Digestion, ligation and transformation of Vh1-pBR322

• 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

• 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

• 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

• 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 Amp^R, E. coli pBBR1MCS-5 Gm^R, V. harveyi JMH626 Cm^R Kan^R and E. coli pRK2073 Spc^R 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 Amp^R: E. coli pBBR1MCS-4 - RFP + V. harveyi JMH626 + E. coli pRK2073
  • Conjugation Gm^R: E. coli pBBR1MCS-5 - RFP + V. harveyi JMH626 + E. coli pRK2073
  • Negative control Amp^R: E. coli pBBR1MCS-4 - RFP + E. coli pRK2073
  • Negative control Gm^R: 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 Amp^R” and “Negative control Amp^R”, Gm and Cmp for “Conjugation Gm^R” and “Negative control Gm^R”. 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.

• 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.

P. pastoris module -- UNDER CONSTRUCTION