WEEK 1

Training

During the first week, we had several training sessions, under the supervision of Gauthier DANGLA-PELISSIER (Instructor), to get acquainted with the lab work, master everyday protocols (PCR/Colony-PCR, competent cells preparation, transformation, cloning, minipreps, PCR clean-ups...), and establish a classic workflow.

Day 1: Lab setup

We recovered lab equipment from our home university, cleaned-up the lab, and had a first meeting to establish the project's workflow.

Day 2: Preparing competent cells stock and transformation

We prepared a stock of competent cells using the following protocol. Afterward, we prepared Petri dishes and we transformed the cells to test their efficiency using a plasmid provided by our instructor.

Day 3: Colony-PCR and agarose gel electrophoresis

The next day we had colonies on our Petri dishes and wanted to check if they acquired the transformed plasmid with the right inserted gene. We picked out some positive colonies and ran a colony-PCR. We then migrated the PCR products on an agarose gel.

Day 4: DNA purification miniprep

We purified the plasmid with the right inserted gene from the positive colonies using a DNA purification kit. In a further step, we digested the purified plasmid with respective restriction enzymes to double-check the presence of the inserted gene.

Day 5: Western-blot

The last day, we checked the induced production rate of the relative protein to the inserted gene (Produced in a preliminary step by our instructor) using the following western-blot protocol.

WEEK 2

For the whole week, we ran into a lot of problems making competent cells, so we spent a lot of time optimizing the protocol.

Day 1

Biobricks design: We designed Hmas, MdlB and MdlC biobricks.
Megaprimers design: We designed megaprimers to add the Tag-his to our biobriks sequences.
Chitinase: We received the chitinase gene sequence from IDT and ran a PCR to amplify the gene.
Methionine-gamma-lyase': We recovered the biobrick from iGEM 2018 kit.

Day 2

pLacI promoter biobrick: We recovered the biobrick from iGEM 2018 kit to assemble with other biobricks.
Chitinase: We took the PCR product and verified the amplification on an agarose gel. We ran into hybridization problems so we had to determine it using a gradient-PCR.

Day 3

Chitinase: We cloned the PCR product in a PSB1C3 plasmid.
Methionine-gamma-lyase: We transformed the biobrick (From the iGEM 2018 kit) into BL21 competent strains beacuse we ran into problems making competent DH5alpha strains .

Day 4

Chitinase: We ran another cloning process on the previous PCR product and transformed it into DH5alpha competent cells.
Methionine-gamma-lyase: We recovered the transformation plates and ran a colony-PCR on selected colonies. Afterward, we launched starters on the positive colonies.
Interlab: We received a training on the measurement devices for the device and created the respective programs.
Biobrick design: we designed a new biobrick (OmpT-AIDAC) a cleavage sequence.
RFP biobrick: We recovered the biobrick (From the iGEM 2018 kit) that will be used for white-red screenings.

Day 5

pLacI promoter biobrick: We transformed the plasmid in DH5alpha competent cells.
Chitinase: We transformed DH5alpha strains with the plasmid containing the chitinase sequence.
Interlab: We recovered the 8 biobricks (From the iGEM 2018 kit) and transformed them into DH5alpha competent cells.
Methionine-gamma-lyase: We purified the DNA using a miniprep kit and stoked it.
MdlB biobrick: We received the biobrick and ran a PCR on it.
RFP: We transformed the biobrick into DH5alpha competent cells.

WEEK 3

Day 1

pLacI promoter biobrick: We recovered the transformation plates and ran a colony PCR on the obtained positive colonies.
RFP biobrick: We recovered the transformation plates and launched overnight starters to purify the plasmid later on (No colony-PCR because the colonies are red: RFP expression).
Chitinase: We recovered the transformation plates and ran a colony PCR on the obtained positive colonies.
We amplified the chitinase IDT gene sequence to recover the stock.
Methionine-gamma-lyase: We recovered the methionine-gamma-lyase biobrick (from the iGEM 2018 kit) and transformed it in DH5alpha competent strains.
InterLab: We recovered the 8 transformations and launched 8 starters in duplicates.

Day 2

pLacI promoter biobrick: We purified the inserted RFP gene using DNA purification kits and restart because the final DNA concentration was too low (13 ng/µL).
RFP biobrick: We purified the inserted RFP gene using DNA purification kits.
Chitinase: We ran a gradient PCR to find the optimal primers hybridization temperature. We then extracted the DNA using a gel extraction kit, digested the gene with respective enzymes, and ran ana overnight ligation in a PSB1C3 plasmid at 16°C.
DH5alpha competent cells: We prepared a new DH5aplha competent cells stock and verified their efficiency using an RFP plasmid.
Methionine-gamma-lyase: We ran a colony-PCR on the positive colonies and verified it on an agarose gel.
InterLab: We launched the 3 calibration measurements and ran the first cell measurements.

