Team:CityU HK/Protocol
Experimental Protocols
1. Genome-wide screening of P450 homologs
Materials:
- Bioinformatic tool: cblaster
Procedure:
1. Carried out in silico analysis of various fungal genomes to search for variecolin biosynthesis gene clusters similar to that of Aspergillus aculeatus ATCC 16872 (CBS 172.66). The structure of the variecolin biosynthetic gene cluster in A. aculeatus is shown below:
2. Installed the cblaster tool in the lab PC along with the download of Python and DIAMOND softwares on the computer for performing local searches.
3. Set up the parameters by
for pre-search configuration.
for pre-search configuration. 4. Ran
using the
module.
using themodule.5. Generated the databases of local sequence from a set of genomes with
module forlocal searches.
module forlocal searches.6. Estimated the genomic neighbourhood size using
the module and generated the cluster heatmap for comparison with the target gene.
the module and generated the cluster heatmap for comparison with the target gene.Figure 1. Cluster heatmap visualization of cblaster search results
Note: A more intense colour (blue) of the heatmap colour bar indicates a higher similarity to the target gene.
7. Selected certain genes with moderate similarity to the target gene for later experiments.
References
1. Gilchrist, C. L. M., Booth, T. J., Wersch, B., Grieken, L., Medema, M. H., Chooi, Y. H. (2020).cblaster: a remote search tool for rapid identification and visualisation of homologous gene clusters. Bioinformatics Advances 1(1), vbab016.
2. Genomic DNA Extraction
Materials:
- Micropipettes
- Micropipette tips
- 1.5 mL microcentrifuge tubes
- Microcentrifuge
- Heating block
- Vortex mixer
- Takara Plant DNA Isolation Reagent (Cat. 9194)
- Emericella variecolor NBRC 32302 for synthesis of variecolin (for vrcA and vrcB gene amplification)
Table 1. Choice of P450 genes from different fungal strains for synthesis of variecolin analogues
Procedure (manufacturer’s instructions):
1. Fungal mycelia were crushed using micropipette tips.
2. Added 400 µL of Extraction Solution 1, and vortexed for 5 seconds. Flicked the tube with a finger to resuspend the fungal mass.
3. Repeated step 2 with addition of 80 µL of Extraction Solution 2 and 150 µL of Extraction Solution 3.
4. Centrifuged at lower speed for 2 seconds and incubated at 50 °C for 15 minutes.
5. Centrifuged at 12,000 rpm for 15 minutes at 4 °C.
6. Collected 400 µL of the upper clear aqueous layer.
7. Added an equal volume of isopropanol to the aqueous layer and mixed gently.
8. Centrifuged at 12,000 rpm for 10 minutes at 4 °C. Discarded the supernatant.
9. Washed the pellet with 1 mL of 70% ethanol.
10. Centrifuged for 3 minutes. Discarded the supernatant.
11. Dried the pellet and resuspended in 50 µL of TE buffer.
3. P450 gene amplification
3.1. PCR of P450 genes
Materials:
Note: The reaction components were stored on ice.
Procedure:
1. Programmed the thermocycler for PCR according to the following thermocycling conditions.
2. Analyzed PCR products by gel electrophoresis at 100 V for 25 minutes with the following components for gel loading.
3. Observed the PCR products in gel documentation system.
3.2. DNA gel extraction
Materials:
- Micropipettes
- Micropipette tips
- 1.5 mL microcentrifuge tube
- Microcentrifuge
- 60 °C incubator
- FastPure Gel DNA Extraction Mini Kit (Vazyme, DC301)
Procedure (manufacturer’s instructions):
1. Cut out the DNA fragment from agarose gel using a clean blade. Transferred the gel piece into a 1.5 mL microcentrifuge tube and recorded the weight of gel.
2. Added an equal volume of Buffer GDP to the tube.
(Note: Volume : weight of gel is 1:1)
3. Incubated the sample at 50-60 °C for 30 minutes. Inverted the tube 3 times during incubation. Vortexed the gel mixture and centrifuged the tube.
4. Transferred the gel solution to a FastPure DNA Mini Columns-G column inserted in a 2-mL collection tube. Centrifuged at 12,000 rpm for 1 minute. Removed the supernatant and returned the column to the collection tube. Repeated the steps with the remaining gel solution.
5. Removed the supernatant. Added 300 µL Buffer GDP to the column. Stand for 1 minute. Centrifuged at 12,000 rpm for 1 minute.
6. Removed the supernatant and put the column back to the collection tube. Added 700 µL of Buffer GW (with ethanol) into the column. Centrifuged at 12,000 rpm for 1 minute.
