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CALENDRIER DESACTIVE EN ATTENDANT DE FINIR lA RETRANSCRIPTION

Day 1 : 06/06/2017

Cleaning the laboratory, then recovery and installation of the equipment. Starting culture of strains TG1 and DH5α. We launched precultures from cryotubes ( sampling with 1 rod and then deposition in an erlenmeyer containing LB media ). Incubation at 37 ° C.

Day 2 : 07/06/2017

Strains cultivation:

Recovery of strains cultivated the day before: DH5α and TG1 To check the concentrations, the OD is measured. For doing so, the culture must be diluted because the optimum measurement of the apparatus is between 0.1 and 0.8. The cultures in the tank are diluted to 1/10 with distilled water.

Our results : TG1: 5.16 / DH5α: 5.21

To calculate the volume to be taken for our 100 mL we do: $$\frac{\text{OD wanted}}{\text{OD obtained before dilution}} \times \text{Volume} = \frac{0.1}{5.16} \times 100 \simeq 2 \text{ mL}$$ 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.

Glycerolization of strains:

Putting the strains in glycerol protect them from the cold, necessary when one will freeze at -80 ° C so that the cells do not collapse. 40% Glycerol in 1.8mL cryotubes → 0.9 of glycerol and 0.9 of strains. We will freeze the cells in exponential phases in order to make our cells competent, ie able to incorporate DNA.

Competent bacteria:

To make competent bacteria, it is necessary to take them in exponential growth phase. A quantity of bacterium is thus taken which is placed in LB medium and then incubated for 1 hour at 37 degree Celsius. An OD value of 0.1 is desired in 100 mL. For TG1-> 1.94 mL For DH5α-> 1.92 mL

Mesuring OD after 1h40: TG1 - 0.7 DH5α - 0.5 The cultures were thus recovered and were divided into falcons (50 ml each) and placed in ice. (For each of the two strains). Preparation of the buffers: Solution TBF1 and TBF2 Preparation of buffer solutions: KAc: 50 mL → 4.9 g MnCl2: 200 mL → 19.79 g KCl: 200 mL → 14.91 g MOPS: 50 mL → 2.09 g Each solution was autoclaved. Then we made two solutions necessary for making the competent cells.

To make competent cells, we ALWAYS work at low temperature (4 ° C) and in sterile medium. The buffers thus prepared were stored in ice. Centrifugation of the cell culture 10 min at 3500 rpm at 4 ° C. The pellet must then be resuspended (gently) in 80 ml of Tbf1 (20 ml as 50 ml of culture in each falcon). 3500 rpm centrifugation for 5 minutes.

Then resuspension of the pellet in 2 mL of Tbf2.

Let incubate for 15 minutes in ice and then aliquot 200 μL of bacteria solution in a cryotube and store at -80 ° C. These aliquots are at I8 for competent DH5 alpha and I9 for competent TG1.

The remainder is stored at -20 ° C in labeled falcons.

After the first centrifugation was carried out in a cold (non-sterile) chamber, we re-started precultures from those in the morning: 100 μL of culture were placed in 10 ml of LB medium and then incubated at 37 ° C. overnight.

The buffer Tbf1 and Tbf2 have also been prepared to be ready so we can restart the manipets in case of failure.

Day 3: 08/06/17

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


Killer red	Crispr Cas9	P3	Génome M13	Measurement	DEPS	QS
Robin	Flora/Soraya	Camille	Camille/Thibault	Lisa	Hussein	Jeremy Flora Ilann

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:

Contact, the cells and plasmids (which are recovered in the iGEM kit)
Heat shock, more permeable membrane that will make the DNA enter: 45 seconds at 42 ° C
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)
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

Day 4: 09/06/17

BROUILLON

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

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).

DAY 7: 14/06/17

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.

	Volume	ECORI HF	SPE1	XBA1	PST1	Buffer	H2O
M13 cut up	3	0,5	0,5	/	/	2	14
EPRI cut up	3,3	0,5	0,5	/	/	2	13,7
EPRC cut up	4,5	0,5	0,5	/	/	2	12,5
EPRC cut down	4,5	/	/	0,5	0,5	2	12,5
GFP cut down	2,2	/	/	0,5	0,5	2	14,8
RBS cut down	1,1	/	/	0,5	0,5	2	15,9

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 as is 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 30 minutes at 135 volt? Which was the limit of the migration, we should have put it only for 25. All fragment under 200 Pb, aren’t observed because it had surpassed the limit of migration. So for EPRI, EPRC cut up and down, we should only see 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 was multiple colonies on the gel.

DAY 8: 15/06/17

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

DAY 9: 16/06/17

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.

DAY 10: 17/06/17

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°

DAY 10: 18/06/17

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°

DAY 11: 19/06/17

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.

DAY 12 : 20/06/17

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.

TABLEAU

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.

DAY 13 : 21/06/17

Lisa and jeremy made 19 petri dishes with Kanamycin.

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

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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