Experiments

Experiments

  • Plate test of CTX against Geotrichum candidum and Penicillium digitatum
  • Test of CTX against Candidatus Liberibacter in leafs
  • Plate test of CTX against potential hosts
  • PCR: Polymerase Chain Reaction
  • NEBuilder
  • Miniprep
  • Digestion
  • Ligation
  • Electrophoresis
  • Bacterial transformation
  • Bacterial expression
  • Chromatography purification
  • SDS-PAGE
  • CTX purification by TEV protease
  • CTX measurement by Nanodrop
  • Filamentous fungi protoplasting
  • Filamentous fungi transformation
  • Filamentous fungi DNA extraction
  • Protein quantification by Lowry assay
  • Yeast transformation
  • V5-Immunodetection
  • Yeast cultivation
  • CTX purification by chemical cleavage
  • Solid-state fermentation using orange peel

Plate test of CTX against Geotrichum candidum and Penicillium digitatum

Materials

  • CTX peptide (2 mg/mL in Milli-Q water)
  • Imazalil fungicide (2 mg/mL)
  • Sterile Milli-Q water
  • BD liquid culture medium
  • Spore suspensions of P. digitatum and G. candidum (1×10⁶ spores/mL)
  • Sterile 96-well plates
  • Sterile Eppendorf tubes
  • BOD incubator (25 °C)

Preparation of Microdilution Solutions

Different final CTX concentrations (10, 25, and 50 µM) were prepared in a final volume of 200 µL per well, starting from a CTX stock solution at 2 mg/mL. Using the standard dilution formula, the following volumes were calculated:

  • 50 µM: 2.0 mg/mL × V1 = 0.115 mg/mL × 0.2 mL → V1 = 11.5 µL
  • 25 µM: 2.0 mg/mL × V1 = 0.0575 mg/mL × 0.2 mL → V1 = 5.75 µL
  • 10 µM: 2.0 mg/mL × V1 = 0.023 mg/mL × 0.2 mL → V1 = 2.3 µL

Samples Preparation

For each treatment, solutions were prepared at a total volume of 600 µL (sufficient for three wells of 200 µL each):

Concentration Volume of Stock (CTX, C+, or Milli-Q) Volume of BD Medium
10 µM 6.9 µL 578.1 µL
25 µM 17.25 µL 567.75 µL
50 µM 34.5 µL 560.5 µL

Three groups of solutions were prepared:

  • CTX (test peptide)
  • C+ (positive control with Imazalil)
  • CN (negative control with Milli-Q water)

Plate Setup

The prepared tubes should be vortexed to ensure proper homogenization. The solutions are then distributed into the wells as follows:

  • 195 µL of the prepared solution (CTX, C+, or Milli-Q water)
  • 5 µL of the spore suspension (1×10⁶ spores/mL)
  • Final volume per well: 200 µL.

After distribution, 5 µL of each fungal suspension is added to the designated wells (see concentrations listed in Materials).

Plate Layout:

1 2 3 4 5 6 7 8 9 10 11 12
Ctx 10 Ctx 10 Ctx 10 H20 H20 H20 C+ 10 C+ 10 C+ 10
Ctx 25 Ctx 25 Ctx 25 H20 H20 H20 C+ 25 C+ 25 C+ 25
Ctx 50 Ctx 50 Ctx 50 H20 H20 H20 C+ 50 C+ 50 C+ 50
medium medium medium
medium medium medium
medium medium medium

Notes: Each condition was tested in triplicate, totaling nine replicates for each fungal treatment (one assay per fungus).

Incubation and Evaluation

The plates were incubated in a BOD chamber at 25 °C, without photoperiod, for an initial period of 48 hours. The first visual assessment was performed after 2 days. In addition, after 7 days, the well contents will be re-inoculated onto solid BD medium to observe fungal growth and determine whether the treatment effect was fungistatic (growth observed after removal of the peptide) or fungicidal (no growth observed).

Test of CTX against Candidatus Liberibacter in leafs.

To evaluate whether the antimicrobial peptide CTX could reduce the bacterial load of Candidatus Liberibacter asiaticus (CLas) in infected citrus leaves.

Materials

  • Citrus leaves naturally infected with CLas
  • CTX peptide solution (100 mM, prepared according to design stage)
  • Tetracycline solution (100 µg/mL; positive control)
  • Sterile 50 mL Falcon tubes
  • Sterile forceps and scissors
  • DNA extraction kit
  • PCR reagents and thermocycler
  • Sterile pipette tips and Eppendorf tubes

Procedure

  1. Sample Preparation
    • Excise infected citrus leaves into ~15 mg sections from the petiole tip (baseline sample).
    • Place remaining leaf tissue into sterile 50 mL Falcon tubes.
  2. Treatment Setup
    • Add treatment solutions as follows:
      • CTX solution (100 mM)
      • Positive control: tetracycline (100 µg/mL)
      • Negative control: sterile buffer without antimicrobial
    • Ensure that each Falcon tube contains the excised tissue fully submerged.
  3. Incubation
    • Incubate tubes for 3 days at room temperature.
    • Keep tubes protected from light to prevent degradation of compounds.
  4. DNA Extraction
    • After 3 days, excise ~15 mg tissue sections again from treated leaves.
    • Extract DNA from both baseline and post-treatment samples using a standard plant DNA extraction kit.
  5. Molecular Analysis
    • Perform conventional PCR to confirm presence of CLas before and after treatments.
    • Use literature-reported tetracycline treatment as positive reference for bacterial load reduction (Bové, 2006).