7. Repeated step 6.
8. Removed the supernatant and centrifuged at 12,000 rpm for 2 minutes.
9. Transferred the column into a clean 1.5 mL EP tube. Added 20 µL of Elution Buffer (Pre-warmed to 55 °C) to the column and stand for 2 minutes. Centrifuged at 12,000 rpm for 1 minute.
10. Discarded the column and stored the purified DNA at -20 °C.
4. DNA cloning by homologous recombination
Materials:
- Lysogeny broth (LB medium)
- E. coli DH5α
- MgCl2
- Nanodrop 2000
- Paper towers
- DMSO
- Liquid nitrogen
- Sterilized tubes
4.1. E. coli DH5α competent cell preparation
Procedure:
1. Pipetted 10 μL competent cells into 10 mL LB medium and incubated at 37 °C with vigorous shaking.
2. Pipetted 1 mL starter culture into a 500 mL sterile flank and add 500 μL MgCl2. Incubated for 5 hours at 30 °C shaker.
3. Transferred the culture vessel to an ice-water bath for 10 minutes when when OD600 is approximately 0.55
4. Harvested the cells by centrifuge at 2500 g at 4 °C for 10 minutes.
5. Poured off medium and drain the remaining medium on a stack of paper towers.
6. Resuspended the cells gently in 20 mL of ice-cold transformation buffer.
7. Harvested the cells by centrifugation at 2500 g for 10 minutes at 4 °C.
8. Poured off medium and drain the remaining medium on a stack of paper towers.
9. Resuspended the cells gently in 5 mL of ice-cold transformation buffer.
10. Added 375 μL of DMSO, mixed and stored it on ice for 10 minutes.
11. Aliquoted cells into a pre-chilled EP tube quickly and snap-frozen the cells with liquid nitrogen.
12. Stored at -80 °C.
4.2. Construction of plasmids
4.2.1 Construction of pAdeA-vrcA plasmid
Materials:
- E. variecolor NBRC 32302
- Vector: pTAex3 and pAdeA,
- ClonExpress Ultra One Step Cloning Kit (Vazyme Biotech Co., Ltd)
Procedure:
1. Amplified vrcA (terpene synthase gene) gene from gDNA of E. variecolor NBRC 32302.
2. Introduced vrcA gene into pTAex3 vector (pTAex3-vrcA) using a ClonExpress Ultra One Step Cloning Kit (Table 1).
3. Amplified the sequence containing the amyB promoter (PamyB)-vrcA-amyB terminator (TamyB) from the pTAex3-varA recombinant plasmid.
4. Ligated the amplified sequence into pAdeA vector and construct a pAdeA-PamyB-vrcA-amyB plasmid (pAdeA-vrcA).
Table 1. Recombination protocol
Note: 50 °C for 15 minutes and hold at 4 °C
4.2.2 Construction of pTAex3-HR-P450 plasmid
Materials:
- pTAex3-HR vector
- P450 genes from different fungal strains (listed in table)
Procedure:
Each P450 gene was ligated into pTAex3-HR by a ClonExpress Ultra One Step Cloning Kit (Table 1).
4.3. P450 gene transformation into E.coli DH5α
Materials:
- Thermocycler
- Gel tank with power supply
- 1.5 mL microcentrifuge tube
- Microcentrifuge
- 65 °C incubator
- FastPure Plasmid Mini Kit (Vazyme Cat. DC201-01)
Procedure:
1. Thawed E.coli competent cells on ice.
2. Transferred the recombination products (5 µL) into EP tube with 80 µL of competent cells and put it on ice for 15 minutes.
3. Heat shocked the mixture at 42 °C for 45 seconds and then put the tube back on ice.
4. Spread 85 µL of bacterial mixture on LB/amp agar plates
5. Incubated the agar plates overnight at 37 °C.
4.4. Colony PCR
1. Picked four separate colonies from each agar plate.
2. Performed colony PCR using the following materials and thermocycling conditions.
3. Performed gel electrophoresis.
4. Picked a single colony that contains a successful construct and inoculated in 10 mL LB broth (with ampicillin) at 37 °C overnight.
4.5. Plasmid extraction
*FastPure Plasmid Mini Kit (Vazyme Cat. DC201-01) (manufacturer's instructions)
1. Prepared triplicates of glycerol bacterial stocks.
2. Centrifuged 5-10 mL overnight cultured bacterial cells at 10,000 × g for 1 minutes and removed the supernatant.
3. Added 250 µL of Buffer P1 (add RNase A before use) and mix thoroughly.
4. Added 250 µL of Buffer P2 and mix thoroughly.
5. Added 350 µL of Buffer P3 to neutralize Buffer P2, mixed gently until a flocculent white precipitate forms, centrifuge at 13,000 × g for 10 minutes.