Day 3

pLacI promoter biobrick: We purified the DNA using a different miniprep kit.
Chitinase: We ran a ligation in PSB1C3 at room temperature.
DH5alpha competent cells: We launched DH5-alpha starter to renew the competent cells stock.
Methionine-gamma-lyase: We purified the DNA using the Promega miniprep kit. Afterward, we launched an overnight megapriming using specific primers to add a Tag-his to our protein so we can purify it.
InterLab: Data analysis and launch of new starters to run a second measurement test.
MdlB biobrick: We ran a ligation in PSB1C3 at room temperature.

Day 4

Methionine-gamma-lyase: We digestion magapriming product by DpnI (To cut the methylated GATC sites), and transformed it into DH5alpha competent strains.
DH5alpha competent cells: we renewed our stock and verified their efficiency using the RFP plasmid.
InterLab: We ran a second measurement test.
MdlB biobrick: We recovered the transformation plates and ran colony-PCR on the positive colonies.
Extracurricular: We made our breaking bugs logo using E. coli strains that express the GFP.

Day 5

DH5alpha competent cells: We double-checked the competent cells efficiency by transforming the RFP plasmid.
MdlB biobrick: We recovered the overnight starters, and purified the DNA using minpreps kits, measured the DNA concentration on the NanoDrop and ran a digestion test.

WEEK 4

Chitinase : 25/06/18 Starter lancé pour une miniprep. 26/06/18 Miniprep : concentration de 225,87 ng/uL ; 1,95  ; 1,82 Envoie au séquençage Mega-priming avec le tag His

27/06/18 Digestion par DpnI du produit de Mega-priming Transformation 28/06/18 Résultats de la transformation sont négatifs : aucune colonie mais après une journée d’incubation à 37°C : des colonies sont apparues mais ne semblent pas être E.coli → Bacillus obtenus car pas meme couleur , odeur et forme (obtenue par isolement ), ne pousse pas bie, à 37°C et prend 2 jours Mega-priming avec le tag His Résultat du séquençage positif 29/06/18 Digestion par DpnI (clivage des sites non méthylés pour la sélection des plasmides d’intérêt pour la transformation) Transformation dans des DH5-alpha de Victoria

MdlB : 25/06/18 Digestion-ligation : [MdlB dig] = 60 ng.µL-1 / 1.89 / 2.04 [RFP dig] = 74 ng.µL -1 / 1.89 / 2.11 Gel avec Plasmide non digéré (digestion totale ?), Plasmide + insert (dig-lig), Plasmide RFP dig (Enzyme ok ?) Transformation dans DH5α : 3 conditions : (plasmide + insert) dig-lig (Gautier) / (Plasmide)dig-lig (V4) / (Plasmide)dig (V4) Q5 PCR sur MdlB pour refaire le stock + clean up + nanodrop + gel 26/06/18 Observation à la MagicBox des DH5α Dig-Lig RFP-MDLB cPCR sur les colonies supposées bonnes pour MDLB Digestion de nouveau de MdlB + clean up [MdlB dig] = 31.40 ng.µL (dilué dans 30 µL) Gel avec produit de la digestion et cPCR Q5 PCR pour refaire les stocks + gel 27/06/18 Ligation de MDLB + RFP (tout deux digérés) à 37°C pendant 3H Transformation du produit de la ligation dans les V4 28/06/18 Boîtes transformés de la veille → pas de colonies blanches le matin mais apparition de colonies blanches le soir. Il ne s’agit pas de DH5α mais d’une autre Bactérie qui est compétente de base.

HYD1 : 26/06/18 Réception de Hyd1 + mise en eau dans 10 µL de PPI Q5 PCR pour l’amplifier + Gel (problème de taille → Migration à relancer) 27/06/18 Gel de Hyd1 qui a été amplifié par Q5 PCR la veille (ce n’est pas Hyd1 qui a été amplifié) Amplification de Hyd1 par Q5 PCR [Hyd1]= 37 ng.µL Migration de Hyd1 → Obtention d’une bande beaucoup trop grande pour qu’elle corresponde à Hyd1. Hypothèses : ™ ne correspond pas, la durée d’élongation est beaucoup trop longue ! 1min = 1kb !! PCR avec Hyd1 en utilisant la Taq DNA Polymérase + ™ 60°C et Élongation 30 secondes à 72°C + Final élongation 5 min. Gel Seconde PCR mais avec un gradient de T°. 28/06/18 Hyd1 clean up [Hyd1 dig]= 8 ng.µL-1 Gel

29/06/18

Ligation + transformation de Hyd1 dans DH5α

RFP : 25/06/18 Transformation (car nous suspectons une contamination du tube de la RFP (issue de la purification) par un autre plasmide car de nombreuses colonies blanches sont obtenues sur boîte : sachant qu’elles devraient être toutes rouges) du tube mère du kit. 26/06/18 Amplification de la RFP Miniprep (40µL à 133 ng/µL)

