Team:Greatbay SCIE/Notebook Method

Experimental Methods

Protein expression

Recombinant plasmid were transformed into E.coli BL21(DE3) Rosetta and grown to OD600 ~0.6 in LB broth containing 50 mg/mL kanamycin and 30 mg/mL chloramphenicol at 37°C. Protein expression was induced with 0.5 mM IPTG at 37°C for 5 h. All mTyr-CNK co-expression strains were incubated at 20 °C and 220 rpm for 20 h.

Protein purification under denature conditions

For CsgA-mfp5, CsgA-mfp5-mfp5, CsgA-mfp5-mfp3, Fp151

1. Cells were collected by centrifugation, and the pellets were stored at −80°C. 1 gram cell pellets were resuspended and lysed by 10 mL extraction solution (8 M guanidine hydrochloride (GdnHCl), 300 mM NaCl, 50mM, pH 7.2). Lysates were incubated at 4°C for 24 h.

2. The insoluble portions of the lysates were removed by centrifuging at 10,000 g, and the supernatants were incubated with 1 mL Ni-NTA resin for 2 h at room temperature. Beads that bound His-tagged protein were then centrifuged, and washed with 20 mL of potassium phosphate buffer (300 mM NaCl, 50 mM K2HPO4/KH2PO4, pH 7.2) for two times. The washed beads were then added with 3 mL potassium phosphate buffer (300mM NaCl, 50 mM K2HPO4/KH2PO4, pH 7.2).

3. The mixed solutions were then loaded on the columns. GdnHCl was further washed away by adding another 12 mL potassium phosphate buffer (300mM NaCl, 50 mM K2HPO4/KH2PO4, pH 7.2). 20 mL washing buffers (50 mM imidazole and 50 mM potassium phosphate buffer, pH 7.2) were passed through the columns to remove contaminated proteins.

4. Proteins were eluted from the column via 1 mL elution buffer (0.3 M imidazole, 300 mM NaCl, and 50 mM potassium phosphate buffer, pH 7.2) appped in six consecutive elution steps.

5. The proteins are made concentrated using dialysis with PBS buffer of pH6.0. Then they are kept in 4℃ for 72h to renaturation.

Protein Purification——Inclusion body extraction using 25% acetic acid

For Fp151

1. Harvested cell pellets were resuspended in 5 ml lysis buffer (10 mM Tris-Cl, 100 mM sodium phosphate, pH 8.0) per gram wet weight. Samples were lysed by ultrasonic decomposition and lysates were centrifuged at 10,000g for 30min at 4℃ and the cell debris was collected for purification.

2. The cell lysate pellet was resuspended in 25% (v/v) acetic acid to extract Fp151 for 30min.

3. The extracting solution was centrifuged at 10,000g for 20 min at 4℃ and supernatant was collected and dialyzed in 5% (v/v) acetic acid buffer at 4℃ using protein concentrator.

Protein purification under native conditions

For rBalcp19K, rBalcp19K-mfp5, mTyr-CNK

1. The harvested cell pellet were rinsed by precooled 10mM phosphate-buffered saline (PBS，pH7.4) and resuspended in 80ml binding buffer (50mM nNa2HPO4, 300mM NaCl and 10mM imidazole, pH 8.0)at a 20-fold concentration.

2. The cells are digested by lysosomes in order to get a soluble protein fraction at a final concentration of 30–50 KU per gram of cell pellets at 30°C for 15min.

3. After that, 40 cycles of sonication at 17 watts is applied for 10s each.

4. The lysates were then centrifuged at14,000 rpm and 4°C for 30min, and the resulting supernatant was co-incubated with Ni-NTA super resins by shaking at 4°C for 4 h.

5. The mixture was then loaded into dispensable plastic columns for chromatography. Non-specific absorbed proteins were rinsed with washing buffer (pH 8.0) (50mM Na2HPO4, 300mM NaCl and 50 mM imidazole）while the specific recombinant proteins were eluted with 12ml eluting buffer (50 mM, 300 mM NaCl and 250 mM imidazole, pH 8.0).

SDS-PAGE

Separating and stacking gels were prepared following protocols. Samples were prepared as below:

1. For each 1 ml cell pellets sample, add 100 μl 1X SDS-PAGE Gel Loading buffer (Sample Buffer) Buffer to the cell pellet and resuspend.