6. Transferred the supernatant from step 4 to the Filtration Column and centrifuged at 13,000 × g for 60 seconds. Discarded the filtrate.
7. Added 600 µL of Buffer PW2 (with ethanol added in) to the Filtration Column and centrifuged at 13,000 × g for 60 seconds. Discarded the filtrate.
8. Repeated step 6.
9. Removed residual washing buffer PW (1,2) by centrifuging the empty Filtration Column for 1 minutes at 13,000 × g.
10. Put the Column into a clean 1.5 mL microcentrifuge tube and added 50 µL of Elution Buffer to the center of the Column membrane, incubated at room temperature for 2 minutes, centrifuge at 13,000 × g for 1 minute.
11. Removed the Filtration Columns, store DNA at -20 °C.
5. Fungal transformation
Materials:
- DPY medium
- PDA-Ade-Arg medium
- TF solution 0
- TF solution 1 (15 mL tube)
- TF solution 2
- TF solution 3
- M medium (bottom layer)+sor
- M medium (top layer)+sor
5.1. pAdeA-vrcA transformation1
Procedure was the same as pTAex3-HR-P450 transformation, except crRNA / tracrRNA / Cas9 was not added.
5.2. pTAex3-HR-P450 transformation2
Procedure:
a) NSAR1 strain transformation preparation
Culture the glycerol stock of NSAR1 strain on DPY medium:
1. Added 100 mL of DPY medium in an autoclaved 500 mL Erlenmeyer flask.
2. Added 100 μL of NSAR1 glycerol stock, and put it in the 30 °C shaking incubator for approximate 4 days.
b) Transformation of NSAR1 strain with target plasmids
Culture the glycerol stock of NSAR1 strain on DPY medium:
1. Added 20 mL of the well-suspended culture into a 20 mL autoclaved cotton-stopped syringe, and filtered the culture to obtain mycelial portion.
2. Added 10 mL of filter sterilized TF solution 1 into a 50 mL Falcon tube and transferred the mycelial portion of the filtered culture into the solution.
3. Incubated the solution for approximately 2 hours in a 30 °C water bath with 60 rpm incubator.
4. Filtered the incubated suspension to obtain the aqueous portion in a clean 50 mL Falcon tube.
5. Centrifuged the aqueous portion at 2000 rpm in 20 °C for 10 minutes.
6. Discarded the supernatant and add 10 mL of TF solution 2 to resuspended the deposit and invert gently.
7. Centrifuged the aqueous portion at 2000 rpm under 20 °C for 10 minutes.
8. Added 15 mL of TF solution 2 to resuspended the deposit after discard the supernatant.
9. Repeated step 7 and add 2 mL of TF solution 2.
10. Loaded 2 μL of the resuspended culture onto bacterial count slide and counted the spore culture.
11. Added 400 μL aliquots of the suspension into 15 mL Falcon tubes. Then added 8 μL of each P450 gene-containing plasmid solution into the culture and mix well.
(Note: It depends. ~5 μg / plasmid, the total plasmid solution should less than 10 μL + crRNA / tracrRNA / Cas9)
12. Incubated the culture at room temperature for approximately 30 minutes.
13. Added 500 μL, 500 μL, and 1700 μL of TF solution 3 into the culture sequentially and mixed every time. Incubated the mixture for an additional 20 minutes, then add 5 mL of TF solution 2 and mix.
14. Centrifuged the mixture at 2000 rpm under room temperature for 10 minutes.
15. Removed the supernatant, and resuspended the deposit with 500 μL of TF solution 2.
16. Added 5 mL of M medium (40 °C) and invert gently.
17. Placed the mixture on M medium plate and incubated it at 30 °C for 4 to 5 days.
18. Picked maximum eight fungal spores from M plate with methionine and sorbitol and place them on an M plate with methionine. Incubated the culture at 30 °C for 3 days.
19. Picked fungal spores from M plate with methioine and placed them on DPY plate. Incubated the cultured at 30 °C for around 3 days.
References
1. Matsuda, Y., Bai, T., Phippen, C. B. W., Nødvig, C. S., Kjærbølling, I., Vesth, T. C., Andersen, M. R., Mortensen, U. H., Gotfredsen, C. H., Abe, I., Larsen, T. O. (2018). Novofumigatonin biosynthesis involves a non-heme iron-dependent endoperoxide isomerase for orthoester formation. Nature Communications, 9(1), 2587–10.