Compétentes : 25/06/18 Transformation avec pLic 60 (Kn10) dans DH5α V3 (toutes premières),V4 (du 18/06) et Version Gautier (pour tester de nouveau la compétence puisque l’existence d’un contaminant est quasi-certaine ) 26/06/18 Mauvais étiquetage des boîtes (résultats NON fiables) Transformation de nouveau des V3 et V4 avec RFP 27/06/18 V3 : pas de colonies V4 : beaucoup de colonies (compétentes) : Bacillus obtenus après 2 jours

29/06/18 Compétentes avec Gothyais (resuspension avec P200 plutot P1000, ne pas trop les laisser dans le mOPS → sinon mort des bactos)

InterLab : 25/06/18 Troisième test interlab et protocol CFU (Dilutions en série) lancé. 26/06/18 Échec du protocol CFU (Erreur de dilution). Lancement de starter sur TD4 pour entrainement sur la dilution en série. 27/06/18 Protocol CFU sur TD4 et analyse de données des 3 test interlab. 28/06/18 Étalement des boites pour un deuxième test CFU. 29/06/18 Deuxième test CFU (Dilutions en série) et mise en incubation overnight. Mgl  : 25/06/18 Envoie au séquençage de MGL tag histidine en c-ter

26/06/18 Analyse du séquençage MGL → la séquence MGL a bien été taguée His. → la semaine pro il faut mitonner pour la slic pas d’envoie au séquençage et assemblage pLacI-Mgl-plasmide RFP Lipase: 25/06/18: Annulation de la séquence Recommande, en ré-optimisant et en coupant en 2 pour pouvoir slicker.

Omp-T AIDA-C: 27/06/18: Annulation de la séquence Recommande, en ré-optimisant et en coupant en 2 pour pouvoir slicker.

VERSION EN ANGLAIS

WEEK 4:

Chitinase: 25/06/18 Starter thrown for a miniprep 26/06/18 Miniprep: concentration of 225,87 ng / uL; 1,95; 1,82 Send to the sequencing Mega-priming with the tag His 27/06/18 Digestion by DpnI of the product of Mega-priming Transformation 28/06/18 Results of the transformation are negative: no colony but after a day of incubation in 37°C: colonies appeared but do not seem a being E.coli Mega-priming with the tag His Positive sequencing 29/06/18 Digestion by DpnI (cleavage of sites not méthylés for the selection of the plasmids of interest for the transformation) Transformation in DH5-alpha of Victoria

Hyd1 : 26/06/18 Reception of Hyd1 + put in water in 10 µL of PPI Q5 PCR to amplify him(it) + Frost(Gel) (problem of size? Migration to be boosted(relaunched)) 27/06/18 Frost(Gel) of Hyd1 which was amplified by Q5 PCR the day before(watch) (it is not Hyd1 who was amplified) Development(Increase) of Hyd1 by Q5 PCR [ Hyd1] = 37 ng. µ L Migration of Hyd1? Obtaining of a far too big band(strip) so that she(it) corresponds to Hyd1. Hypotheses: ™ does not correspond, the duration of strain is far too long! 1min = 1kb!! PCR with Hyd1 by using Taq DNA Polymérase + ™ 60°C and Strain 30 seconds in 72°C + Final strain 5 min. Frost

Second PCR but with a gradient of T °.

28/06/18 Hyd1 squeaky clean up [ Hyd1 dig] = 8 ng. µ L-1 Frost 29/06/18 Ligation + transformation of Hyd1 in DH5a

MdlB:

25/06/18 Digestion-ligation: [ MdlB dig] = 60 ng. µ L-1 / 1.89 / 2.04 [ RFP dig] = 74 ng. µ L 1 / 1.89 / 2.11 Frost with not digested Plasmide (total digestion?), Plasmide + insert ( dig-lig ), Plasmide RFP dig (Enzyme OK?) Transformation in DH5a: 3 conditions: ((Gautier) plasmide + insert) dig-lig / ( Plasmide) dig-lig ( V4) / ( Plasmide) dig ( V4) Q5 PCR on MdlB to redo the stock + squeaky clean up + nanodrop kick + frost

Plasmide RFP: 25/06/18 Transformation (because we suspect a contamination of the tube of the RFP (stemming from the purification) by another plasmide because numerous white colonies are obtained on box: knowing that they should be quite red) of the tube mother of the kit. 26/06/18 Amplification of the pRFP Miniprep (40µL in 133 ng / µ L)

Competent: 25/06/18 Transformation with pLic 60 ( Kn10) in (very first) DH5a V3, V4 (of 18/06) and Gautier version (to test again the competence because the existence of a contaminant is quasi-certain)