uL H2O 10,5uL Deps (79,5ng): Buffer T4 2uL Deps 7uL pSBIC3 5uL T4 ligase 0,5uL H2O 5,5uL p3Xfa (36ng): Buffer T4 2uL p3Xfa 2uL pSBIC3 5uL T4 ligase 0,5uL H2O 10,5uL Ligation ON at 16°C

I also tried to ligate LacI-RBS to pA0744. As LacI-RBS has already been cut down S/P (the date wasn’t written), I only need to digest the pcr clean up of pA0744 (pcr cleanup from 01/08) w/ XbaI and PstI. 2.1 buffer 2uL pA0744 2,5uL XbaI 0,2uL PstI 0,2uL H2O 15,1uL 3h of digestion at 37°C and 20 min at 80°C. After that I tried the ligation Buffer T4 2uL PA0744 2uL LacI-RBS_C3 2uL T4 ligase 0,5uL H2O 13uL

Ligation ON at 16°C

08/08/17

Camille: Transformation the 20µL of ligation from 07/08 (Deps_C3, p3D3_C3, p3Xfa_C3 and LacI-RBS-PA0744_C3) with 100µL of DH5a competent cells (for each). 20 min on ice, then 42°C for 1min, then 5min on ice, addition of 800µL of LB and 1h at 37°C.

Marie: The starters of yesterday didn't grow much so the concentrations of the mini prep I did this morning were low (7 and 25ng/μL). After that, I launched the digestion with (around 100ng of plasmid, 2μL of CutSmart, 0,5μL of BamHI and qsp 20μL of water). The restriction site is not supposed to be there anymore so it shouldn't be digested. The gel showed nothing, because the mini prep were not concentrated enough. I re launched the starters using a more nutritive medium.

09/08/17 Marie: I did the mini prep of the starters I launched yesterday (M13KO7Δp3 colonies 27 and 48) and also the mini prep of Robin (p3SS GFP, Xpr GFP, Xpr KR and Xpr SNV). I obtained . Then I launched a digestion with BamHI with around 200ng of pplasmid on the gel we don't see anything.

Hussein: the colonies done by camille previously were kept to incubate, the rfp take more time to maturate the colonies donne friday 4 are being tested by a pcr using the oligo vf and vr the colonies tested are deps 1, deps 10, p3xfa 2, and p3vcc 2 which were done following the protocol recommended by Prima Valerie. the electrophoresis shows only one viable candidate and that was p3vcc 2 the migration profile put the band of p3vcc 2 between a 1000 pb and 1500 pb the length of p3vcc is 825 pb and the pcr add 300 pb, 150 pb for each oligo adding for a total of 1125 pb.

the positive control of rfp shows a band higher meaning the band of p3vcc isn’t an rfp and the transplantation is white and not red. p3vcc 2 was launched for an overnight starter.

an gel extract for psb1c3 backbone was also done to recuperate only an linearized plasmid. the final concentration of the gel clean up was of 15 ng/ul. the transformation of camille which was left to mature were transplanted on a petri dish 10 colonies were chosen only 4 were tested by pcr. the result of the gel were promising 1 of each 4 colonies were positive see gel.

10/08/17

Hussein: a starter for deps, p3xfa and p3d3 was launched. 17 petri dishes with chloramphenicol antibiotic were cast as well 17 petri dishes with kanamycin antibiotic. the starters launched yesterday for p3vcc was mini-prepped and the final concentration was 62.94ng/ul 6 colonies of each construction laci-RBS Multi Tag and Laci-RBS-P3SS done by robin the previous day were tested by pcr. the pcr product was then migrated on agarose gel the results are: all colonies tested negative we can see on the gel that no fragment passed the 600 pb mark all colonies tested were between 400 and 600 meaning there was no insertion other colonies should be tested.

11/08/17 Hussein:

the starters for deps, p3xfa and p3d3 were mini-prepped and the final concentration was as follow 99 ng/ul for deps , 82 ng/ul for p3xfa and 179 ng/ul for p3d3

100 ng of each mini-prep as well for p3vcc were digested with 0.1 ul of ecori-HF and 0.1 of pstI in final of 10 ul. the digest was then migrated on agar gel. the result are shown on the gel in summary there were 3 bands for the plasmid at 2000 pb the plasmid is 2070 pb deps showed a band at 1500 pb, and deps is 1590 pb p3xfa showed a band at 1000 pb, and p3xfa is 978 pb p3d3 showed a band at 600 pb and p3d3 is 616 pb all 3 sequences are positive and have the appropriate restriction sites for ecori and pstI these were then sent for sequencing.