2. Wei, X., Matsuyama, T., Sato, H., Yan, D., Chan, P. M., Miyamoto, K., Uchiyama, M., & Matsuda, Y. (2021). Molecular and computational bases for spirofuranone formation in setosusin biosynthesis. Journal of the American Chemical Society, 10.1021/jacs.1c08336.
6. HPLC, GC-MS, and NMR analysis of metabolites from transformants
6.1. Metabolites extraction
Materials:
- Ultrasonic sonicator
- Sonication tubes
- Eppendorf vials
- Micropipettes
- Micropipette tips
- Microcentrifuge
- Vortex mixer
Procedure:
1. Cut a slice of agar from each transformant and put into a 15 ml tube.
2. Added ethyl acetate (EtOAc) to immerse agar.
3. Sonicated the tubes for 1 hour.
4. Poured the solutions out and transferred into 1.5 mL Eppendorf tubes.
5. Evaporated the solutions with nitrogen gas.
6. Added 100 µL of methanol to dissolve the samples. Vortexed the samples.
7. Centrifuged the samples at 13,500 rpm for 4 minutes.
8. Pipetted supernatant for HPLC analysis.
6.2. High Performance Liquid Chromatography (HPLC)
analysis of metabolites
Materials:
- HPLC autosampler vials
- Analytical HPLC instrument: Dionex Ultimate 3000 UHPLC system (Thermo Scientific)
- Column: Kinetex C18 column (100 Å, 2.6 µm, 2.1 i.d. x 100 mm; Phenomenex)
- Column temperature: 40 °C
- Flow rate: 0.4 mL/min
- Solvent: 20 mM formic acid (Solvent A) and acetonitrile containing 20 mM formic acid (Solvent B)
Procedure:
1. Ran the HPLC program using the following composition of mobile phase.
6.3. Gas Chromatography - Mass Spectrometry (GC-MS) analysis of metabolites
Materials:
- n-hexane
- GC syringe
- GC instrument: Agilent 7890A/5975C system
- Column: HP-5ms capillary column (0.25 mm i.d. × 30 m, 0.25 μm film thickness)
- Carrier gas: Helium
- Average velocity: 27.573 cm/s
- Electron impact ionization: 70 eV
Procedure:
1. Ran the GC program. Held the temperature at 100 °C for 3 minutes.
2. Gradually increased the temperature at a rate of 14 °C/min until reaching 268 °C. Held at this temperature for 6 minutes.
6.4. Nuclear Magnetic Resonance spectroscopy (NMR)
Materials:
- Chloroform solvent (CDCl3)
- Bruker Ascend Avance III HD spectrometer
Procedure:
Each purified compound was dissolved in chloroform solvent (CDCl3) and our department’s technician, Dr. M. K. Tse, conducted the NMR analysis for us. Briefly, NMR spectra were obtained 600 MHz (1H)/150 MHz (13C) with a Bruker Ascend Avance III HD spectrometer, and chemical shifts were recorded with reference to solvent signals (1H NMR: CDCl3 7.26 ppm, 13C NMR: CDCl3 77.0 ppm).
7. Large scale growth of fungal mass for extraction of natural products
Materials:
- 30 °C incubator
- Ultrasonic Sonicator
- Rotary evaporator
- Preparative HPLC instrument:
- Column: XBridge BEH C18 OBD Prep Column (100 Å, 5 µm, 19 i.d. x 250 mm; Water Corporation)
- Flow rate: 10 mL/min
- Solvent: Acetonitrile and water
Procedure:
1. Cultivated 150 plates of each transformant at 30 °C for one week.
2. Crushed fungal cultures and agar medium into pieces and immersed in ethyl acetate.
3. Extracted the metabolites twice by sonication.
4. Filtered the extract and removed ethyl acetate with a rotary evaporator.
5. Separated the crude extract by flash chromatography or silica-gel column chromatography.
6. Purified each target compound by preparative HPLC using different methods as shown below:
8. Antimicrobial assay
Materials:
- LB agar
- Mueller Hinton (MH) Broth
- 2 mL Eppendorf tube
- Ampicillin
- 96 well plates
- Microplate reader
Bacteria used:
Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Escherichia coli
Procedure:
a) Preparation of bacterial cultures
1. Thawed bacterial glycerol stock and plating each bacterial strain into individual LB agar plate. Incubated overnight at 37 °C.
