Design and orders

• Design of the gBlocks and launching of the synthesis order for IDT
• Design of the PCR oligos

Design and orders

• Design of the gBlocks and launching of the synthesis order for IDT
• Design of the PCR oligos

Taking care of V. harveyi

• Reception and storage of V. harveyi BB120 (WT) and V. harveyi JMH626 (ΔcqsA ΔluxQ ΔluxN) CmR at -80 °C.
• Investigation of V. harveyi antibiotics resistances.

Amplifying pSB1C3

• Reception of IDT gBlocks and storage
• Preparative work with the cloning vectors : Amplification and stock of pSB1C3

Taking charge of the lab and of V. harveyi

• Growth kinetic assay in order to determine the adequate NaCl concentration for V. harveyi growth.
• Reception of IDT gBlocks and storage
• Transformation of DH5α competent cells with pYFP, pDsRed, pPIZα, pBR322 and amplification of the plasmids. The plasmids are double digested with the appropriate restriction enzymes.

Cloning

• pPICzα, jmp62-DsRed (pDsRed) and jmp62-YFP (pYFP) were transformed into E.coli DHAα in order to amplify them.  
• Bacteria grown were then put into liquid culture to do miniprep.
• Digestion BamHI/EcoRI to check pPICzα gel migration profile
  Digestion XbaI/NheI to check jmp62-DsRed gel migration profile
  Digestion BamHI/NheI to check jmp62-YFP gel migration profile

Plasmids preparation

• Transformation of DH5α competent cells with pSB1C3 and amplification of the plasmids. The plasmids are double digested with the appropriate restriction enzymes.
• Scale up of the preparative digestion, in order to have more DNA matrix.
• Reception of the PCR primer.

Plasmids preparation

• Transformation of DH5α competent cells with pGAP, pSB1C3 and amplification of the plasmids. The plasmids are double digested with the appropriate restriction enzymes.
• Scale up of the preparative digestion, in order to have more DNA matrix.
• Reception of the PCR primer.

Cloning

• Preparative digestion of pPICZα with BamHI/BglII to clone our gene followed by a gel extraction to purify the open plasmid.
• Glycerol stocks of E.coli DHAα containing pPICZα, pDSRed and pYFP were made after liquid culture.

Cloning VhCqsA

• Amplification of the IDT gblocks and optimisation of the PCR cycle condition for VhCqsA part
• Digestion of the PCR amplicons and ligation with the appropriate cloning vector.

Assembly of V. harveyi gene circuit : starting the sub-cloning work

• Amplification of the IDT gblocks and optimisation of the PCR cycle condition for VhCqsA part
• Digestion of the PCR amplicons and ligation with the appropriate cloning vector.
• Cloning attempt #1 and #2 to sub-clone  Vh1, Vh2 and  Vh3 in their respective vector.  

Cloning

• PCR of DsRed and YFP with primers containing restriction sites. PCR products for these genes have right size. PCR products are purified.
• PCR of our parts (cOT2 ; leucrocine ; DNY15; Odr10) from IDT GBlocks. All PCRs succeeded.
• cOT2 ; leucrocine ; DNY15 ; Odr10 were digested BamHI/BglII to be inserted in pPICZα.
• Transformation of pPICZα-cOT2 ; pPICZα-leucrocine ; pPICZα-Odr10 and pPICZα-DNY15 in E. coli DH5α
• Miniprep of liquid culture transformants
• Digestion BmaHI/BglII to check the gel migration profile.
  → It appears that the plasmid was well digested but the insert does not appear. There is an intense band around 1300bp that might correspond to supercoiled plasmid. To conclude it seems like pPICZα closed itself without the insert: BamHI and BglII are compatibles.
• New primers were ordered replace by PCR the BglII site by KpnI site to remove the possibility for the plasmid to close itself.

Cloning VhCqsA

• Optimization of PCR cycle
• Cloning of VhCqsA into pSB1C3 : positives clones grew 

Assembly of V. harveyi gene circuit : continuing the sub-cloning work

• Assembly of V.harveyi gene circuit: Analysis of the transformants of V.harveyi module (attempt #1 and #2) by checking of the restriction map.
• New attempt (attempt #3) to sub-clone Vh1, Vh2 and  Vh3 in their respective vector.  