2. Take 16 μl of supernatant and mix with 10 μl 5X SDS-PAGE Gel Loading buffer (Sample Buffer).

3. Boil for 10 minutes and load 5–10 μl per well.

Gels were run at 120V and When the dye front is nearly at the bottom of the gel it is time to stop the run. Removal the gel from the cassette. The plates are separated and the gel is dropped into a staining dish containing deionized water. After a quick rinse, the water is poured off and stain added.

NBT staining

DOPA residues were detected in mature hybrid amyloid fibres with the nitro blue tetrazolium (NBT) staining assay; only the modified samples turned purple due to redox-cycling of DOPA residues.

1. 60 μL of 50 μg/mL adhesive protein samples were spotted onto Protran BA83.

2. nitrocellulose membranes (Whatman) manually.

3. The membranes spotted with protein solutions were incubated.

4. in 20 mL freshly made 0.6 mg/mL NBT solution in 2 M potassium glycinate buffer (pH =10.0) at room temperature in the dark (covered with aluminum foil) for 45 mins.

5. The membranes were washed with 10 mL 0.16 M sodium borate solution twice and soaked in another 20 mL sodium borate solution overnight. Imaging of the stained membranes was carried out with a scanner.

Surface coating analysis

Surface coating analysis was used to qualitatively assess the adsorption ability of recombinant adhesive proteins.

1. The hydrophobic tissue culture polystyrene plates (Corning, NY, USA) were directly used without any pretreatments, while the hydrophilic glass slides were bathed in 75% (v/v) ethanol overnight, rinsed with Milli-Q water, and dried in air.

2. To perform surface coating analysis, 10 μl of 0.5 mg/ml recombinant adhesive proteins were dissolved in 1×PBS (pH=6.0) was added onto the two substrates.

3. After fully adhering to the substrates, the recombinant proteins on the two surfaces was rinsed with Milli-Q water and the absorbed proteins were visualized by Coomassie brilliant blue staining. Bovine serum album (BSA) was used as negative controls. The experiments were performed with at least three independent replicates.

Fluorescence and growth curve measurement

1. 25-ml cultures of E. coli BL21(DE3) bearing sfGFP fusion constructs were inoculated in 250ml flasks with 25ml LB medium containing kanamycin (50 mg/ml) overnight.

2. Overnight cultures were diluted at 1000-fold in 200-μL LB medium with 50 mg/ml Kanamycin in 96 well plates. Cultures in 96 well plates were incubated at 37℃, 900rpm, with cover. 6 repeats were conducted for each samples.

3. 500μM IPTG was added into each wells when OD600nm reach 0.2/0.5/0.8 OD600 nm.

4. OD600nm and fluorescence was measured (488-nm excitation, 530-nm emission,10-nm band pass for GFP) with a Microplate Fluorescence Reader (THERMO Varioskan Flash) continuously for 22 h.

Molecular cloning in Pichia pastoris

(modified from protocol shared by NJTech, see Collaboration)

  • 5–10 μg of each construct for each transformation
  • YPD Medium
  • 50-ml conical polypropylene tubes
  • 1 L cold (4°C) sterile water (place on ice the day of the experiment)
  • 25 mL cold (4°C) sterile 1 M sorbitol (place on ice the day of the experiment)
  • 30°C incubator
  • Electroporation device and 0.2 cm cuvettes
  • YPDS plates containing 500 or 1000 μg/ml Zeocin™

Preparing Pichia cells

1. Grow 5 ml of your Pichia pastoris strain in YPD in a 50 ml conical at 30°C overnight.

2. Inoculate 500 ml of fresh medium in a 2 L flask with 0.1–0.5 ml of the overnight culture. Grow overnight again to an OD600 = 1.3–1.5.

3. Centrifuge the cells at 1,500 × g for 5 minutes at 4°C. Resuspend the pellet with 500 ml of ice-cold, sterile water.

4. Centrifuge the cells as in Step 3, then resuspend the pellet with 250 ml of ice-cold, sterile water.

5. Centrifuge the cells as in Step 3, then resuspend the pellet in 20 ml of ice-cold 1 M sorbitol.

6. Centrifuge the cells as in Step 3, then resuspend the pellet in 1 ml of ice-cold 1 M sorbitol for a final volume of approximately 1.5 ml. Keep the cells on ice and use that day. Do not store cells.

