1. PCR

- Annealing temperature: primer Tm value -5
- Enzyme: PfuDNA polymerase
- Extension time：15s/kb

2. DNA Gel purification

- Measure DNA concentration after purification

3. Goldengate assembly

- Using restriction enzymes BsaI and T4 DNA ligase
- Vector-segment ratio of 1:3

4. Transformation

- All goldengate products are transformed
- DH5α strain for plasmid cloning and
- BL21 for protein expression and synthesis

5. Colony PCR

- Pick single colony as template for colony PCR
- Annealing temperature: primer Tm value -5
- Enzyme: TaqDNA polymerase
- Extension time：15sec/kb

6. Sanger DNA sequencing

- Send positive bacterial strains tested by colony PCR to Rui Biotech Ltd. Guangzhou for DNA sequencing for confirmation of correct sequence

7. Strain preservation

- Preserve strain by mixing bacterial liquid with glycerol in 1:1 ratio (e.g. 100ul bacterial liquid mix with 100ul glycerol)

8. Plasmid extraction

- Alkaline lysis method

List of Primers

List of Plasmids & Strains

1. Activation

- Pick a single colony from the plate and inoculate it into a 4 mL LB medium containing K-antibiotic in a shaking tube.
- Prepare 400 mL LB medium in a 1 L shaking flask (No need for a pET-28a control)

2. Scale-up Culture

- Add 200 µL of K-antibiotic to the 400 mL shaking flask, then add the entire 4 mL culture from Day 3. Incubate at 37°C with shaking at 220 rpm for 2 hours.

3. Induction

- When the OD600 of the culture in the shaking flask reaches 0.6-0.7 (the liquid becomes semi-opaque, such that your fingers appear slightly blurred when viewed through it), add 400 µL of 1M IPTG and incubate at 37°C for 3.5-4 hours

4. Harvesting Cells

- Collect the culture in a 200 mL centrifuge bottle and centrifuge at 8500 rpm for 10-15 minutes. Discard the supernatant and add 40 mL of 20 mM Tris-HCl

5. Cell Disruption

- Disrupt the cells at 80% amplitude for 25 minutes

6. Centrifugation

- Centrifuge at 8500 rpm for 10 minutes at 4°C to separate the inclusion bodies (pellet) and the cell lysate (supernatant)

1. Reagent preparation

- Washing solution: 20mM Tris HCl+20mM imidazole
- Eluent: 20mM Tris HCl+200mM imidazole
- 20% ethanol

2. Material preparation

- Fill with new column material, 3mL denaturation-resistant column material.
- The column material can be reused and stored in 20% ethanol in a 4℃ refrigerator
- Open the stopper and let the ethanol in the column material flow out naturally
- Add 20mM Tris-HCl, fill it up, let it flow out naturally, repeat 3 times, and wash the residual ethanol in the column material
- Plug the stopper for use

3. Purification

- Add the broken cell lysate (supernatant) to the column material, collect the effluent liquid in a 50 mL centrifuge tube, and the collected liquid is the flow-through liquid (FT). Observe whether the FT becomes clear. If not, add it to the column material again until the flow-through liquid becomes clear. The more flow-through times, the better
- Fill the column material with cleaning solution, open the stopper, add new cleaning solution after it flows out, repeat twice, and mark the collected cleaning solution as W
- Add 2~3 times the volume of elution solution to the column material (3 mL column material plus 8 mL elution solution), open the stopper, discard the 1 mL of liquid that flows out first, and then start collecting the effluent, marked as E

4. Post-treatment of column material

- Wash with elution solution for another 2-3 times the column volume to remove residual protein
- Wash with 20 mM Tris-HCl for 4-5 times the column volume
- After washing with 20% ethanol 2-3 times the column volume, fill it with 20% ethanol and store it in refrigerator at 4

5. Ultrafiltration* (not recommended, protein will precipitate)

- Add the eluted solution in the 15 mL centrifuge tube to the upper layer of the ultrafiltration tube and fill it up to the mark with Tris-HCl
- Place the ultrafiltration tube in a precooled centrifuge below 10°C and centrifuge at 6500 rpm for 10 min
- Transfer the filtrate from the lower layer of the ultrafiltration tube to a new 50 mL centrifuge tube, mark it as filtrate, and collect 1 mL into a clean 1.5 mL centrifuge tube
- Fill the upper layer of the ultrafiltration tube with Tris-HCl to the mark. (If there is little filtered liquid, use a micropipette to blow the ultrafiltration membrane to resuspend the insoluble particles)
- Repeat b-d until the filtrate volume reaches 50 mL
- Transfer the upper layer of the ultrafiltration tube to a new 15 mL centrifuge tube, and be careful to transfer the insoluble particles on the filter membrane together, and mark it as concentrated

6. Run the flow-through, washing, elution, concentration, and filtrate through a protein gel

7. Dialysis (we recommend gradient dialysis)

- Prepare dialysate
#1: final concentration of 100 mM imidazole dissolved in 20 mM TrisHCl Dialysate
#2: final concentration of 50 mM imidazole dissolved in 20 mM TrisHCl Dialysate
#3: final concentration of 25 mM imidazole dissolved in 20 mM TrisHCl
- Soak the dialysis bag in pure water and boil for 1 minute, pour out the boiling water, add pure water, and check whether the dialysis bag is leaking
- Pour out the pure water, add dialysate #1, wait for the dialysis bag to cool down, carefully pour the eluted protein into the dialysis bag, clamp the clamp, and check for leakage
- Soak the dialysis bag completely in the dialysis solution and place it in a 4℃ refrigerator for 2h
- Pour out the dialysis solution #1, add the dialysis solution #2, and place it in a 4℃ refrigerator for 2h
- Pour out the dialysis solution #2, add the dialysis solution #3, and place it in a 4℃ refrigerator for 2h
- Pour out the dialysis solution #3, add pure water, and place it in a 4℃ refrigerator for 2h

8. SUMO digestion

- Use the BCA protein assay kit to determine the protein concentration
- Configure the SUMO digestion system on ice
  - Sumo-tag protein, 20 ug
  - 10×sumoBuffer (+Salt/-Salt), 20 uL
  - Ulp1 protease (+Salt/-Salt), 0.5 uL
  - ddH20, up to 200 uL
- 4℃ overnight reaction
- Protein electrophoresis verification

- Inoculate Staphylococcus aureus and Escherichia coli into 5 mL of antibiotic-free LB, 220 rpm, 37°C overnight
- Take 200 uL of bacterial solution and evenly spread it on the antibiotic-free LB plate
- After the bacterial solution on the plate evaporates, poke a hole with a 1 mL sterile pipette tip
- Drop the antimicrobial drug to be tested into the small hole and let it evaporate
- Invert the plate in a 37°C incubator overnight

Collagen/chitosan binding

- Configure 4.5% collagen/chitosan water solution, stir and dissolve at 70℃ ~30min
- Add 4.5% sodium alginate to the prepared collagen solution, stir and dissolve at 90℃ for ~3h until completely dissolved, and leave it overnight for swelling
- Add the binding domain-fluorescent fusion protein to the collagen mixture, stir and dissolve at room temperature ~ 10min, until the colour is uniformly displayed and no dominant sediment presented. Vacuum dry the solution ~1h
- Evenly distribute the mixture on the median dish, add 0.4% calcium chloride solution for solidification, the target hydrogel will be obtained overnight
- Observe and compare protein binding efficiency under blue-light

Cellulose binding

- Add 2 rows of protein (different binding domain proteins linked to chromoprotein and chromoprotein as control) to 48-well plate
  - Row 1: gauze soaked in protein
  - Row 2: protein only
- Soak overnight at 4°C
- Transfer gauze in Row 1 to isopropanol
- Dry at 40°C
- Wash with water
- Observe and compare protein binding efficiency under blue-light

Pichia pastoris competent cell preparation

1. Inoculate a single colony of yeast recipient strain on YPD plate and culture at 30°C for 2 days;
2. Pick up the single colony from the plate and inoculate it into 5mL YPD liquid medium, shake at 30°C overnight;
3. After overnight culture, inoculate at an initial OD600 of 0.2 into 50mL YPD medium and shake culture to an OD value of 1.2~1.5 (about 5h+);
4. Centrifuge at 4°C, 5000rpm for 5 minutes to collect the sedimented cells, and resuspend the cells in 50ml of pre-cooled sterile water;
5. Centrifuge at 4°C, 5000rpm for 5 minutes to collect the sedimented cells, and resuspend the cells in 50ml of pre-cooled sterile water;
6. Centrifuge again at 4°C, 5000rpm for 5 minutes to collect the sedimented cells, and resuspend the cells in 10ml of 1mol/L sorbitol;
7. Solubilize the cells in 500ul of 1mol/L pre-cooled sorbitol, do not add glycerol, and store at -80°C for a few hours until transformation.

