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Revision as of 15:01, 6 October 2017
Experiments
One colony has grown O/N on the Petri dish. This colony was picked in liquid LB media culture with Cm.
Possible explanations for the low amount of colonies:
Another ligation was carried out today, using more DNA and another Cm concentration. The ligation was processed as follows:
Ligation protocol
A ratio 1:10 of plasmid/insert was chosen:
Competent cells transformation was processed following the transformation protocol.
NB: because of the lack of a right 42°C water bath, the heatshock step was made at 37°C for 40 sec.
2 different plates were made: one at [Cm] = 25 g/L and the other at [Cm] = 12.5 g/L
6 Clones, 2 from CM25 (25-1 and 25-2) and 4 from Cm 12.5 (12-1, 12-2, 12-3 and 12-4 ) that appear on Cm25 and Cm12.5 were grow on plate and on liquid media (5mL) LB + Cm 25 in order to miniprep them with the Miniprep protocol
Digestions were made on the plasmids obtained with the digestion protocol.
In order to confirm the insert, two more digestion were made, with PvuII (which cut once on pSB1C3, and not on the insert) and SacI (which cut once on the insert, and once on pSB1C3) following the digestion protocol
The gel extraction from the 08/10 was digested using EcoRI and SpeI with the digestion protocol. The quantities for QSP 100 were:
No heat inactivation were made. Instead, a PCR puriffication was processed using Sigma GenEtutTM PCR Clean Up kit. Afterward, a DNA quantification gel were made containing:
Consequently, the ligation was carried out and lasted 50min. The followig quantities were used :
Finally, Top 10 cells were transformed using the transformation (RbCl-method) protocol. Three aliquots were used:
After O/N incubation, the followging observation were made on the Petri dishes:
Hence, eight transformants from each plate were put on liquid LB-Cm medium for subsequent mini prep. They were named A 1 to 8 and B 1 to 8. The transformants named were minipreped. The resulting DNA solutions were stored at -20°C.
After a quick analysis gel of all the miniprep, without any digestion (data not shown), 6 transformants seems to have the right plasmid lenght: A3, A4, A5, B3, B5, B6.
Thus, these transformants were digested with PvuII and E/P to assess the right insertion of Vc CqsA.
Each tube was put in a water bath at 37°C for 1h. The resulting digestion were put on a 1% agarose gel as follows:
Clones A4 and A5 were named Vc CqsA 1 and Vc CqsA 2 and put in liquid culture to be stored at -80°C the next day.
Four E. coli precultures were made in LB-Cm (5 mL):
Each tubes were complemented with 208 µL of a sterile glucose solution to reach a final concentration of 10 g/L. The tubes were put at 37°C O/N. In addition, two V. harveyi precultures were made:
They were put at 30°C O/N. OD of the O/N precultures were taken at 8.06 am. Therefore, fresh LB-Cm-Glc flasks of 10 mL were inoculated to reach OD = 0.1. (final glucose concentration in the medium: 10 g/L). Time of inoculation: 8.32 am. As OD = 0.3 had already been passed at 10.40 am, IPTG induction was made immediatly (final concentration : 0.5 mM). At 1 pm, the cultures were at the end of their exponential phase. Thus, the supernatants could be retrieved following the next steps:
OD of the V. harveyi O/N precultures were checked again at 11.10 am: JMH626: 4.15, Vh WT:3.11
(x5) LB flasks of 10 mL were inoculated with JMH626 to reach OD = 0.1. In addition, (x1) LB flask of 10 mL was inoculated with Vh WT, also at OD = 0.1. Time of inoculation: 11.28 am. When the OD was around 0.7, each of the 10 mL cultures were centrifugated at 4500 rpm for 6 min. The resulting supernatants were discarded while the pellets were resuspended with 5 mL of fresh LB medium and 5 mL of SN (one flask = one SN). Then, the cultures were put at 30°C. The whole process of resuspension was over at 3.30 pm.
In addition, 80 µL of the resuspended JMH cultures were dropped-off on a Petri dish and put at 30°C O/N. Acting as a landmark of bioluminescence, 80 µL of a Vh WT culture were also put on the Petri dish. For liquid cultures, the positive control showed bright bioluminescence as expected. Meawhile, all the cultures with the clones SN stayed dark. Regular checkings didn't bring more information. The Petri dish incubated O/N at 30°C was observed.
Additional experiments need to be performed to conclud on these bioluminescence essays. Particularly, bioluminescence of JMH626 without any supplementation has to be tested.
PCRs were processed on the pPICZαA coming from miniprep (cf section 1): PCR protocol
The aim of this PCR was to obtain a linear plasmid with 2 specific restriction sites at its extremity (BamHI and KpnI).
Once the PCR was over a gel migration (at 100 V, during 30 min) was performed: Gel migration protocol
pPICZα was not pure but we decided to do a gel extraction of the band of interest after the digestion. So PCR products went through a PCR purification.
Nanodrop of the PCR product: [pPICZαA] = 260,5 ng/µL
Once we knew the DNA concentration we decided to do the preparative digestion (BamHI-KpnI-HF): Digestion protocol
A gel migration (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol
The band of interest at 1800 pb was purified via the Gel extraction protocol.
PCRs was processed on the part pGAP-Odr10-pFUS1-cOT2 received from IDT: PCR protocol
Once the PCR was over a gel migration (at 100 V, during 30 min) was performed: Gel migration protocol
Odr10-cOT2 was not pure but we decided to do a gel extraction of the band of interest after the digestion. So PCR products went through a PCR purification.
Nanodrop of the PCR product: [Odr10-cOT2] = 277,3 ng/µL
Once we knew the DNA concentration we decided to do the preparative digestion (BamHI and KpnI-HF): Digestion protocol
A gel migration (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol
The band of interest at 2800 pb was purified via the Gel extraction protocol.