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WEEK 11 : 14/08 → 18/08

12/08/17 Hussein : minprep of puca 18 and laci-rbs-pa0744 PCR amplification for laci-rbs PCR clean of the PCR product for laci-rbs Digestion of p3ss pcr amplification and multi tag PCR amplification with xbaI and pstI Digest of laci-rbs PCR amplification with EcorI and speI Digest of psb1c3-rfp with ecoRI and pstI Gel extract to recuperate the band at 2000pb and discard the RFP band at 1000pb. Gel clean up of gel extract for psb1c3. Ligation of laci-rbs with multi tag and psb1c3 linearized plasmid. Ligation of laci-rbs with p3ss and psb1c3 linearized plasmid. Transformation with 100ul of dh5alpha. Spread on chloramphenicol Petri dishes. The digestion and ligation today were done using a microwave oven. We tested the digestion of the psb1c3 plasmid with ecorI and pstI at different time interval first at 20 second with the microwave oven set at 600 watt the power of the microwave stayed constant. With just 20s we didn’t observe any bands on the gel. With 50 s we saw 3 bands, one at 3000pb meaning the plasmid was cut just at one side, a second band at 2000 pb the plasmid without the insert and a third band at 1000 pb the insert RFP size, the gels photo showing the partial digestion wasn’t taken.

With 1 minute 20 second we saw 2 bands on the gel meaning a full digestion of the plasmid.

these results put us at ease and more testing should be done but we shouldn’t be afraid of using this in conjunction of the classic protocol to accelerate the rate of our work.

13/08/17 14/08/17

Camille: Transformation of LacI-RBS_C3 in DH5alpha

Camille + Marie : Quick change of M13KO7p2 in order to delete p3 gene. We went to see Erick to modified our quick change protocoles. We wanted to test 3 differents temperatures of hybridation (47,9°C/53,5°C/57°C).

1µL pfu turbo 2,5µL dNTP (10uM) 1,25µL M13p3r 1,25µL M13p3f 2,5µL pfu buffer 15µL H2O (qsp 50µL)

95°C 2 min

95°C 30 sec 30 cycles XX°C 30 sec 68°C 25 min 68°C 20 min

16°C hold

15/08/17

Camille : In the morning Erick put 0,5uL of DpnI in each PCR tube and incubate them at 37°C for 5 hours. Transformation of PCR quikchange in TG1 E.coli cells. 10µL of PCR for 200µL of bacteria (1h 4°C, 45s 42°C, 800µL LB, 1h at 37°C). Then they are put in Kanamycin petri dish, at 37°C ON.

LacI RBS transformation grow well.

16/08/17 Marie: The transformation was successful. On the first Petri dish (47,9℃), there was 5 big colonies and a lot a small colonies. On the second Petri dish (53,5℃), there was 3 big colonies. And on the last Petri dish (57℃), there was no colony. So I did a repiquage and a verification PCR of 19 colonies. On the gel, we don’t see anything. I prepared 7 starters, one per colony, with a kanamycin antibiotics.

17/08/17 Marie : I did the mini prep of the starters I prepared yesterday, I obtained 90.3ng/µL, 112.3ng/µL, 98.35ng/µL, 110ng/µL, 112ng/µL, 100ng/µL, 115.5ng/µL. Then I did a digestion using the microwave (50 sec at 600 watt) of the colonies 2,4 and 5. On the gel, we only see that the colony 2 has two stripes so there is still p3. But we don’t see anything for the two other colonies. So I started again (with a new gel red), I did the digestion of the colonies 3,4 and 6. We don’t see anything for the colony 6. And for the two others, we see only one stripe but it’s not the right lenght (5500bp and 10000bp instead of 7300bp).

18/08/17 Marie: I did a digestion of the colonies 1,3,4,5,7 from the PCR cycle with Pfu Turbo and also of M13KO7p2. I made 3 tubes per colony : 1 with no enzyme, 1 with PstI/BamHI-HF and the last one with PstI/NgoMIV. On the gel, we only see the stripes of the 3 tubes containing M13KO7p2. But for all the others, we see that the plasmids are all degraded. We need to start everything again. I launched a PCR cycle with Pfu Turbo. This time I put it at 52°C. ⋍100ng of M13KO7 0,5µL of DMSO 3µL dNTP (10uM) 1,25µL M13p3r 1,25µL M13p3f 2,5µL pfu buffer 0,5µL pfu turbo 15µL H2O (qsp 25µL)

95°C 2 min

95°C 30 sec 30 cycles 52°C 30 sec 68°C 20 min 68°C 20 min

16°C hold

________________________________________ WEEK 12 : 21/08 → 25/08

21/08/17 Marie: I put 0,5µL of DpnI and put the tube in the incubator 37°C for 4h. Then I started 2 transformations in TG1 cells. I put 10µL in 200µL of bacteria cells each: 1h in the ice, 45s at 42°C, 5mn in the ice, added 900µL of LB, 1h at 37°C, centri, remove 850µL of supernatant then re suspend the pellet and spread each tube on a petri dish with kanamycin antibiotics.