Pichia pastoris electroporation

1. Prepare 80 µL of yeast competent cells and mix them with 5-10 µg of purified linearized plasmid on ice for 15 minutes (ice bath for pre-cooling the electroporation cup).
2. Electroporate the mixture (1500 V, 400 Ω, 25 F).
3. After electroporation, quickly add 1 mL of sorbitol (pre-cooled) and culture on a shaker at 30°C for 2 hours. Then, plate the cells on MD plates.
4. Grow the cells on MD medium at 30°C for 3-4 days and perform identification.

Selection for multi-copy transformants following electroporation

1. A spreader was used to suspend the transformants in sterile water.
2. After measuring cell density, cells were diluted accordingly so that the cells were plated at a density of 10 cells per standard 8.5 cm petri dish on YPD agar containing G418 (0 to 2 mg/ml).
3. Since the number of resistant colonies drops significantly above 0.5 to 1 mg/ml G418, a range of concentrations of G418 (e.g., 0.25, 0.5, 1 and 2 mg/ml) was used. It took two to five days for resistant transformants to appear.

Analyze samples by SDS-polyacrylamide gel electrophoresis

1. Thaw cell pellets quickly and place on ice.
2. For each 1 mL sample, add 100 uL Breaking Buffer to the cell pellet and resuspend.
3. Add an equal volume of acid-washed glass beads (size 0.5 mm). Estimate equal volume by displacement.
4. Vortex 30 seconds, then incubate on ice for 30 seconds. Repeat for a total of 8 cycles.
5. Centrifuge at maximum speed for 10 minutes at 4°C. Transfer the clear supernatant to a fresh microcentrifuge tube.
6. Take 50 uL of supernatant (cell lysate) and mix with an appropriate volume of denaturing PAGE Gel Loading buffer (Sample Buffer).
7. Heat the sample as recommended and load 10-20 uL per well. Thickness of the gel and number of wells will determine volume loaded. You may store the remaining sample at -20°C for western blots, if necessary. You may store the cell lysates at -80°C for further analysis.

Yeast Extract Peptone Dextrose Medium (1 liter)

1. Dissolve 10 g yeast extract and 20 g of peptone in 900 mL of water. Note: Add 20 g of agar if making YPD slants or plates.
2. Autoclave for 20 minutes on liquid cycle.
3. Add 100 mL of 10X D.
4. Store the liquid medium at room temperature. Store the YPD slants or plates at 4°C. The shelf life is several months.

1. Preparation of Inoculum:

- Grow the microbial strain overnight in appropriate broth at the optimal temperature.
- Dilute the overnight culture to achieve a final concentration of approximately 1 x 10^6 CFU/mL (colony-forming units per milliliter).

2. Preparation of Antimicrobial Dilutions:

- Prepare a stock solution of the antimicrobial substance at a concentration that is 10 times higher than the expected MIC.
- Perform serial dilutions of the stock solution in the appropriate broth to achieve a range of concentrations (e.g., 0.5, 1, 2, 4, 8, 16, 32, 64 µg/mL).

3. Setting Up the Microtiter Plate:

- Dispense 100 µL of the diluted antimicrobial solutions into the wells of a 96-well microtiter plate.
- Include wells with only broth as negative controls and wells with the microbial inoculum and no antimicrobial as positive controls.

4. Inoculation:

- Add 100 µL of the standardized microbial inoculum (1 x 10^6 CFU/mL) to each well containing the antimicrobial dilutions.
- Mix gently by pipetting up and down or using a vortex mixer.

5. Incubation:

- Incubate the microtiter plate at the appropriate temperature for the specific microorganism (usually 24 hours for bacteria at 35-37°C or 48 hours for fungi at 25-30°C).

6. Reading the Results:

- After incubation, check each well for microbial growth, which can be indicated by turbidity or color change (if using indicator dyes).
- The MIC is defined as the lowest concentration of the antimicrobial agent that prevents visible growth in the well.

7. Data Analysis:

- Record the MIC for each antimicrobial tested.
- Repeat the experiment at least twice to confirm results and calculate the average MIC.