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
The gel is not that clear: pSB1C3 without the insert is to high on the gel, but our transformant is a the good weight length : 3k3 bp. We will need to investigate further in order to be sure that we really have pSB1C3 + VhCqsA.
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.
We identified a insert on our native pSB1C3, of around 600 bp, matching with the previous gels that we had. Also, 3 clone are ok: Clone1, 25-1 and 25-2. All of them have the wanted digestion: 2000 bp + 1300 bp. We will further investigate using PvuII, cutting twice, once on the insert, once on the vector.
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 3 plasmids were cut three time, as it was expected for the one containing CqsA sequence. Those 3 clone are validated, and have been stored on glycerol 25%.
Their name will become: CqsA Anna (25-1), CqsA Maxant (C1) and CqsA Teo (25-2 )
Cloning of Vc_CqsA
The gel extraction from the 08/10 was digested using EcoRI and SpeI with the digestion protocol. The quantities for QSP 100 were:
2.5µL of EcoRI
2.5µL of SpeI
30µL of DNA template
10µL of Cutsmart
55µL of water mQ
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:
2µL of ladder 1kb
1µL of loading dye + 5µL of digested DNA
1µL of loading dye + 5µL of digested pSB1C3
First, it is clear that the gel extraction worked fine. Second, it seems that the insert is twice the concentration of the digested plasmid.
Consequently, the ligation was carried out and lasted 50min. The followig quantities were used :
2µL of ligase buffer
1µL of ligase
10.8µL of water mQ
1.4µL of pSB1C3 digested E/S
4.8µL of insert
Finally, Top 10 cells were transformed using the transformation (RbCl-method) protocol. Three aliquots were used:
100µL of competent celles + 10µL of DNA: plate A
100µL of competent cells + 5 µLof TE buffer + 5µL of DNA: plate B
100µL of competent cells + 9µL of TE buffer + 1µL of pSB1C3 non digested (positive control): plate C
After O/N incubation, the followging observation were made on the Petri dishes:
Plate A: 12 transformants
Plate B: 19 transformants
Plate C: cell mat (even with a dilution x10)
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.
Premix for PvuII (for 8 digestions):
2 µL of pSB1C3 E/S + 8 µL of PvuII premix
2 µL of A3 miniprep + 8 µL of PvuII premix
2 µL of A4 miniprep + 8 µL of PvuII premix
2 µL of A5 miniprep + 8 µL of PvuII premix
2 µL of B3 miniprep + 8 µL of PvuII premix
2 µL of B5 miniprep + 8 µL of PvuII premix
2 µL of B6 miniprep + 8 µL of PvuII premix
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:
All of the transformants have the same profil and have both of the expected bands. Finally, the 6 transformants are 6 right clones Vc CqsA.
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.
Solid Bioluminescence assay
Four E. coli precultures were made in LB-Cm (5 mL):
Vh CqsA Max MG1655
Vh CqsA Anna MG1655
Vh CqsA Teo MG1655
pSB1C3 empty MG1655
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:
LB: Vh WT (5 mL)
LB: JMH626 (10 mL)
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:
pipeting of 10 mL of each of the cultures in (x5) eppendorf tube of 2 mL
centrifugation max speed, 5 min
pooling of each resulting supernatants
filtration through a 0.2 µm filter
storage at -20°C
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.
Test plan of the Petri dish (The Vh WT drop is the bioluminescence landmark)
The positive control, JMH626 culture supplemented with Vh WT SN is as bright as the Vh WT drop. Thus, SN from Vh WT can restore bioluminesence to its normal state, as far as human eyes can tell. The negative control shows faint bioluminscence, corresponding either to a basal expression of quorum sensing, or an activation of the quorum sensing by the HA1 ad AI-2 molecules produced naturally by E. coli. The second option seems quite unlikeky as LuxN and LuxQ (receptors of HA1 and AI-2 respectively) are knocked out in the JMH626 genome. SN of clones A and T show a bioluminescence as strong as the negative control, no real activity of C8-CAI-1 can be observed. SN of clone M seems to restore bioluminescence to normal state, as for the positive control.
Additional experiments need to be performed to conclud on these bioluminescence essays. Particularly, bioluminescence of JMH626 without any supplementation has to be tested.
Digestion, ligation and transformation of Vh1-pBR322
Analytical gel: digestion control (07/17)
06/20: pBR322 (amplified the 06/19) was digested with EcoRI-HF and PstI-HF. Gel migration and gel extraction were performed to keep the vector.
07/17: Vh1 part (PCR from IDT part) was digested with EcoRI-HF and PstI-HF, and purified with PCR purif kit.
07/20: Ligation with T4 DNA Ligase and associated buffer from New England Biolabs was performed with pBR322 and Vh1 both digested with EcoRI-HF and PstI-HF, and the ligation mix was transformed into E. coli Stellar competent cells.
07/21: 6 transformants were observed.
07/24: The 6 transformants were grown on liquid culture for plasmid extraction.
07/25:Plasmids were extracted with miniprep kit. Analytical digestion was performed with EcoRI/PstI and PvuII/XhoI. Three transformants had the expected digestion profile and were stored for Vh1-Vh2-pBR322 cloning.