Cloning

• While waiting for the new primers, another try was made using for the ligation a 1:10 ratio (pPICZα:insert).
• Transformants appeared in all plates except for Odr10.
• Miniprep and digestions of transformants to check the gel migration profile.
• We obtained 2 clones pPICZα-DNY15. The gel confirmed that D1 and D2 are clones with the entire plasmid pPICZa A open only in BamHI site with one insert in this site. D1 has the insert in the good direction and D2 has the insert in the opposite direction.
• Clones D1 and D2 were stored in glycerol.
• At the end of the week, PCR with new primers containing KpnI instead of BglII were made for all the parts (cOT2 ; leucrocine ; DNY15 ; Odr10). All the PCR worked perfectly, PCR tubes were pooled. Odr10 has secondary products which need gel extraction.
• Digestion of plasmid and parts with KpnI and BamHI

Assembly of V. harveyi gene circuit : continuing the sub-cloning work

• Assembly of V.harveyi gene circuit: Analysis of the transformants of V.harveyi module (attempt #1 and #2) by checking of the restriction map.
• New attempt (attempt #3) to sub-clone Vh1, Vh2 and  Vh3 in their respective vector.  

Cloning

• Ligation and transformation of pPICZα-cOT2 ; pPICZα-leucrocine ; pPICZα-Odr10 and pPICZα-DNY15.
• Miniprep of transformants and gel migration were made. The ladder was clear but there was no DNA on tranformants colums.
• Nanodrop analysis showed that there was not enough DNA.
  → The hypothesis is that the liquid culture was too long so E. coli lost the plasmid.

Experiments with P. pastoris

• We did plates of YPD with increasing concentration of zeocin to select P. pastoris clones which integrated the most copies of the construction.
• Preparation of competent yeast (Pichia pastoris)
• Digestion of D1 clone (pPICZα-DNY15) with HindIII to integrate the plasmid construction at the AOX1 promotor in P. pastoris genome.
• Electroporation of competent yeast with the digested DNA.

Expressing VhCqsA (2nd)

• Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)
• SDS page fo the protein overexpression of our clones (fail)

Assembly of V. harveyi gene circuit : A new strategy

• Amplification of the IDT gblocks, digestion of the PCR amplicons.
• Amplification and digestion of the sub-cloning vectors.
• Ligation and transformation in E. coli STELLAR competent cells (attempt #8).

Cloning

• This time transformation were made with E. coli XL1 Blue which are supposed to be more efficient.
• No DNA after gel migration like the previous week.

Experiments with P. pastoris

• Colony PCR of P. pastoris clone which grew on YPD zeocin. → All clones have the gene construction in their genome!
• The 11 clones of Pichia were grown on plates with 250 µg/mL, 500 µg/mL and 1000 µg/mL to see which one has the best integration of the construction. All clones grew onto 250, 500 and 1000 µg/mL zeocin.

Expressing VhCqsA (3rd)

• Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)

Assembly of V. harveyi gene circuit : The first transformant

• TA cloning (attempt #9): amplification of the IDT gblocks with KAPA2G polymerase , digestion of the PCR amplicons. Ligation with pGEM vector TA cloning vector.
• Analysis of the transformants (attempt #8 and #9)  by checking the restriction map.
• The part 1 and 2 were successfully cloned in pBR322 and pGEM.  

Cloning

• PCR were made again of all parts in order to have enough materials to do ligation.
• This time lot of DNA was digested.
• Several strategies were tried to obtain pPICZα-cOT2 ; pPICZα-leucrocine  and pPICZα-Odr10 with DHAα and XL1 Blue strains.
• At the end of the week we got 2 clones of pPICZα-Odr10 in E. coli DHAα

Experiments with P. pastoris

• The supernatant of P. pastoris containing the DNY15 gene was extracted and then concentrated via rotavapor and freeze-drying.
• In a 24 wells plate, kinetic growths of V. harveyi were followed with or without P. pastoris supernatant to check the bactericide effect of DNY15. → this first try was not perfectly made
• Inhibition test with the same supernatant was made on petri dish. → this first try was not perfectly made

Expressing VhCqsA (aborted) + Cloning of VcCqsA

• Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR aborted : no growth of bacteria

Step 2 : reconstruction of CqsS Rc by ligating Vh2 to Vh1 in pBR322.

• Storage of the transformants with Vh1 and Vh2 in glycerol at -80°C.
• Amplification, digestion of pBR322-Vh1,  pGEM-Vh2 and ligation in E. coli TOP10 competent cells (attempt A)
• Transformation of Vh3 (attempt #10) in E. coli DH5α competent cells
• Analysis of the transformants (attempt #A and #10)  by checking the restriction map.

Cloning

• Different strategies were made again to obtain pPICZα-cOT2 and pPICZα-leucrocine in E.coli. → Several clones of pPICZα-cOT2 and pPICZα-leucrocine were obtained!!!
• Liquid culture of DHAα containing pSB1C3 was made to get enough material to do cloning of our parts in it.
• Ligation of YFP in pPICZα-Odr10 and in pPICZα-DNY15 instead of the AMP gene.
• Integration of AMP genes in pSBIC3 (=iGEM parts).

Experiments with P. pastoris

• Cytotoxicity test of DNY15 was tried again with kinetic growths of V. harveyi.
• Cytotoxicity test of DNY15 was tried again on petri dish: No effect of DNY15 can be seen.
• Genomic integration of pPICZα-cOT2 , pPICZα-leucrocine and pPICZα-Odr10.

Expressing VhCqsA (4th time) + VcCqsA cloning

• Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)
• PCR on VcCqsA

Step 2 CqsS Rc assembly in progress…still no success for Vh3

• Amplification, digestion of pBR322-Vh1  pGEM-Vh2 and ligation in E. coli TOP10 competent cells (attempt B and C)
• Transformation of Vh3 (attempt #11 to #13) in E. coli TOP10 competent cells using an alternative restriction enzyme :  PstI-HF restriction enzyme is remplaced  by SpeI.

Cloning

We obtained the YFP gene after the GAP promotor and the FUS1 promotor in E. coli.

Experiments with P. pastoris

• pGAP-YFP and pFUS-YFP were transformed in P. pastoris.
• P. pastoris clones were obtained with pPICZα-Odr10 ; pPICZα-leucrocine and pPICZα-cOT2 but the PCR colony didn’t work. After several trials and controls we concluded that the DNA extraction the way we did it was not enough to do a PCR of the GAP promotor in the pichia genome.
• A kinetic study of V. harveyi growth was made using pichia supernatant. → A difference was observed but the experiment needs to be done again.

Cloning of VcCqsA

• From ligation to transformation : transformants grew.

Step 2 CqsS Rc assembly in progress, preparation of the conjugation

• Analysis of the transformants (attempt #13)  by checking the restriction map : the part 3 is successfully cloned in pSB1C3. The transformant is stored  in glycerol at -80°C.
• Amplification of pBBR1MCS-4 and pBBR1MCS-5 for cloning of Vh3 and for a conjugation assay.
• Ligation of Vh3 in pBBR1MCS-4 and transformation in E. coli TOP10 (attempt α).
• Amplification, digestion of pBR322-Vh1  pGEM-Vh2 and ligation in E. coli TOP10 competent cells (attempt D to F)

• P. pastoris clones were obtained with the YFP reporter behind both promotor: GAP and FUS1.
• Fluorescence test was made but it didn’t work.
• A cytotoxicity test was made on V.harveyi with pichia-AMP supernantant but we had trouble because of volumes used.
• We tried a new DNA extraction protocol to do the PCR colony on P. pastoris and it did work.

Confirming VcCqsa clone + Expressing VhCqsA (5th time)

• Transformant were good
• Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)

• Analysis of the transformants (attempt #F)  by checking the restriction map : the part Vh1+Vh2 is successfully cloned in pBR322. The transformant is stored  in glycerol at -80°C.
• Amplification, digestion of pBR322-Vh1+Vh2, pSB1C3-Vh3. Ligation and transformation (attempt #1 and #2) in E. coli TOP10 competent cells.
• Ligation of Vh3 in pBBR1MCS-4 and transformation in E. coli TOP10 (attempt β and γ)
• Vh3 part validation : diacetyl production assay by NMR detection with E. coli BL21.

• Cytotoxicity test was made again but this time on a petri dish. We had a halo for the antibiotic but P. pastoris’ supernatant didn’t stop V. harveyi’s growth.
• We’ve tried to characterize pFUS1 promoter in different media but we got no positive result.

