Our Experiments

SOP #1 Gene cloning/construction of plasmids

Construction of plasmids by in vivo recombination of DNA fragments with overlapping ends

All the plasmids in this study were assembled by E. coli in vivo assembly of 3 DNA fragments with perfectly matched overlapping ends in the adaptor sequences. After analysis by gel electrophoresis, DNA fragments from PCR reactions were used directly without either DpnI digestion or purification for transformation into high efficiency competent Saccharomyces cerevisae cells

Materials
  • S.cerevisae competent cells
  • DNA fragments
  • Micropippete
  • Heat block
  • Microtube
  • Ice block
  • SOC medium
Methods
  1. DNA fragments (0.6 μL from each of PCR reactions) are mixed prior to transformation.
  2. 1µl of the DNA mixture are added to and gently mixed with 10 μL competent cells, and the cells incubated for 30 min on ice.
  3. Following the standard 30 s heat shock at 42˚C on a metal thermal block and 2 min incubation on ice, 120 μL of SOC medium was added to each transformation.
  4. After vigorously shaking the cells for 1 h at 37˚C, all the cells from each transformation were plated onto a single petri dish, and incubated for 20 h at 37˚C.
Colony analysis
Materials
  • Camera
  • Buffer
  • Magnesium chloride
  • dNTPs
  • Taq DNA polymerase
  • Primers
  • Heat block
Methods
  1. Developed colonies from each transformation are photographed and the number of colonies counted manually on a computer screen.
  2. For the plasmids, colony PCR of randomly selected colonies is performed to assess cloning accuracy.
  3. A stock solution was made to contain 1X buffer, 2.5 mM MgCl2, 0.25 mM dNTPs, 0.05 U/μLTaq DNA polymerase, and 0.2 μM primers.
  4. Cells from colonies are hand-picked up by pipette tips and released to the PCR tube by rubbing the tip against the bottom plastic wall.
  5. Fifteen microliters of the PCR stock solution is added to each tube, followed by heating 95˚C for 10 minutes and 30 standard PCR cycles.
  6. Agarose gel electrophoresis is then used to analyze the result.
  7. PCR primers are designed to reside within 2 separate DNA fragments so that a positive PCR band of expected size reflected the correct joining of 2 DNA fragments.
  8. For effective colony PCR and subsequent analysis by agarose gel, PCR product sizes of around 200 bp will be optimal.
Sequence analysis
Materials
  1. Plasmids from randomly selected colonies of each construct are isolated.
  2. Restriction digestion followed by agarose gel electrophoresis is used to confirm cloning accuracy.
  3. Additionally, 2 isolated plasmids from each construct is sequenced at joint regions by the Sanger method.
  4. Finally, whole plasmid sequencing by Next Generation Sequencing (NGS) on two plasmids–one each from randomly selected plasmid strain.
Notes

The gene sequences (AiOSC1, AiCYP71D2 and candidate Melia ) were extracted from the NCBI database from a study that obtained the sequences using cDNA obtained from RNA extracted from the A.indica plant (Hou et al). The gene fragments were synthesized from Twist Bioscience.

SOP #2 Yeast strain construction

For constructing an efficient dihydroniloticin-producing strain, two strains are to be made, one control strain with the AiOSC1 (2,3 oxidosqualene cylase) and AiCYP71D2 (azadirachta indica cytochrome oxidase) fragment containing plasmid and the AiOSC1 and candidate Melia azederach cytochrome oxidase fragment containing plasmid.

Materials
  • Constructed plasmids
  • Water bath
  • CSM-Ura agar plates
  • Incubator
  • Sterile water
  • Shaking incubator
  • Centrifuge
  • Microcentrifuge
  • Falcon tubes
  • Vortexer
  • Micropipette and pipette tips
  • Polyethylene glycol
  • Lithium acetate
  • Microcentrifuge tubes
  • Spreader
Methodology
  1. Prepare an overnight culture
  2. After 12-16 hrs of growth, the OD600 of the yeast culture is determined and back diluted in 5mls of fresh YPD at OD2
  3. Incubate the falcon tube in the shaking incubator at 30C and 200 rpm for around 4 hours
  4. Harvest the cells by centrifugation at 3000g for 5 mins and resuspend the pellet in 5ml of sterile water
  5. Centrifuge at 3000g for 5 mins to pellet the cells and resuspend pellet in 5mls of sterile water
  6. Transfer the cell suspension to a 1.5mls microcentrifuge tube, centrifuge for 30s at 13,000g and discard the supernatant.
  7. Resuspend the cells in 1.0 ml of sterile water. Centrifuge in a microcentrifuge at 13,000g for 30s and remove the supernatant
  8. Make up sufficient transformation mix as shown in the table below;
Transformation mix Volume (ul)
PEG 3350 (50% w/v) 240
LiAc 1.0M 36
LiAc 1.0M 36
Constructed Plasmid plus sterile water 34
Total Volume 310
Note:

Only 34µl of DNA should be used and the excess supplied with water. To calculate the amount of DNA needed, concentrations should be:

  • 100ng of BB1
  • 100ng of BB2
  • 500ng of donor DNA

10. Add 360ml of transformation mix to each transformation tube and resuspend the cells by vortexing vigorously. Note: Include a negative control tube containing no plasmid

11. Place the tubes in a water bath at 42°C and incubate for 40 minutes.

12. Centrifuge the tubes at 13,000g for 30s in a microcentrifuge and remove the supernatant

13. Add 1ml of sterile water and vortex vigorously

14. Plate 200µl of the cell suspension onto the appropriate CSM-URA plate. Spread with a stick thoroughly and wait for it to get dried a bit and then put it upside down and incubate in a 30°C incubator

15. The positive colonies will be selected on CSM-URA plates to obtain S. cerevisiae strains for dihydroniloticin production.

SOP #3 Functional analysis of candidate cytochrome oxidases

Materials
  1. CM-Ura agar media
  2. Primers
  3. 2% glucose
  4. Galactose
  5. Centrifuge
  6. Tissue grinding machine
Methodology
For Culture:
  1. The positive colonies were selected on CM-Ura plates.
  2. They will then be confirmed by PCR amplification and sequencing.
  3. The recombinant cells were incubated at 30 °C in CM-Ura medium with 2% glucose for 6days.
  4. Subsequently, glucose was substituted with galactose at the logarithmic phase, and the cells were grown at 30 °C for 5 days
    5. For metabolite extraction,
  5. 600 μL of the cell culture was centrifuged at 10,625 × g for 10 min.
  6. The cell pellet was grinded by using a tissue grinding machine for 20 min at 60 Hz and 4 °C with an equivalent amount of EA and 0.5 g of zirconia beads (0.5 mm) and
  7. The mixture will be spun at 12,000g for 5 min and100 μL of the supernatant was extracted for derivatization.
  8. The derivatization using 100 μL of N-methyl-N-[trimethylsilane]-trifluoroacetamide was conducted at 80 °C for 30 min to identify the components of the upper organic phase by GC−MS.

SOP #4 Purification of dihydronilocticin produced from the two strains

Materials
  • Culture plates
  • KOH
  • EtOH
  • EA solution
  • Dichloromethane
  • Silica gel
  • N-hexane
  • Column chromatography apparatus
Methodology
  1. Yeast fermentation liquid from the two constructed strains is cultured and then centrifuged at 5000×g for 10 min.
  2. The pellet is resuspended in 20%KOH/50% EtOH (1:1) for 1 h at 95 °C.
  3. The lysate is extracted twice with the same volume of EA (60−90 °C).
  4. The EA solution is then gathered and then dried under vacuum conditions to produce the yeast extract.
  5. 5 g yeast extracts collected before are dissolved with the least amount of dichloromethane, and then, 5 g of silica gel is weighed and mixed with samples.
  6. The size of the column was 35/20,φ 32 mm, and length 457 mm, and the proportion of mixed silica gel:blank silica gel = 1:10 is used for wet loading.
  7. The silica gel ispoured into the chromatography column and washed with 300mL of n-hexane: EA (15:1, V/V).
  8. Then, 400 mL (about 5 times column volume) is rinsed with n-hexane: EA = 10:1, 5:1, and 4:1.
  9. Then, the yeast extract is rinsed with n-hexane: EA (3:1, V/V) until the dihydroniloticin elution was complete.
  10. The eluent was collected ineach 10 mL tube, and 100 μL of the eluent is dried, derivated, and analyzed by GC−MS.

SOP #5 GC-MS analysis

Materials
  • Gas chromatograph system linked to mass spectrometer with a capillary column
  • Helium (carrier gas)
Methodology
  1. The metabolites in yeast strains are analyzed using a gas chromatograph system linked to the mass spectrometer, equipped with an HP-5 capillary column.
  2. The injection temperature is maintained at 250 °C and the interface temperature at 280 °C.
  3. The temperature of the EI ion source is set as 230 °C and the voltage as70 V.
  4. The temperature of the column is raised at the rate of 20 °C/min from 80 to 280 °C and maintained for 20 min. Helium serves as the carrier gas at a flow rate of 1.5 mL/min.
  5. The scans are recorded for a mass range from 60 to 1000 Da.
References:
  1. Huang F, Spangler JR, Huang AY. In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR. PLoS One. 2017;12(8):1–21.
  2. Hou K, Yu W, Wang X, Liu J, Liu Y, Liu J, et al. Metabolic Engineering of Saccharomyces cerevisiae for de Novo Dihydroniloticin Production Using Novel CYP450 from Neem (Azadirachta indica). J Agric Food Chem. 2022;70(11):3467–76.

Inspirations