measurement

Describe how and why you chose your iGEM project.

To establish successful yeast protein expression and display system, we developed protocols to transform our host strain, Pichia pastoris GS115, culture and induce protein expression in the yeast, detect protein expressions and analyze the enzyme activities to address optimal temperature and pH levels, as well as stability and resistance. We conducted gene cloning and some enzyme activity assays at the BioLab of Mingdao High School. The majority of the experiments were carried out at the Institute of Molecular Biology of National Chung Hsing University under the supervision of tenured distinguished Prof. Hung-Jen Liu.


Abbreviations

  1. YPD: Yeast Peptone Dextrose
  2. YPDS: Yeast Peptone Dextrose Sorbitol
  3. BMY: Buffered Minimal YNB (Yeast Nitrogen Base)
  4. OD: optical density (e.g., OD600)
  5. BSA: Bovine Serum Albumin
  6. IFA: Immunofluorescence Assay
  7. PBST: Phosphate-Buffered Saline with Tween 20
  8. FITC: Fluorescein Isothiocyanate, which emits a bright green fluorescence at the wavelength of excitation/emission = 495/520 nm
  9. SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
  10. DNS: 3,5-dinitrosalicylic acid

YEAST TRANSFORMATION

Preparation of Competent Cells

  1. Retrieve Cells: Take P. pastoris GS115 from -80°C freezer.
  2. Streak Plating: Streak on YPD agar and incubate at 28°C for 16-24 hours.
  3. Inoculation: Inoculate a single colony into 10 ml YPD broth and incubate at 28°C, shaking at 120 rpm for 14-18 hours.
  4. First Centrifugation: Centrifuge culture at 4°C, 1800 xg for 10 minutes, discard supernatant.
  5. Resuspension: Resuspend cells in 10 ml sterile water at 4°C.
  6. Second Centrifugation: Repeat centrifugation and resuspension steps once.
  7. Sorbitol Addition: Add 10 ml of 1 M sorbitol, centrifuge, discard supernatant, and repeat once.
  8. Final Resuspension: Resuspend cells in suitable volume of 1 M sorbitol, aliquot 70 μl per tube, store on ice for use on the same day.

Electroporation Transformation

  1. DNA Preparation: Digest 10 μg of the vector with SacI to recover linear plasmid DNA, dissolve in 30 μl sterile water.
  2. Mixing: Add linear plasmid DNA to the competent cells (70 μl), mix and keep on ice.
  3. Electroporation: Place mixture in a sterile electroporation cuvette at 4°C. Electroporate at 25 μF, 1.5 kV, and 0.5 msec using ECM 830 Square Wave Mode in BTX Gemini System.
  4. Recovery: Transfer electroporated cells to a 50 ml centrifuge tube, rinse electroporation cuvette 5 times with 1 ml of 1 M sorbitol each, combine rinses in the tube.
  5. Incubation: Incubate at 28°C for 2-4 hours.
  6. Final Centrifugation: Centrifuge at 1800 xg, 4°C for 10 minutes, remove 900 μl supernatant, resuspend cells in remaining supernatant.
  7. Plating: Plate cells on YPDS agar plates with 2000 μg/ml Zeocin, incubate at 28°C for 3-5 days.
  8. Storage: Store plates at 4°C after colonies reach appropriate size.

YEAST PROTEIN INDUCTION AND GROWTH CURVE

Protein Induction in P. pastoris GS115

  1. Select Single Colony: Pick a single colony from a YPDS plate containing 2000 μg/ml Zeocin.
  2. Initial Culture: Inoculate in 10 ml YPD broth and incubate at 28°C with shaking at 120 rpm for 12-16 hours.
  3. First Centrifugation: Centrifuge at 2200 xg, 4°C for 10 minutes, then remove the supernatant.
  4. Resuspension in BMY: Add 10 ml of BMY and centrifuge at 2200 xg, 4°C for 10 minutes. Remove the supernatant and repeat this step once.
  5. Induction Preparation: Adjust the final cell suspension to an OD600 of 1 with an appropriate volume of BMY.
  6. Induction: Add glucose to a final concentration of 0.005% to induce expression. Incubate at 28°C with shaking at 120 rpm.
  7. Maintenance: Add 0.005% glucose every 24 hours.
  8. Harvest: Collect induced culture every 24 hours for growth curve analysis and activity analysis.