hussein: i diluted AgeI-HF with 9 ul of diluent A

22/08/17 Marie: The transformation did not work very much but I still got one little colony, so I let the dishes in the incubator. In the afternoon, the colony I saw in the morning was bigger and some very little colonies had appeared. So I did a verification PCR of 10 colonies (including the big one). I used oligos 109 and 110 that binds to p3 domain, so if the QuickChange worked there should be no amplification. On the gel, we can see that the colony 1 is amplified so there is p3 in it. But all the other colonies seem good. Thus, I prepared 4 starters with the colonies 2, 3, 4 and 5.

23/08/17 Marie: The starters didn’t grow so I let them in the incubator. I talked with Laetitia and she advised to put less Kanamycin in the petri dishes and the starters. So I prepared new starters with the colonies 11, 12, 13 and 14. And I put two of them in the incubator 37°C and the other two in the incubator 30°C. I prepared, also, some petri dishes, 8 with Kanamycin (half than usual), 3 with Knanmycin (normal) and 9 with Chloramphenicol.

24/08/17 Marie: The starters didn’t grow neither did the repiquage from the 22th. So I did a repiquage again, I repiqued 20 colonies (kanamycin half than usual) and I let the starters from yesterday in the incubator. I also launched a QuickChange, one tube for the mutation A and another for the mutation B. 1µL pfu turbo 6µL dNTP (10uM) 2,5µL Primer Bup or Aup 2,5µL Primer Bdw or Adw 5µL pfu buffer 1µL DMSO 32µL H2O (qsp 50µL)

95°C 2 min

95°C 30 sec 29 cycles 55°C 30 sec 68°C 20 min(2 min per kb) 68°C 2 min

16°C hold

25/08/17 Marie: The repiquage has grown so I did a colony PCR on the 19 colonies. I put 1µL of DpnI in each of the QuickChange tube and let it incubate for 4h at 37°C. Then I started a transformation by putting 10µL in 100µL of TG1 cells. I used only 20µL per PCR tube. Then I spread the mixture in 4 petri dishes with kanamicyn antibiotics (half than usual), 2 for M13KO7mutA and 2 for M13KO7mutB. And then I put one M13KO7mutA and one M13KO7mutB in the incubator 37°C and the other 2 petri dishes at 30°C. I launched 2 starters of 10mL with kanamycin antibiotics (half than usual). One starter was put in the incubator 37°C and the other in the incubator 30°C.

Camille: I did the 2 mini prep of the starters prepared by Marie on Friday. I obtained 161,6ng/μL (37℃) and 192ng/μL (30℃).

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WEEK 12 : 28/08 → 31/08 28/08/17 Marie: I launched the digestion of the 2 mini prep from yesterday plus M13KO7p2 (original) with the enzymes PstI and BamHI. There should be only one stripe because Δp3 don't have BamHI restriction site. M13KO7 original is here as control, it should be digested twice. We see 2 stripes for M13KO7p2 but nothing for either of the two M13KO7Δp3. There is no plasmid. Also, the Petri dishes of the QuickChange from Thursday showed multiples colonies (mutB has more than mutA and 30℃ has more than 37℃ but there is a high possibility of contaminant).

29/08/17 Marie: I did half the mini prep of the starters I prepared yesterday, I did the first tenth. I obtained 72.9ng/µL, 75.8ng/µL, 76.7ng/µL, 59.25ng/µL, 63.8ng/µL, 62ng/µL, 59ng/µL, 61ng/µL, 66.25ng/µL and 63.8ng/µL. Then I digest them (microwave 600W 50s) with PstI/AvrII for the colonies 1 to 5 and PstI/BspEI for the colonies 6 to 10. On the gel, I also put the plasmid alone. And we see nothing, there is no plasmid.

30/08/17 Marie: I prepared 19 petri dishes with Chloramphenicol antibiotics. Laetitia did the mini prep of the colonies 11 to 20 (that I didn’t do yesterday). But there was nothing in the mini prep. So I did a transformation with 10µL of each tube from the last QuickChange (I didn’t use all of them last time) in 100µL of TG1 cells. And I spread it on 2 petri dishes with Kanamycin antibiotics (normal dose).

31/08/17 Marie: I did the mini prep of LacI-RBS-p3Ec and LacI-Deps, I obtained 124 ng/µL, 159 ng/µL, 235.5 ng/µL and 244 ng/µL.