Expressing VhCqsA (6th time) + Bioluminescence

• Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)
• Optimisation of Vibrio harveyi JMH626 bioluminescent assay

• 2nd  diacetyl production assay by NMR detection with E. coli BL21.
• Success of the conjugation assay of pBBR1MCS-4.

• This time, we tested the production of DNY15 in YPD with 40g/L of glucose instead of 30g/L because publications which use pGAP as a promotor to produce AMP sometimes have a higher production with more glucose.
• Cytotoxicity tests using the supernatant from the 40g/L glucose culture media was made. Supernatants were filtered beforehand and half was freeze-dried.  We got a weak halo for the DNY15 production in glucose 40 and supernatant freeze-dried!

Expressing VhCqsA (7th time)

• Production of C8-CAI-1 in minimal media, and analysis on MS (fail) extration on LLE dichloromethane and analysis on NMR (fail)

• 3rd  Diacetyl production assay by NMR detection with E. coli MG1655.
• Amplification of pBBR1MCS-4 and pBBR1MCS-5.

• Because we still had trouble integrating some parts in P. pastoris’ genome we tried the integration via electroporation and via chemical way at the same time.
• We launched one again P. pastoris culture in YPD 40g/L and 50g/L glucose to try producing more DNY15.

Bioluminescence assay

• Trial on the expression of C8-CAI-1 with induction on bioluminescence on liquid media : fail

• 4th diacetyl production assay by NMR detection with E. coli MG1655 in M9 media supplemented with xylose and pyruvate.
• Transformation of E. coli TOP10 competent cells with GFP from iGEM kit
• Ligation and transformation of pBBR1MCS-4 to RFP from iGEM kit in E. coli TOP10 competent cells for conjugation to V. harveyi.
• Preparation of the clones for sequencing.

• We prepared sample to sequence the AMP genes we wanted to give to the iGEM registry.
• Culture media from P. pastoris culture of last week were freeze-dried. And the cytotoxicity test on petri dish was made again but this time, even the positive control with antibiotic didn’t work.
• P. pastoris clones were obtained with pAOX1-leucrocine, pAOX1-cOT2, Odr10-pFUS-RFP.

Bioluminescence assay

• Trial on the expression of C8-CAI-1 with induction on bioluminescence on plate : success

• Results of the 4th Diacetyl production assay by NMR detection: a small peak of diacetyl is visible.
• 5th diacetyl production assay by NMR detection with E. coli MG1655 in M9 media supplemented with xylose and pyruvate.
• Results of ligation of RFP in conjugative plasmid: all transformants are red. Two transformants are stored at -80°C in order to be used for conjugation assay.
• Preparative work for the ligation of Vh3 in pBBR1MCS-4 with fresh material

• AMP genes were sent to be sequenced.
• P. pastoris producing DNY15 was cultured to do a RT-qPCR experiment to compare it to the WT P. pastoris.
• New cytotoxicity tests on petri dish were made but this time it didn’t work because of the paper disk we used. Once again the positive control didn’t work.
• Cultures in BMMY adding methanol every 24h for 120h were made for both pAOX1-leucrocine and pAOX1-cOT2 P.pastoris clones. Half of each supernatant was stored at 4°C and the other half was evaporated to concentrate 10 times.

• Observation by microscopy of V. harveyi (rfp) transformant
• Ligation of pBBR1MCS-4 and Vh3 at 16°C overnight, and transformation in competent E. coli (Stellars)

• New cytotoxicity tests on petri dishes were made with the leucrocine and cOT2 supernatants. This time with appropriate discs. Positive control did not work again.
• New cytotoxicity tests on petri dishes were made but using pure chloramphenicol. A weak halo was seen. We found out that the V. harveyi strain we used to do cytotoxicity test was maybe! the JMH626 one which was chloramphenicol-resistant.
• RT-PCR experiment was made.
• To test the Odr10-RFP system and activate pFUS, nitrogen depletion cultures were done and two sources of nitrogen were tested: sulfate ammonium and glutamine. The fluorescence following was made in a plate reader.
• New cytotoxicity tests on petri dishes were made starting from a fresh V. harveyi cuture. Leucrocine, cOT2 and D-NY15 (40g/L and 50g/L glucose) productions were tested. Halos are clearly visible for D-NY15 40g / L not concentrated and concentrated 10 times.
• A 24 well plate was launched by adding leucrocine, cOT2 and D-NY15 supernatant to V. harveyi cultures in exponential phase.