Growth Curve Analysis

  1. Collection: Collect 100 μl of the culture every hour.
  2. Preparation: Mix the 100 μl culture sample with 900 μl of BMY to dilute the sample.
  3. Calibration: Calibrate the spectrophotometer using 1 ml of BMY.
  4. Measurement: Measure the OD600 value of the diluted sample in a cuvette.
  5. Calculation: Multiply the OD600 value by 10 to get the actual absorbance value of the induced culture.

PROTEIN EXPRESSION

Immunofluorescence Assay (IFA)

  1. Collection of Yeast Cell: Collect 1 ml of induced culture in an Eppendorf. Suspend cells in 50 mM Tris-HCl (pH 8.0) and centrifuge at 4°C, 2200 xg for 10 minutes. Discard the supernatant.
  2. Cell Fixation: Resuspend cells in 50 mM Tris-HCl (pH 8.0) and centrifuge again at 4°C, 2000 xg for 10 minutes. Remove the supernatant and add 200 μl of 10% formaldehyde solution. Drop cells onto a coverslip and let the formaldehyde evaporate completely in a chemical fume hood.
  3. Blocking: Rinse coverslips twice with 2 ml 1xPBS. Add 2 ml blocking buffer and incubate for 2 hours at room temperature.
  4. Antibody Incubation: Remove blocking buffer and add 160 μl of primary antibody (Anti-6xHis antibody, diluted 300x in blocking buffer). Incubate at 4°C for 12-16 hours.
  5. Washing: Wash coverslips 4-6 times with 1xPBST containing 0.05% Tween 20, each time lasting for 10-15 minutes.
  6. Secondary Antibody Incubation: Add 160 μl of secondary antibody (Goat anti-mouse IgG-FITC, diluted 300x in blocking buffer). Incubate at 4°C for 12-16 hours.
  7. Final Washes: Wash coverslips 4-6 times with 1xPBST, each wash lasting for 10-15 minutes.
  8. Mounting: Let the coverslip dry, add 10 μl of fluorescence enhancer, and seal edges with nail polish. Store in a slide box at 4°C until ready for fluorescence microscopy. Images taken with the help of Prof. Hung-Jen Liu at National Chung Hsing University using an Olympus FV1000 laser confocal microscope.

Protein Assay Dye Reagent Concentrate (Bio-Rad)

  1. Standards: Dilute 10 mg/ml BSA to create standard solutions of 0, 1, 2, 4, and 8 μg/m. Measure the OD595 of each standard solution. Plot the values on a graph to create a standard curve (ensure R2 > 0.99).
  2. Samples: Take 2 μl of the sample and mix with 798 μl of sterile ddH2O. Add 200 μl of Protein Assay Dye Reagent to the sample mixture. Incubate at room temperature for 5 minutes. Measure by reading at OD595. Apply the OD595 value to the standard curve formula to calculate the protein concentration of the sample.

SDS-PAGE Electrophoresis Analysis

  1. Cell Preparation: Mix cells with an equal volume of glass beads. Lysis by vortexing for 20 seconds, then rest on ice for 30 seconds; repeat at least for 20 minutes. Then, centrifuge at 4°C, 13,200 xg for 10 minutes and collect the supernatant. Mix with an equal volume of 5x sample dye. Heat at 100°C on a dry bath for 10 minutes. Cool on ice for 10 minutes. Store the samples at -20°C if necessary.
  2. Gel Preparation: Clean the electrophoresis glass plates and assemble the gel apparatus. Prepare 10% separating gel and 10% stacking gel (provided by the lab of Prof. Hung-Jen Liu). Insert a comb, allow the gel to polymerize, then load samples and protein marker.
  3. Running, Staining and Destaining: Run at 70 V, 455 mA for 150 minutes. Disassemble the glass plates and apparatus. Remove excess gel. Stain with Coomassie brilliant blue for 12-16 hours at room temperature. Destain with a solution of methanol and acetic acid, repeating 3-4 times until clear bands are visible.

DNS ASSAY FOR REDUCING SUGAR (β-Xylanase Enzyme Activity)

Principle of 3,5-dinitrosalicylic Acid (DNS) Assay

  1. Substrate Hydrolysis: Xylan is hydrolyzed by β-Xylanase, producing xylose and other oligosaccharides.
  2. Colorimetric Detection: The aldehyde groups of the sugars reduce DNS (3,5-dinitrosalicylic acid) to 3-amino-5- nitrosalicylic acid, changing the solution's color from yellow to reddish-brown.
  3. Absorbance Measurement: The intensity of the color, corresponding to the concentration of reducing sugars, is measured at 540 nm.

Preparation of Reagents

  1. 50 mM Tris-HCl (pH 8.0): Dissolve 2.424 g Tris-base in ddH2O, adjust pH to 8.0 with HCl, and make up to 400 ml.
  2. 5% Xylan Solution: Dissolve 0.05 g xylan in 1 ml 50 mM Tris-HCl, ensuring complete dissolution.
  3. DNS Solution: Heat 50 ml ddH2O to 50°C, gradually add 0.315 g DNS, 10 ml 5N NaOH, followed by potassium sodium tartrate, sodium sulfite, and phenol. Make up to 100 ml and store protected from light at room temperature.
  4. Buffer Solutions at Various pH Levels: Prepare 50 ml of 0.1 M buffers for pH levels from 3 to 8 using specific amounts of citric acid, sodium citrate, and phosphate salts (Tables 1 and 2).

Table 1: Citric Acid and Sodium Citrate Buffer System

Image 2

Table 2: Sodium Dihydrogen Phosphate and Disodium Hydrogen Phosphate Buffer System

Image 2

Standard Curve

  1. Preparation: Dissolve 0.03 g xylose in 1 ml 50 mM Tris-HCl to make a 200 mM solution, then dilute to create standards ranging from 0 to 1800 μmol/ml.
  2. Reaction: Mix 200 μl of each standard with 300 μl DNS solution, heat in boiling water for 15 minutes, cool on ice, add 500 μl ddH2O, and measure OD540 to plot the standard curve.
Image 3

Measuring β-Xylanase Activity

  1. Cell Harvesting and Washing: Centrifuge 1 ml of sample at 4°C, 2200 xg for 10 minutes, wash by resuspending in 1 ml Tris-HCl, and repeat once. Finally, resuspend cells in 100 μl Tris-HCl.
  2. Enzyme Reaction: Mix 40 μl of this suspension with 120 μl Tris-HCl and 40 μl of 5% xylan, incubate at 37°C for 15 minutes.
  3. Reaction Termination: Following centrifuge, collect 180 μl of supernatant, add 270 μl DNS solution, heat at 100°C for 15 minutes, then cool, followed by adding of 450 μl ddH2O.
  4. Measure: Read at OD540 using a spectrophotometer.
  5. Calculate: Compare the absorbance to the standard curve to determine enzyme activity (units defined as nmol xylose released per minute).
Image 4

Optimal Condition, Shelf Life, Stability and Resistance tests

  1. Optimal Temperature and pH: Test enzyme activity at various temperatures (15°C to 85°C) and pH levels (using citric acid-sodium and sodium phosphate buffers), determining the highest activity as 100% relative activity.
  2. Shelf-Life Study: Analyze the enzyme activities in freeze-dried yeast powder stored at 37°C over a period of 2 to 16 weeks.
  4. Stability Tests: Analyze the thermal stability of enzyme activities in freeze-dried yeast powder across varying temperatures ranging from 60°C to 90°C.
  5. Resistance Tests: Evaluate the enzyme's resistance to gastrointestinal conditions using pepsin and trypsin.