PCRs were processed on the parts pGAP-leucrocine, pGAP-cOT2, pGAP-DNY15 received from IDT: PCR protocol
pGAP-AMP = pGAP-leucrocine, pGAP-cOT2, pGAP-DNY15
A gel migration was performed, in order to check the PCRs. Gel migration protocol
The PCR products of the pGAP-AMPs were pure so a PCR purification was made after the different PCR had been pooled.
Nanodrop of the PCR products:
The 3 pGAP-AMP were digested with KpnI-HF and BamHI : Digestion protocol
Enzymes were removed while using a PCR purification kit.
pPICZαA and Odr10-cOT2 coming from a gel extraction were quantify thanks to a gel because the nanodrop couldn’t give precise quantity.
Their concentration was estimated at 30 ng/µl while pGAP-AMP went through the nanodrop after the PCR purification.
Finally, competent cells transformation of E. coli DH5α was processed following the protocol: Transformation protocol. (NB: competent cells were plated on LB medium [zeo] = 25 g/L.)
Several clones were put in liquid culture of 5 ml of LB with [zeo] = 25 g/l.
Plasmids extraction was performed on the previous culture of 5 mL E. coli transformants grown on LB liquid media with zeocin : Miniprep protocol
Digestion were then processed as follows: Digestion protocol
We do have:
There is no mutation in the αfactor-AMP sequences so these clones were used to clone AMP in pSB1C3 for iGEM registry.
We have 3 AMP genes to clone in pSB1C3.
Digestions were processed as follows (SpeI-HF and EcoRI-HF): Digestion protocol
A gel migration (at 100 V, during 30 min) was performed with all digestions mix, in order to separate the DNA fragments: Gel migration protocol
The Gel extraction protocol has been followed.
Ligation & transformation of DNY15; cOT2 and leucrocine in pSB1C3 (Ligation Protocol with T4 DNA Ligase (M0202))
Finally, competent cells transformation in E. coli DH5α was processed following the protocol: Transformation protocol. NB: competent cells were plated on LB medium [cm] = 25 g/L. 6 clones of each plate were grown into LB+Chloramphenicol (5 ml each tube).
Plasmids extraction was performed on the previous culture of 5 mL E. coli transformants grown on LB liquid media with chloramphenicol.
Miniprep was then processed as follows: Miniprep protocol
Digestions were then processed as follows: Digestion protocol
A gel migration (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol. All clones with good gel migration profile were kept.
To integrate a genic construction in P. pastoris genome, the first step is to linearize the plasmid at the localization of the integration (for instance in our case we linearize in the GAP promotor to have an integration in this promotor).
Each genic construction was digested as follows: Digestion protocol
Once digested, each construction is electropored in P. pastoris following the Electroporation protocol.
Yeast after transformation were plated on YPD + [zeo] = 250 g/l. Every transformants were then replated on YPD + [zeo] = 1000 g/l to select clones with a higher rate of genomic integration.
pGAP-DNY15 clones on [zeo] = 1000 g/l
The PCR colony protocol was used to verify that every clone had the insert.
pGAP-DNY15: A gel migration to amplify the pGAP-DNY15 gene integrated in pichia genome (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol
Odr10-cOT2: A gel migration to amplify the Odr1 0-cOT2 gene integrated in pichia genome (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol
The P. pastoris strains containing pGAP-DNY15 or the empty plasmid were grown in 50mL YPD 40g/L of glucose for 4 days at 30°C in an agitating incubator. RNAs of both were extracted and a RT-PCR experiment has been done.
D-NY15 production was performed with the P. pastoris clone E obtained previously. Two cultures were carried out: one for D-NY15 E and the other for the negative control (P. pastoris transformed with pPICZα without insert). Each clone was inoculated in 50 mL YPD 40 g/L glucose and grown for 4 days at 30 °C in an agitating incubator. 15mL of each supernatant culture were stored at 4°C while 35mL were freeze-dried and then resuspended in 3.5mL of water.
Cytotoxicity test on plate were made using the disc diffusion technique. 200µL of V. harveyi WT (OD600 around 0.5) 100 times diluted were spread on LB agar. Discs were soaked with supernatant, placed on the petri dish and incubated overnight at 30°C.
The same experiment was made again spreading 200µL of V. harveyi WT no diluted:
Ligation, transformation of Vh_quorum
Ligase buffer 2x
2µL
Vector
1.2µL
Insert
5µL
Ligase
1µL
Purified water
10.8µL
Cloning of Vc_CqsA
Premix for PvuII (for 8 digestions):
Solid Bioluminescence assay
Digestion, ligation and transformation of Vh1-pBR322
Digestion, ligation and transformation of Vh2-pBR322
Digestion, ligation and transformation of Vh3-pSB1C3
Digestion, ligation and transformation of Vh1-Vh2-pBR322
Digestion, ligation and transformation of RFP-pBBR1MCS-4 and RFP-pBBR1MCS-5
Conjugation of RFP-pBBR1MCS-4 and RFP-pBBR1MCS-5 in Vibrio harveyi JMH626
Each mix was deposed on a membrane upon a LB plate. The plates were incubated overnight at 30°C.
PCR of the plasmid pPICZαA
PCR of the insert Odr10-cOT2
PCR of the inserts pGAP-AMP
Quantification of DNA concentration for ligation
The Ligation Protocol with T4 DNA Ligase (M0202) has been followed.
Restriction map of transformants
Sequencing of one clone of each pGAP-AMP
Clonage of antimicrobial peptides in pSB1C3 iGEM parts
Integration of genic construction in P. pastoris genome
RT-PCR experiment
AMP production and cytotoxicity tests