2. Inoculated a single bacterial colony from LB agar plate to MH broth and cultured overnight at 37°C.
3. Adjusted the optical density (O.D) of bacterial culture with MH broth to 0.5 MH (equivalent to 1.5 × 108 colony-forming unit /mL) which can be stored at 4 °C for 15 days without loss of cell viability.
b) Preparation of samples (variecolin analogues)
1. Dissolved a certain amount in 100 μL DMSO.
2. Transferred 50 μL of sample into another EP tube.
3. Added 50 μL of DMSO to make a 2-fold dilution.
4. Repeated step 3 to prepare serial 2-fold dilutions of each sample.
c) 1.5 MIC measurement
1. Added 5 μL of different concentrations of sample into each well (the black box) except column 11 and 12.
2. Added 95 μL of diluted bacterial suspension into each well (the red box) and mix gently.
3. Placed the 96 well plate at 37 °C for 24 hours.
4. Measured the absorbance (O.D) at 600 nm with a microplate reader.
Table 1 Final concentration (μg/mL) of sample in each well
9. Anticancer activity screening
9.1. Cell seeding
Materials:
- CO2 incubator
- Micropipettes
- Micropipette tipss
- Multichannel pipette
- 96-well microplates
- Haemocytometers
Procedure:
1. Pre-warmed the media and trypsin at 37 °C.
2. Used microscope to observe the breast cancer cell MCF-7 in T-75 flask to ensure no contamination.
3. Removed the medium by GMEM and washed flask with 1 mL EDTA.
4. Added 3 mL of trypsin and left the flask in incubator for 4 minutes to allow the cells to detach.
5. Used microscope to check the state of the cells. Gently tapped the flask if cells were not detached completely.
6. Added 7 mL media and mixed it gently to neutralize the trypsin.
7. Transferred the mixture into a 15 mL tube and mixed gently.
8. Transferred 15 μL mixture into a 1.5 mL tube and added 15 μL of 1X erythromycin. Mixed well by reverse pipetting.
9. Used a haemocytometer to count the cells.
10. Calculation for cell counts was done using the equation C1V2 = C2V2 [C1 = cells counted, V1 = volume of cell mixture, C2 = 6 × 104 cells/mL (6000 cells per well), V2 = total volume of microplates + excess].
11. Mixed well the cell stock before diluting the cells. Added 1.3 mL V1 into media till the whole mixture volume is 21 mL (V2).
12. Added the cell mixtures into the central part of the microplates using a multi-channel pipette.
13. Added water into the unfilled wells.
14. Closed the microplates and labeled it. Put the microplates into incubator for 24 hours.
9.2. Addition of drug (variecolin analogues) to MCF-7 cells
Materials:
- 96-well microplates
- Micropipettes
- Micropipette tips
- Multichannel pipette
Procedure:
1. Placed the media into 40 °C water bath.
2. Used equation C1V1 = C2V2 to calculate the volumes of drug and media.
Note: V2 = 0.9 mL for 3 replicates
3. Checked the condition of cells using a microscope.
4. Added 900 μL media into tubes. Total 6 tubes for 6 drugs.
5. Removed V1 μL of media and added V1 μL of the drug to a concentration of C2.
6. Inverted the tube to mix well.
7. Removed all the old media of MCF-7 by aspiration.
8. Added the media into the microplates except the first column which is for drug.
9. Added 200 μL drug into first column.
10. Preformed serial dilution by transferring 100 μL drug from first column into second column and mixed well, then transferred the 100 μL solution from second to the third column and repeated the step for other columns except the last two columns, which are used as control.
11. Put microplates into incubator.
9.3. Colorimetric microculture assay
Materials:
- MTT reagent
- DMSO solution
- 96-well microplates
- Micropipettes
- Micropipette tips
- Multichannel pipettes
- Pipette controller
- Aspiration system
Procedure:
1. Mixed 36 mL of medium with 4 mL of MTT reagent.
2. Removed the old media from the wells.
3. Added 100 μL of the mixture of media and MTT reagent to the each of the wells.
4. Waited for 1 hour. Observed the colour change of MTT reagent.
5. Performed aspiration to remove the solution.
6. Added DMSO solution to make cells evenly distributed.
7. Measured the absorbance in a spectrophotometer to determine the colour intensity of the samples.
References
1. Sîrbu, A., Palamarciuc, O., Babak, M. V., Lim, J. M., Ohui, K., Enyedy, E. A., Shova, S., Darvasiová, D., Rapta, P., Ang, W. H., Arion, V. B. (2017). Copper(II) thiosemicarbazone complexes induce marked ROS accumulation and promote nrf2-mediated antioxidant response in highly resistant breast cancer cells. Dalton Transactions : An International Journal of Inorganic Chemistry, 46(12), 3833–3847.
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