Plate test of CTX against potential hosts.

To evaluate the potential toxicity of the antimicrobial peptide CTX against the host organisms Escherichia coli, Saccharomyces cerevisiae, and Aspergillus oryzae, and to determine the minimum inhibitory concentration (MIC) that affects their growth.

Materials

  • CTX peptide (stock solution as provided by Prof. Eduardo Festozo)
  • E. coli culture (OD-adjusted inoculum)
  • S. cerevisiae culture (OD-adjusted inoculum)
  • A. oryzae spore suspension (1×10⁶ spores/mL)
  • Sterile 96-well microplates
  • Appropriate growth media for each organism
  • Incubators set at:
    • 37 °C for E. coli
    • 30 °C for S. cerevisiae and A. oryzae
  • Sterile pipette tips and Eppendorf tubes

Procedure

1. Preparation of Inocula

  • Adjust E. coli and S. cerevisiae cultures to ~0.1 OD₆₀₀.
  • Prepare A. oryzae spore suspension at 1×10⁶ spores/mL.

2. Preparation of CTX Solutions

  • Dilute CTX stock solution to the working concentrations defined during the design stage (e.g., 10 µM, 25 µM, 50 µM).

3. Plate Setup

  • Dispense treatments into wells of a sterile 96-well plate (final volume: 200 µL per well).
  • Inoculate wells as follows:
    • E. coli: add inoculum corresponding to ~0.1 OD₆₀₀.
    • S. cerevisiae: add inoculum corresponding to ~0.1 OD₆₀₀.
    • A. oryzae: add 5 µL of spore suspension (1×10⁶ spores/mL).
  • Include positive and negative controls (medium without CTX and/or reference antifungal if applicable).

4. Incubation

  • Incubate plates under optimal conditions for each host:
    • E. coli: 37 °C
    • S. cerevisiae and A. oryzae: 30 °C
  • Maintain incubation without shaking unless otherwise required by culture conditions.

5. Growth Monitoring

  • Assess microbial growth visually at defined time points.
  • Compare biomass development between treated and untreated controls to determine inhibition.

PCR: Polymerase Chain Reaction

To perform routine PCR using Phusion™ DNA Polymerase, following the recommended conditions for optimal amplification. This protocol is suitable for standard templates; however, amplification of GC-rich templates, sequences with strong secondary structure, low template concentrations, or long amplicons may require further optimization. The NEB Tm calculator should be used to determine accurate annealing temperatures (source).

Materials

  • Phusion™ DNA Polymerase
  • 5X Phusion HF Buffer or GC Buffer
  • 10 mM dNTPs
  • Forward and reverse primers (10 µM each)
  • Template DNA (< 250 ng)
  • DMSO (optional, for difficult templates)
  • Nuclease-free water
  • Mineral oil (if PCR machine does not have a heated lid)
  • Thermocycler preheated to 98 °C

Reaction setup

Component 20 µL Reaction 50 µL Reaction Final Concentration
5X Phusion HF or GC Buffer 4 µL 10 µL 1X
10 mM dNTPs 0.4 µL 1 µL 200 µM
10 µM Forward Primer 1 µL 2.5 µL 0.5 µM
10 µM Reverse Primer 1 µL 2.5 µL 0.5 µM
Template DNA variable variable ≤ 250 ng
DMSO (optional) 0.6 µL 1.5 µL 3%
Phusion DNA Polymerase 0.2 µL 0.5 µL 1 U / 50 µL
Nuclease-free water to 20 µL to 50 µL —

Thermocycler setup:

STEP TEMP TIME
Initial Denaturation 98°C 30 seconds
25-35 Cycles 98°C
Annealing °C
72°C
5-10 seconds
15 seconds
15-30 seconds/kb
Final Extension 72°C 5-10 minutes
Hold 4-10°C

NEBuilder® : HiFi DNA Assembly for dsDNA Fragments

This protocol is designed for assembling standard dsDNA fragments (>120 bp) with overlaps of 15–30 bp using the NEBuilder® HiFi DNA Assembly Master Mix (cloning kit or bundle for large fragments).

  • For fragments smaller than 120 bp, alternative methods such as ssDNA bridge or annealed oligonucleotide protocols are recommended.
  • To generate a custom protocol with optimal fragment concentrations and volumes, use the NEBuilder® Protocol Calculator (online tool).

Optimal DNA Quantities

  • For 2–3 fragments: 0.03–0.2 pmols total DNA
  • For 4–6 fragments: 0.2–0.5 pmols total DNA
  • Assembly efficiency decreases with increasing number or length of fragments.

Reaction Setup

Prepare the assembly reaction on ice:

Component 2–3 Fragment Assembly* 4–6 Fragment Assembly** NEBuilder Positive Control
Recommended DNA molar ratio vector:insert = 1:2 vector:insert = 1:1 —
Total amount of DNA fragments 0.03–0.2 pmols (X µL) 0.2–0.5 pmols (X µL) 10 µL
NEBuilder HiFi DNA Assembly Master Mix 10 µL 10 µL 10 µL
Nuclease-free water 10 – X µL 10 – X µL 0
Total Volume 20 µL 20 µL*** 20 µL
* For 2–3 fragment assemblies.
** For 4–6 fragment assemblies.
*** Total volume may vary depending on fragment input.

Plasmid Miniprep

All miniprep were performed using Monarch® Plasmid DNA Miniprep Kit.

Notes:

  • All centrifugations at 16,000 × g (~13,000 RPM).
  • Keep B3 (Neutralization Buffer) at 4 °C (contains RNase A).
  • Do not vortex after lysis or neutralization → prevents shearing chromosomal DNA.

Step-by-Step

  1. Pellet cells
    • Spin 1–5 mL culture (≤15 OD units) for 30 s.
    • Discard supernatant.
  2. Resuspend
    • Add 200 µL Resuspension Buffer (B1, pink). Vortex until no clumps remain.
  3. Lyse
    • Add 200 µL Lysis Buffer (B2, blue/green).
    • Gently invert 5–6× until solution is dark pink/clear.
    • Incubate 1 min (no longer!).
  4. Neutralize
    • Add 400 µL Neutralization Buffer (B3, yellow).
    • Gently invert until yellow + precipitate forms.
    • Incubate 2 min.
  5. Clarify lysate
    • Spin 2–5 min.
  6. Bind DNA
    • Transfer supernatant to spin column.
    • Spin 1 min (or 30 s to save time). Discard flow-through.
  7. Wash 1
    • Add 200 µL Wash Buffer 1.
    • Spin 1 min (30 s if saving time).
  8. Wash 2
    • Add 400 µL Wash Buffer 2.
    • Spin 1 min.
    • Dry column
    • Optional: re-spin 1 min to remove ethanol/salts.
  9. Elute DNA
    • Add ≥30 µL Elution Buffer (or nuclease-free H₂O, pH 7–8.5) to center of column.
    • Wait 1 min, spin 1 min.
    • For plasmids ≥10 kb: preheat buffer to 50 °C, incubate 5 min before spin.

DNA Digestion with Restriction Enzymes

Recommended DNA Input

  • Digest 0.2–1.5 µg DNA with a 2–10× excess of enzyme.
  • Standard reaction volume: 20 µL.

Reaction Setup (20 µL)

Add components in the following order:

  • Nuclease-free water: 16–16.5 µL
  • 10X Restriction Enzyme Buffer: 2 µL
  • Substrate DNA: 1 µL (~1 µg)
  • Restriction Enzyme: 0.5–1 µL (5–10 units)
  • Total Volume: 20 µL

Procedure

  1. Assemble reaction on ice.
  2. Mix gently and briefly spin down.
  3. Incubate at the optimal temperature for the enzyme (typically 37 °C) for 1–16 hours.

DNA Insert Ligation into Vector DNA

Reaction Setup (20 µL total)

  • Linear vector DNA: 20–100 ng
  • Insert DNA: 1:1 to 5:1 molar ratio over vector
  • 10X T4 DNA Ligase Buffer: 2 µL
  • T4 DNA Ligase: 1 Weiss Unit
  • Nuclease-free water: to 20 µL

Agarose Gel Electrophoresis (with SYBR Safe)

Materials & Equipment

  • Agarose
  • 1X TAE or TBE buffer
  • SYBR Safe DNA Gel Stain
  • DNA samples + loading buffer
  • DNA ladder
  • Casting tray + comb
  • Electrophoresis chamber + power supply
  • Microwave
  • Blue-light transilluminator

Procedure

  1. Preparing the Gel
    1. Weigh 1 g agarose and mix with 100 mL 1X TAE.
    2. Microwave until dissolved (~1–3 min, swirl intermittently).
    3. Cool to ~50 °C.
    4. Add SYBR Safe (per manufacturer’s instructions, geralmente 1:10,000 → 10 µL por 100 mL).
    5. Pour into the casting tray with a comb.
    6. Let solidify (20–30 min at RT or 10–15 min at 4 °C).
  2. Loading the Gel
    1. Place gel in an electrophoresis box and cover with 1X TAE.
    2. Add loading buffer (1:5 ratio) to each DNA sample.
    3. Load:
    4. Lane 1: DNA ladder
    5. Other lanes: samples
    6. Ensure no bubbles enter wells.
  3. Running the Gel
    1. Run at 80–150 V until the dye front reaches ~75% of gel length (~1 h).
    2. Remember: DNA runs to red (positive electrode).
  4. Visualization
    1. Place gel on a blue-light transilluminator (preferred, minimizes DNA damage).
    2. Alternatively, use UV light (short exposure).
    3. Compare sample bands to DNA ladder to estimate fragment sizes.

Bacterial Transformation (Heat Shock)

Equipment

  • Shaking incubator at 37 °C
  • Stationary incubator at 37 °C
  • Water bath at 42 °C
  • Ice bucket with ice
  • Microcentrifuge tubes
  • Sterile spreading device

Reagents

  • LB agar plates (with appropriate antibiotic)
  • LB or SOC medium (without antibiotic)
  • Competent cells (stored at −80 °C)
  • DNA to transform (10 pg – 100 ng)

Procedure

  1. Preparation
    • Thaw competent cells on ice (20–30 min).
    • Warm antibiotic agar plates to room temperature (optional: incubate at 37 °C).
  2. DNA Mixing
    • Add 1–5 µL DNA to 20–50 µL competent cells.
    • Mix gently by flicking tube (do not pipette).
  3. Incubation on Ice
    • Incubate DNA–cell mixture on ice for 20–30 min.
  4. Heat Shock
    • Place bottom ½–⅔ of tube in 42 °C water bath for 45 sec.
    • Immediately return to ice for 2 min.
  5. Outgrowth
    • Add 250–1000 µL LB or SOC medium (no antibiotic).
    • Incubate in shaking incubator at 37 °C for 45 min.
  6. Plating
    • Plate 50 µL on one LB agar plate (with antibiotic).
    • Plate the remaining volume on a second plate.
    • (Optional) If volume is too large, centrifuge gently and resuspend in smaller volume before plating.
  7. Incubation
    • Incubate plates overnight at 37 °C.
    • Expect colonies the next day.

Cultivation & Induced Expression (IPTG)

Objective

To express a recombinant protein (e.g., sfGFP–CTX from pIGEM001) in E. coli BL21(DE3) using IPTG induction under controlled cultivation conditions.

Materials

  • E. coli BL21(DE3) carrying the expression plasmid (e.g., pIGEM001–sfGFP–CTX)
  • LB medium (liquid and agar)
  • Kanamycin (50 µg/mL final; adjust to match your selection marker)
  • Sodium phosphate buffer (50 mM, pH 7.0; optional, for pH stabilization)
  • IPTG (1 M stock solution)
  • Baffled Erlenmeyer flasks (≥5× culture volume)
  • Shaking incubator (30–37 °C)
  • Spectrophotometer (OD₆₀₀ measurement)

Culture Setup (reference volumes)

Stage Volume Medium Additives
Starter 5 mL LB Kan (50 µg/mL)
Pre-culture 25–50 mL LB Kan (50 µg/mL)
Main culture 250–1000 mL LB + 50 mM sodium phosphate (pH 7.0) Kan (50 µg/mL)

Procedure

  1. Starter Culture (Overnight)
    1. Inoculate 5 mL LB + Kan (50 µg/mL) with a single colony.
    2. Incubate at 37 °C, 200 rpm, 12–16 h.
  2. Pre-Culture (Optional but Recommended)
    1. Inoculate 1:100 from starter into 25–50 mL LB + Kan.
    2. Grow at 37 °C, 200 rpm until OD₆₀₀ ≈ 0.4–0.8 (~2–4 h).
  3. Main Culture
    1. Inoculate 1:50–1:100 into 500 mL LB + 50 mM phosphate buffer + Kan in a 2–3 L baffled Erlenmeyer flask.
    2. Incubate at 37 °C, 200–220 rpm until OD₆₀₀ ≈ 0.6.
  4. Induction
    1. Add IPTG to 0.5 mM final (from 1 M stock: 0.5 mL/L).
    2. Shift incubation temperature to 30 °C.
    3. Express for 16 h (overnight) at 200 rpm.

      4. Optimization Tips

      • For improved solubility: lower induction temperature (16–25 °C) and/or reduce IPTG (0.05–0.2 mM); extend induction to 16–20 h.
      • For faster but riskier expression: induce at 37 °C for 2–4 h (higher risk of inclusion bodies).
      • For fluorescent proteins (e.g., sfGFP), monitor emission directly during culture.
  5. Harvest
    1. Measure final OD₆₀₀; collect a sample for SDS-PAGE.
    2. Centrifuge culture at 4,000–6,000 × g, 10–15 min, 4 °C.
    3. Discard supernatant; freeze pellets at −20/−80 °C or proceed directly to lysis and purification (e.g., IMAC).

Protein purification by IMAC

Objective

To purify sfGFP–CTX fusion protein from E. coli lysates using immobilized metal affinity chromatography (IMAC) with cobalt resin.

Materials

  • Talon cobalt resin (1 mL)
  • Ultrapure water
  • Binding buffer: 20 mM sodium phosphate (pH 7.0), 100 mM NaCl, 5 mM imidazole
  • Elution buffer: 20 mM sodium phosphate (pH 7.0), 100 mM NaCl, 500 mM imidazole
  • Lysate containing sfGFP–CTX
  • Centrifuge tubes
  • SDS–PAGE reagents and equipment

Procedure

  1. Resin Preparation
    • Wash 1 mL of cobalt resin with 30 mL ultrapure water.
    • Equilibrate with a 20 mL binding buffer.
  2. Protein Binding
    • Incubate equilibrated resin with clarified lysate for 15 min under gentle mixing.
    • Collection of Flowthrough
    • Collect the unbound fraction (flowthrough).
  3. Resin Wash
    • Wash resin with a binding buffer to remove nonspecific proteins.
  4. Protein Elution
    • Elute bound protein using an elution buffer (20 mM sodium phosphate, pH 7.0, 100 mM NaCl, 500 mM imidazole).
    • Collect eluted fractions.
  5. Analysis
    • Analyze eluted fractions by SDS–PAGE to confirm presence and purity of sfGFP–CTX.

Protein Analysis by SDS–PAGE

Materials

  • Distilled water
  • SDS–PAGE sample buffer (Laemmli buffer)
  • Protein molecular weight marker
  • Heating block or water bath (95 °C)
  • SDS–PAGE apparatus and power supply
  • Staining solution (e.g., Coomassie Brilliant Blue)
  • Destaining solution

Procedure

  1. Sample Preparation
    1. For each sample, mix:
      • ~10ug protein sample (Max vol. is 20 uL)
      • Complete it with H20 if necessary
      • 5 µL sample buffer
    2. Heat at 95 °C for 5 min to denature proteins.
  2. Gel Loading
    • Load 20 µL of each prepared sample into separate wells.
    • Load 7 µL of the molecular weight marker into one well.
  3. Electrophoresis
    • Run the gel at 110 V for 20 min.
    • Increase to 130 V and continue for 1 h.
  4. Staining and Destaining
    • Stain the gel in Coomassie solution for 4 h.
    • Destain for 24 h until background is clear in desilated H2O
  5. Analysis
    • Visualize the protein bands.
    • Compare the protein profiles across samples to assess expression or purification over time.

CTX Purification by TEV Protease Cleavage

Objective

To release CTX peptide from the sfGFP–CTX fusion protein using TEV protease and separate it from the fusion tag and enzyme.

Materials

  • Elution fractions containing sfGFP–CTX fusion protein
  • Tris buffer (for buffer exchange)
  • TEV protease (NextVitro)
  • Centrifugal concentrators (for buffer exchange)
  • 10 kDa MWCO centrifugal filter units
  • Incubator or water bath (30 °C)

Procedure

  1. Buffer Exchange
    • Exchange elution fractions into Tris buffer using centrifugal concentrators.
  2. Proteolytic Cleavage
    • Add TEV protease at a 1:50 (w/w) enzyme-to-protein ratio.
    • Incubate the mixture at 4 °C for 24 h.
  3. Separation of CTX Peptide
    • Pass the cleavage reaction through a 10 kDa MWCO centrifugal filter.
    • Collect the flowthrough, which contains the released CTX peptide (~2.3 kDa).
    • Larger proteins (sfGFP fusion tag and TEV protease) remain in the retentate.

Measurement by NanoDrop

Objective

To quantify purified CTX peptide using UV absorbance on a NanoDrop spectrophotometer.

Materials

  • CTX peptide sample (filtrate after purification)
  • NanoDrop spectrophotometer
  • Tris buffer (or buffer used for final elution) as blank

Procedure

  1. Instrument Setup
    • Turn on the NanoDrop spectrophotometer.
    • Select Protein A₂₈₀ measurement mode.
  2. Blank Calibration
    • Load 2 µL of buffer (same buffer as CTX sample).
    • Run blank measurement to zero the instrument.
  3. Sample Measurement
    • Load 2 µL of CTX peptide sample onto the pedestal.
    • Measure absorbance at 280 nm.
  4. Concentration Calculation
    • The NanoDrop automatically calculates concentration using Beer–Lambert’s law.
    • For CTX, use ε = 5,500 M⁻¹ cm⁻¹ (extinction coefficient).
  5. Data Recording
    • Record concentration in mg/mL or µM.

Protoplastization of Aspergillus oryzae (ORY7)

Objective

To generate viable protoplasts from Aspergillus oryzae ORY7 for downstream applications such as genetic transformation.

Materials

  • A. oryzae ORY7 spores (from agar plates)
  • MMG or YPD medium
  • APB buffer (1.1 M MgSO₄, 1 M Na₂HPO₄, 1 M NaH₂PO₄, pH 5.8)
  • Lysozyme (VINOTASTE, 40 mg/mL)
  • ATB buffer (1.2 M sorbitol, 50 mM CaCl₂, 20 mM Tris, 0.6 M KCl)
  • ½ ATB buffer (diluted):
  • Miracloth
  • 250 mL Erlenmeyer flasks
  • 50 mL Falcon tubes
  • Centrifuge (capable of 3000 × g with adjustable acceleration/deceleration)
  • Shaking incubator (30 °C)
  • Neubauer chamber
  • Micropipettes and sterile tips
  • Ice for storage steps

Procedure

  1. Biomass Production
    1. Harvest spores directly from agar plates using 20 mL MMG or YPD medium.
    2. Inoculate into a 250 mL Erlenmeyer flask containing 100 mL medium.
    3. Incubate at 30 °C, 180 rpm for ~15 h.
  2. Mycelial Collection
    1. Filter the culture through Miracloth.
    2. Collect the resulting mycelial “cake.”
    3. Wash with ~20 mL APB buffer.
  3. Enzymatic Digestion
    1. Transfer mycelial cake to a 50 mL Falcon tube containing 20 mL APB + lysozyme at 40 mg/mL (800 mg total).
    2. Incubate at 30 °C, 150 rpm for 2–3 h.=
    3. Check protoplast formation under the microscope at ~1 h 45 min and again at 3 h to confirm digestion.
  4. Protoplast Recovery
    1. Filter the digestion mixture through Miracloth.
    2. Adjust the flow-through to 40 mL with ATB buffer.
    3. Slowly add 5 mL diluted ½ ATB, creating a two-phase system.
    4. Centrifuge at 3000 × g (acc 9, dec 4) for 12 min.
      • Protoplasts will be located in the upper phase (~5 mL).
    5. Carefully collect the upper phase with a micropipette into a fresh 50 mL Falcon tube.
  5. Washing and Concentration
    1. Bring suspension to 40 mL with ATB buffer.
    2. Centrifuge at 3000 × g (acc 9, dec 9) for 12 min.
    3. Discard supernatant and gently resuspend the pellet in 1 mL ATB buffer.
  6. Counting and Storage
    1. Count protoplasts using a Neubauer chamber.
      • A concentration >10⁶ protoplasts/mL is recommended for transformation.
    2. Prepare 100 µL aliquots in 0.6 mL microtubes.
    3. Use immediately for transformation experiments.

Transformation of Aspergillus oryzae Protoplasts

Objective

Introduce donor DNA and CRISPR guide plasmid into A. oryzae protoplasts using PEG–CaCl₂ mediated transformation.

Materials

  • Protoplast suspension (~1 × 10⁶ protoplasts/mL, stored at −80 °C)
  • Donor DNA and CRISPR guide plasmid (3–10 µg total DNA)
  • PCT buffer (pre-chilled, 4 °C) (50% (w/v) PEG 8000, 50 mM CaCl₂, 20 mM Tris, 0.6 M KCl.)
  • ATB buffer
  • Selective regeneration medium supplemented with 1.2 M sorbitol
  • Sterile Petri dishes
  • Incubator (30 °C)

Procedure

  1. Preparation
    • Thaw 150 µL of protoplast suspension (~1 × 10⁶ protoplasts/mL) on ice.
  2. DNA Addition
    • Add 3–10 µg of total DNA (donor DNA + CRISPR guide plasmid).
    • This should correspond to 25–50% of the final reaction volume.
  3. PCT Buffer Addition
    • Slowly add 200 µL of cold (4 °C) PCT buffer along the tube wall.
    • Do not pipette mix; gently let the buffer flow in to avoid damaging protoplasts.
    • Incubate at room temperature for 10 min.
  4. ATB Buffer Addition
    • Carefully add 250 µL of ATB buffer using the same slow approach.
    • Avoid direct mixing to preserve protoplast integrity.
  5. Plating
    • Spread the transformation mixture onto a selective regeneration medium containing 1.2 M sorbitol.
  6. Incubation
    • Incubate plates at 30 °C until colonies appear.