Digestion, ligation and transformation of Vh2-pBR322
Analytical gel before ligation (07/18)
06/20: pBR322 (amplified the 06/19) was digested with EcoRI-HF and PstI-HF. Gel migration and gel extraction were performed to keep the vector.
17/07: Vh2 part (PCR from IDT part) was digested with EcoRI-HF and PstI-HF, and purified with PCR purif kit.
18/07: Ligation with T4 DNA Ligase and associated buffer from New England Biolabs was performed with pBR322 and Vh1 both digested with EcoRI-HF and PstI-HF, and the ligation mix was transformed into E. coli Stellar competent cells.
07/26: Observed transformants were grown on liquid culture.
07/26: Plasmids were extracted with miniprep kit. Analytical digestion was performed with EcoRI/PstI and PvuII/XhoI. All the transformants had the expected digestion profile and were stored for Vh1-Vh2-pBR322 cloning.
Digestion, ligation and transformation of Vh3-pSB1C3
Analytical gel before ligation (07/12)
08/12: Vh3 part (PCR from IDT), previously digested EcoRI-HF and PstI-HF, was digested with EcoRI-HF and SpeI-HF and purified with PCR purif kit. pSB1C3-Ter (BBa_1006) was digested with EcoRI-HF and SpeI-HF and gel extracted. Ligation with T4 DNA Ligase and associated buffer from New England Biolabs and transformation were performed. E. coli Top10 competent cells previously prepared were used.
08/14: Eight transformants were observed on the transformation plates and six of them were grown on liquid culture.
08/15: Plasmid extraction with miniprep kit and analytical digestion of the 6 transformants with EcoRI/PstI and ApaI/NcoI. The two transformants with the expected digestion profile were stored for cloning into conjugative plasmid and for diacetyl production in E. coli.
Digestion, ligation and transformation of Vh1-Vh2-pBR322
08/07: Vh1-pBR322 and Vh2-pBR322 were digested with EcoRI-HF/XhoI (EX) to try the ligation Vh1 part into Vh2-pBR322. Gel migration and gel extraction were performed with these digestion mix.
08/17: Ligations was performed: Vh1 EX (insert) with Vh2-pBR322 EX (vector). T4 DNA Ligase and associated buffer from New England Biolabs were used. E. coli Top10 competent cells previously prepared were used for transformation of ligation mix.
08/18: Transformants were observed on the transformation plate. They were grown on liquid culture for plasmid extraction and analytical digestion.
08/19: Plasmid extraction was performed with miniprep kit for all the transformants. The plasmids were then digested with EcoRI-HF/PstI-HF and BamHI/XhoI. All the transformants had the expected profile. They were stored for a Vh1-Vh2-Vh3 cloning.
Digestion, ligation and transformation of RFP-pBBR1MCS-4 and RFP-pBBR1MCS-5
Analytical gel before ligation (13/09)
pBBR1MCS-4 (1) and pBBR1MCS-5 (1) were used for ligation
08/15: Plasmid extraction was performed using miniprep kit. Preparative digestion of the conjugative plasmids pBBR1MCS-4 and pBBR1MCS-5 with EcoRI-HF and SpeI-HF (ES) from New England Biolabs was performed.
09/14: Ligation with T4 DNA ligase and associated buffer from New England Biolabs was performed with pBBR1MCS-4 ES and pBBR1MCS-5 ES (vector) with RFP ES (BBa_J04450, insert). The RFP was initially digested by the Pichia pastoris module for its own constructions. The ligation mix was transformed into E. coli Top10 competent cells previously prepared.
09/19: Red transformants were observed on the transformation plates for ligation RFP + pBBR1MCS-4 and RFP + pBBR1MCS-5. They were grown on liquid culture, and the two strains (one for pBBR1MCS-4 and one for pBBR1MCS-5) which seem to have the higher RFP activity were used for conjugation.
Conjugation of RFP-pBBR1MCS-4 and RFP-pBBR1MCS-5 in Vibrio harveyi JMH626
09/21: E. coli pBBR1MCS-4 AmpR, E. coli pBBR1MCS-5 GmR, V. harveyi JMH626 CmR KanR and E. coli pRK2073 SpcR helper were grown overnight on 5 mL liquid culture with appropriate antibiotics. E. coli were grown at 37°C and V. harveyi at 30°C.
09/22: Centrifugation and resuspension in LB was performed for each liquid culture. The following conjugation mix were prepared:
Conjugation AmpR: E. coli pBBR1MCS-4 - RFP + V. harveyi JMH626 + E. coli pRK2073
Conjugation GmR: E. coli pBBR1MCS-5 - RFP + V. harveyi JMH626 + E. coli pRK2073
Negative control AmpR: E. coli pBBR1MCS-4 - RFP + E. coli pRK2073
Negative control GmR: E. coli pBBR1MCS-4 - RFP + E. coli pRK2073
Each mix was deposed on a membrane upon a LB plate. The plates were incubated overnight at 30°C.