Electroporation of Pichia cells

1.Mix 80 μl of the cells from Step 6 (previous page) with 5–10 μg of linearized DNA (in 5–10 μl sterile water) and transfer them to an ice-cold 0.2 cm electroporation cuvette.

2.Incubate the cuvette with the cells on ice for 5 minutes.

3.Pulse the cells using the manufacturer’s instructions for S. cerevisiae.

4.Immediately add 1 ml of ice-cold 1 M sorbitol to the cuvette. Transfer the cuvette contents to a sterile 15-ml tube and incubate at 30°C without shaking for 1 to 2 hours.

5.Spread 10, 25, 50, 100, and 200 μl each on separate, labelled YPDS plates containing 100 μg/ml Zeocin™. Plating at low cell densities favours efficient Zeocin™ selection.

6.Incubate plates from 3–10 days at 30°C until colonies form.

7.Pick 10–20 colonies and streak for single colonies on fresh YPD or YPDS plates containing 100 μg/ml Zeocin™.

Determine the Mut phenotype

1.Using a sterile toothpick, pick one colony and streak or patch one ZeoR transformant in a regular pattern on both an MMH plate and an MDH plate, making sure to patch the MMH plate first.

2.Use a new toothpick for each transformant and continue until 10 transformants have been patched (1 plate).

3.To differentiate Mut+ from MutS, make one patch for each of the controls (GS115/MutS Albumin and GS115/pPICZ/lacZ Mut+) onto the MDH and MMH plates.

4.Incubate the plates at 30°C for 2 days.

5.After 2 days or longer at 30°C, score the plates. Mut+ strains will grow normally on both plates, while MutS strains will grow normally on the MDH plate but show little or no growth on the MMH plate.

Colony PCR Analysis of Pichia Integrants

Mut+ colonies were used for colony PCR to determine if the gene of interest has integrated into the Pichia genome. Amplify gene of interest with the α-factor primer (for pPICZα only) or 5’ AOX1 primer paired with the 3´ AOX1 primer. If screening Mut+ integrants using 5’ and 3’ AOX1 primer, you should see two bands, one corresponding to the size of your gene of interest, the other to the AOX1 gene (approximately 3.0 kb). If you use the α-factor primer as a PCR primer, you will not see a band with GS115. This is because there is no α-factor signal associated with the chromosomal AOX1 gene. Sometimes there will be ghost bands appearing in your PCR. These do not seem to be significant as they have not been shown to be a problem.

Protein expression in Pichia pastoris

Materials

BMGY/BMMY (buffered complex glycerol or methanol medium)

BMGH/BMMH (buffered minimal glycerol or methanol medium containing histidine)

baffled flasks

Small scale expression

1. Using a single colony, inoculate 25 ml of BMGY in a 250 ml baffled flask. Grow at 28–30°C in a shaking incubator (250 rpm) until culture reaches an OD600 = 2–6 (approximately 16–18 hours).

2. Harvest the cells by centrifuging at 1,500–3,000 × g for 5 minutes at room temperature. Decant supernatant and resuspend cell pellet to an OD600 of 1.0 in BMMY medium to induce expression (approximately 50 ml).

3. Place culture in a 250 ml baffled flask. Cover the flask with 1 layer of sterile gauze nd return to incubator to continue growth.

4. Add 100% methanol to a final concentration of 0.5% methanol every 24 hours to maintain induction.

5. At each of the times indicated below, transfer 1 ml of the expression culture to a 1.5 ml microcentrifuge tube. Use these samples to analyse expression levels and determine the optimal time post-induction to harvest. Centrifuge at maximum speed in a tabletop microcentrifuge for 2–3 minutes at room temperature. Time points (hours): 0, 6, 12, 24 (1 day), 36, 48 (2 days), 60, 72 (3 days), 84, and 96 (4 days).

6. For secreted expression, transfer the supernatant to a separate tube. Store the supernatant and the cell pellets at –80°C until ready to assay.

7. Analyse the supernatants and cell pellets for protein expression by Coomassie-stained SDS-PAGE.