gDNA Extraction of Filamentous Fungi

  1. Biomass Collection
    • The 100 mg of biomass was removed from 72 hours of static cultivation in 10 mL dish plates in YPD 1% glucose + 2% maltose.
  2. Protein Check
    • The remaining liquid medium was used to check presence of protein in SDS–PAGE.
  3. Grinding Biomass
    • The biomass was ground to a fine powder using a cotton stick in liquid N₂ and resuspended in 500 µL of DNA extraction buffer (0.5% SDS, 0.2 M Tris-HCl pH 8, 0.025 M EDTA pH 8, 0.25 M NaCl).
    • Vortex for 30 s.
  4. Cooling
    • Suspension was cooled on ice for 5 minutes.
  5. Potassium Acetate Treatment
    • Add 100 µL of 8 M potassium acetate and mix by inversion ~8–10 times.
  6. Centrifugation
    • Spin at 13,000 rpm for 10 min.
  7. Repeat Step
    • Transfer supernatant to another tube and repeat step 5 and 6.
  8. DNA Precipitation
    • Add 300 µL of 100% isopropanol to the supernatant and centrifuge at 13,000 rpm for 15 min.
  9. Washing Pellet
    • Wash pellet with 1 mL of cold 70% ethanol (do not resuspend) and centrifuge at 13,000 rpm for 5 min.
  10. Drying Pellet
    • Discard supernatant and dry the pellet at 42 °C for 20 min.
  11. Resuspension
    • Resuspend in 100 µL warm H₂O.
  12. RNA Removal (Optional)
    • Add 2 µL of RNase (10 mg/mL) and incubate at 65 °C for 30 min.
  13. Storage
    • Store DNA at −20 °C

Total Protein Quantification by Lowry Method

Materials

  • MilliQ water
  • Solution A
  • Solution B
  • Microtiter plate
  • Pipettes and sterile tips
  • Microplate reader (Gen5 3.02)

Procedure

  1. Sample Preparation (in triplicate)
    • Line A (blank): 20 µL MilliQ water.
    • Line B (sample): 20 µL of sample at original concentration.
    • Line C (dilution): 5 µL of sample + 15 µL water (4× dilution).
  2. Reaction Setup
    • Add 10 µL of Solution A to each well.
    • Mix gently by pipetting up and down.
    • Add 80 µL of Solution B to each well.
    • Mix again by pipetting up and down.
  3. Incubation
    • Incubate the plate for 15 min in the dark.
  4. Absorbance Measurement
    • Measure absorbance at 750 nm using the Gen5 3.02 microplate reader.
    • Record values.

Notes on Absorbance

  • If absorbance > 0.5 → repeat test with more diluted sample.
  • If absorbance > 0.2 → use the “high” standard curve.
  • If absorbance < 0.2 → use the “low” standard curve.
  • Quantify samples every 2 days.

Data Analysis

  1. Calculate the blank average (Line A).
  2. Subtract blank average from each sample triplicate.
  3. Calculate the average of the corrected triplicates.
  4. Correct for dilution: multiply average by dilution factor (e.g., 4× if used Line C).
  5. Apply the standard curve equation:
    6. $$x = \frac{y-0.0404}{0.0003}$$ home standard curve
    • y = corrected absorbance value
    • x = protein concentration (µg/mL)
  6. Convert to µg/µL by dividing by 1000.

Yeast Transformation using PEG/Lithium Acetate

This protocol describes the transformation of Saccharomyces cerevisiae using the PEG/LiAc/ssDNA method, a widely used approach for introducing DNA into yeast cells by facilitating it uptake through chemical treatment and heat shock.

Materials

  • Saccharomyces cerevisiae competent cells (log-phase culture)
  • Plasmid DNA (1–5 µg)
  • Single-stranded carrier DNA (ssDNA, salmon sperm DNA, denatured by boiling and chilled on ice)
  • 50% (w/v) Polyethylene glycol (PEG 3350 or 4000) solution
  • 1.0 M Lithium acetate (LiAc) solution, sterile
  • TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5)
  • Sterile distilled water
  • Microcentrifuge tubes (1.5 mL)
  • Sterile pipette tips
  • Water bath or heat block set at 42 °C
  • Appropriate selective agar plates

Procedure

  1. Preparation of Competent Cells
    • Grow yeast overnight in rich medium (YPD) at 30 °C with shaking.
    • Inoculate fresh YPD to OD600 ~0.3–0.4 and grow until mid-log phase (~0.8 OD600).
    • Harvest cells by centrifugation (3000 g, 5 min) and wash once with sterile water.
    • Resuspend pellet in 1X LiAc solution and incubate 30 min at 30 °C to increase competence.
  2. Transformation Mixture Setup
    • In a sterile microtube, combine:
      • 50 µL competent yeast cells
      • 240 µL 50% PEG solution
      • 36 µL 1.0 M LiAc
      • 25 µL boiled ssDNA (2 mg/mL)
      • DNA of interest (1–5 µg plasmid DNA)
    • Mix gently by pipetting up and down or flicking the tube (avoid vortexing).
  3. Incubation
    • Incubate the transformation mixture at 30 °C for 30 min.
    • Perform a heat shock at 42 °C for 15–20 min.
  4. Recovery and Plating
    • Centrifuge cells briefly and resuspend pellet in 200 µL sterile water or YPD medium.
    • Plate on selective agar plates (appropriate auxotrophic marker or antibiotic resistance).
    • Incubate plates at 30 °C for 2–3 days until colonies appear.

V5-tag Detection by Immunostaining

Objective

To detect V5-tagged proteins using monoclonal mouse anti-V5-FITC antibody (Invitrogen).

Materials

  • Samples expressing V5-tagged protein
  • Monoclonal mouse anti-V5-FITC antibody (Invitrogen)
  • Phosphate-buffered saline (PBS) or appropriate washing buffer
  • Microcentrifuge tubes or chamber slides (depending on sample type)
  • Pipettes and sterile tips

Procedure

  1. Sample Preparation
    • Prepare cells or protein samples to be stained.
    • Wash once with PBS to remove residual medium or buffer.
  2. Antibody Incubation
    • Dilute the monoclonal mouse anti-V5-FITC antibody 1:500 in PBS or staining buffer.
    • Add antibody solution to the samples.
    • Incubate at 25 °C for 1 h in the dark.
  3. Washing
    • Wash samples 3× with PBS to remove unbound antibody.
  4. Detection
    • Visualize fluorescence under a fluorescence microscope (FITC channel).
    • Compare tagged vs. untagged control samples.

Yeast Cultivation

Strains

  • Experimental: S. cerevisiae BY4742 + pCYctx-snac
  • Control: S. cerevisiae BY4742 + pCY002 (empty vector control)

Culture Conditions

  • Medium: YNB –Ura (synthetic dropout medium lacking uracil)
  • Volume: 150 mL
  • Vessel: 500 mL Erlenmeyer flask
  • Duration: 2 days
  • Temperature & shaking: 30 °C, 200 rpm

CTX Purification by Chemical Cleavage

Objective

To release and quantify CTX peptide from yeast expressing Aga1p–CTX by chemical cleavage and subsequent measurement.

Materials

  • Yeast biomass (OD₆₀₀ ≈ 2, 10 mL)
  • Cleavage buffer (1:1 ratio with OD₆₀₀): (1 mM NiCl2, 0.1 M CHES buffer, pH 8.6, 0.1 M NaCl.)
  • 50 mL Falcon tubes
  • Shaker incubator (25 °C, 200 rpm)
  • Centrifuge (up to 10,000 rpm)
  • Concentration device (centrifugal filter or equivalent)
  • NanoDrop spectrophotometer

Procedure

  1. Cleavage Reaction
    • Resuspend 10 mL of yeast biomass (OD₆₀₀ ≈ 2) in 10 mL cleavage buffer (1:1 ratio with OD₆₀₀).
    • Incubate at 25 °C, 200 rpm for 18 h.
  2. Separation
    • Centrifuge at 10,000 rpm for 10 min.
    • Collect the supernatant.
  3. Concentration
    • Concentrate the supernatant approximately 30× using centrifugal filters.
  4. Quantification
    • Measure peptide concentration with a NanoDrop spectrophotometer.
    • Compare results between CTX-expressing and control samples to confirm peptide release.

Cultivation of Aspergillus oryzae (ORY7) in Orange peel

1. Substrate Preparation

Materials

  • Orange waste (provided by Prof. Gabriela Macedo)
  • Blender
  • Tyler sieve 10 (1.68 mm)
  • Tyler sieve 42 (0.35 mm)

Procedure

  1. Process orange waste in a blender using the pulse function to break fibers and homogenize the mixture.
  2. Pass the mixture sequentially through Tyler sieve 10 (1.68 mm) and then Tyler sieve 42 (0.35 mm) to obtain uniform particles.
  3. Collect and store the processed substrate for subsequent steps.

2. Preparation of Ammonium Sulfate Solution & Inoculation

Materials

  • Distilled water
  • Ammonium sulfate ((NH₄)₂SO₄), 5 g/L
  • 125 mL Erlenmeyer flasks
  • Processed orange peel substrate
  • ORY7 inoculum (spores at 10⁶/mL)

Procedure

  1. Dissolve 0.675 g ammonium sulfate in 135 mL distilled water.
  2. Adjust the solution to pH 7.0 with acid or base as needed.
  3. In each 125 mL Erlenmeyer flask, add:
    • 30 mL of diluted ammonium sulfate solution
    • 2.4 g of processed orange peel substrate (particle size 0.35–1.68 mm).
  4. Autoclave at 121 °C for 20 min.
  5. After cooling, supplement with uracil and uridine (required for ORY7 growth).
  6. Inoculate each flask with 1 mL of ORY7 spore suspension (10⁶/mL).

3. Extraction of Secreted Proteins (Secretome)

Materials

  • Sodium acetate buffer solution (20 mM, pH 5)
  • Fermented substrate
  • Paper filter

Procedure

  1. Add sodium acetate buffer to the fermented substrate at a ratio of 10 mL buffer per 1 g substrate (~25 mL for 2.5 g substrate).
  2. Shake at 200 rpm for 1 h.
  3. Filter the mixture through paper filter.
  4. Collect the aqueous phase (secretome).
  5. Store the secretome for further analysis.