09/23: The membranes were resuspended into water and the suspensions were spread on a new LB plate with double antibiotic selection: Amp and Cmp for “Conjugation AmpR” and “Negative control AmpR”, Gm and Cmp for “Conjugation GmR” and “Negative control GmR”. The plates were incubated overnight at 30°C.
09/24: An uncountable quantity of colonies was observed on the two conjugation plates. No colonies were observed on the negative controls, which proves that conjugation worked. The cells were not red yet, and the contact inhibition on the plate (because of the too high quantity of colonies) may alter it aspects. Thus, the conjugation cells were replated on another plate with the same double antibiotic selection.
09/25: The replated Vibrio harveyi cells showed a red fluorescent activity compared to a plate with V. harveyi JMH626 without conjugation.
Plates obtained from conjugation Left: negative control / center: conjugation / right: replated conjugation colonies Above: with pBBR1MCS-4 / Below: with pBBR1MCS-5
Comparison of JMH626 from conjugation (RFP-pBBR1MCS-4) and JMH626 (no conjugation)
Comparison of JMH626 from conjugation (RFP-pBBR1MCS-5) and JMH626 (no conjugation)
09/26: V. harveyi JMH626 with pBBR1MCS-4, V. harveyi JMH626 with pBBR1MCS-5, V. harveyi JMH626 without plasmid (control) were grown on liquid culture overnight with antibiotics and LB at 30°C.
PCR of the plasmid pPICZαA
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.
PCR of the insert Odr10-cOT2
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.
PCR of the inserts pGAP-AMP
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:
[pGAP-cOT2] = 185 ng/µl
[pGAP-leucrocine] = 185 ng/µl
[pGAP-cOT2] = 277 ng/µl
The 3 pGAP-AMP were digested with KpnI-HF and BamHI : Digestion protocol
Enzymes were removed while using a PCR purification kit.
Quantification of DNA concentration for ligation
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.
[pPICZα] = 30 ng/µl
[Odr10-cOT2] = 30 ng/µl
[pGAP-AMP] = 70 ng/µl
The Ligation Protocol with T4 DNA Ligase (M0202) has been followed.
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.
Restriction map of transformants
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:
2 clones containing pGAP-DNY15
3 clones containing pGAP-cOT2
4 clones containing pGAP-leucrocine
2 clones containing Odr10-cOT2
Sequencing of one clone of each pGAP-AMP
Result of the sequencing of the Clone n°1 of pGAP-DNY15, the clone n°4 of pGAP-leucro and the clone n°5 of pGAP-Odr10-cOT2.
There is no mutation in the αfactor-AMP sequences so these clones were used to clone AMP in pSB1C3 for iGEM registry.
Clonage of antimicrobial peptides in pSB1C3 iGEM parts
We have 3 AMP genes to clone in pSB1C3.
Odr10-cOT2 is digested to extract the pPICαA containing pFUS1 and cOT2.
pGAP-leucro is digested to extract the pPICZαA containing pGAP and leucro.
pGAP-DNY15 is digested to extract the DNY15 gene.
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.
The 4 400 pb band of Odr10-cOT2 was purified: pPICZα-pFUS1
The 400 pb band of Odr10-cOT2 was purified: cOT2
The 2 400 pb band of pGAP-leucro was purified: pPICZα-pGAP
The 300 pb band of pGAP-leucro was purified: leucro
The 300 pb band of pGAP-DNY15 was purified: DNY15
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.
Integration of genic construction in P. pastoris genome
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
pGAP-DNY15
pGAP-leucro
pGAP-cOT2
Odr10-cOT2
pPICZα without insert was used as a negative control.
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
RT-PCR experiment
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.
AMP production and cytotoxicity tests
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:
Protocols
PCR
Introduction
We used the Thermo Scientific Phusion High-Fidelity DNA Polymerase. Amplification of templates with high GC content, high secondary structure, low template concentrations or long amplicons may require further optimization.
Materials
PCR thermocycler
PCR tubes
nuclease-free water
dNTP
Phusion HF Buffer (X5) or GC Buffer
Primers (both forward and reverse)
Template DNA
Phusion polymerase
Procedure
All components should be mixed and centrifuged prior to use. It is important to add Phusion DNA Polymerase last in order to prevent any primer degradation caused by the 3´→ 5´ exonuclease activity.
Phusion DNA Polymerase may be diluted in 1X HF or GC Buffer just prior to use in order to reduce pipetting errors.
Use of high quality, purified DNA templates greatly enhances the success of PCR.
We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C).
Component
50 μL
final concentration
Nuclease-free water
qs 50 μL
Buffer Phusion HF (5X)
10μL
1X
10 mM dNTPs
1 μL
200 μM
10 μM Forward primer
2.5 μL
0.5 μM
10 μM Reverse Primer
2.5 μL
0.5 μM
DNA template (10 ng/μL)
1 μL
10ng
Phusion DNA Polymerase
0.5 μL
1.0 U/0.5 μL of reaction
Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary
Transfer PCR tubes from ice to a PCR machine with the block preheated to 98°C and begin thermocycling:
