Experiments

Overview

All laboratory procedures were standardised to ensure reproducibility and to support the engineering cycles described on our Engineering page. Below are the detailed wet lab protocols that enabled construction, verification, protein expression, and functional testing of our tyrosinase-based dyeing system.

PCR amplification of gene fragments

PCR reaction setup: Assemble all reaction components on ice. Gently mix all the components of the reaction before use.

Mix well and quickly transfer the reactions to a thermocycler:

Plasmid Extraction

1. Prepare reagents: Ensure RNase A is added to Buffer P1; ethanol is added to Wash Solution; check Buffer P2 and P3 for precipitates.
2. Centrifuge 1.5–5 mL overnight bacterial culture at 8,000 × g for 2 min; discard supernatant.
3. Resuspend pellet in 250 μL Buffer P1.
4. Add 250 μL Buffer P2; invert gently 5–10 times; incubate at room temperature for 2–4 min.
5. Add 350 μL Buffer P3; invert gently 5–10 times.
6. Centrifuge at 12,000 × g for 5–10 min; transfer supernatant to adsorption column; centrifuge at 8,000 × g for 30 s; discard flow-through.
7. Add 500 μL Wash Solution; centrifuge at 9,000 × g for 30 s; discard flow-through.
8. Repeat step 7.
9. Centrifuge empty column at 9,000 × g for 1 min.
10. Transfer column to a clean 1.5 mL tube; add 50 μL Elution Buffer; incubate at room temperature for 1 min; centrifuge at 12,000 × g for 1 min. Measure DNA concentration and store at −20 °C.

Agarose Gel Electrophoresis

1. Dissolve 0.50 g agarose in 50 mL 1× TAE buffer; microwave until dissolved. Add nucleic acid dye according to the manufacturer's instructions to prepare a 1% gel.
2. Place comb, pour gel, and allow it to solidify (~30 min).
3. Place gel in electrophoresis tank; cover with 1× TAE buffer (1–2 mm above gel).
4. Load PCR product and DNA marker into wells.
5. Run at 120 V for 30–40 min; image gel and identify target band.

Gel Extraction

1. Cut target DNA band under UV light.
2. Transfer gel slice to a 1.5 mL tube; add 500 μL gel dissolution buffer.
3. Incubate at 56 °C until dissolved; vortex every 3 min.
4. Pre-treat adsorption column with equilibrium solution.
5. Add dissolved gel to column; incubate 1 min; centrifuge at 12,000 rpm for 30 s; discard flow-through.
6. Add 500 μL WB; centrifuge at 12,000 rpm for 30 s; discard flow-through.
7. Repeat step 6.
8. Centrifuge empty column at 12,000 rpm for 2 min.
9. Transfer column to clean tube; add 30 μL EB; incubate 2 min; centrifuge at 12,000 rpm for 1 min to collect DNA.

Double enzyme digestion of vector and PCR product

Incubate at 37 °C for 30 min.

Ligation of target gene and vector

Incubate at 4 °C overnight.

Seamless Cloning

Incubate at 50 °C for 20 min; place on ice for 2 min immediately.

Transformation of E. coli

1. Thaw competent cells on ice.
2. Aliquot 50 μL cells into pre-chilled tubes; add 5 μL ligation product; mix gently.
3. Incubate on ice for 30 min; heat shock at 42 °C for 45 s; place on ice for 2 min.
4. Add 500 μL antibiotic-free LB; shake at 37 °C, 200 rpm for 1 h.
5. Spread 200 μL on LB + ampicillin plate; incubate upside down at 37 °C overnight.
6. Pick single colonies; seal plate; store inverted at 4 °C.

Colony PCR

Pick individual colonies and inoculate into LB broth with 100 μg/mL ampicillin. Incubate the cultures and prepare the PCR mix:

Mix well and set the following PCR program:

Analyze products by agarose gel electrophoresis.

Protein Expression

1. Transform 50 μL of E. coli Rosetta (DE3) competent cells with ~100 ng of TyrBm_pET-21a(+), TyrBm-L1-CipA_pET-21a(+), CipA-L1-TyrBm_pET-21a(+), TyrBm-L2-CipA_pET-21a(+), and TyrBm-L3-CipA_pET-21a(+) plasmid separately.
2. Perform colony PCR to identify transformants.
3. Inoculate PCR-verified clones into 5 mL LB medium with 100 μg/mL Ampicillin; shake at 37 °C, 200 rpm overnight. Transfer 1 mL to 50 mL LB medium with 100 μg/mL Ampicillin; grow to OD₆₀₀ = 0.6–0.8.
4. Induce with 0.1, 0.5, and 1.0 mM IPTG; shake at 20 °C or 37 °C overnight.
5. Centrifuge, discard supernatant, and resuspend pellet in PB (pH 7.4). Repeat wash once.
6. Sonicate on ice (30 min, 40% power, 3 s pulse, 7 s pause).
7. Centrifuge; collect supernatant and inclusion body fractions.

SDS-PAGE Electrophoresis

12% Separation Gel

Pour 3 mL gel; polymerize for 30 min.

Stacking Gel

1. Pour gel; insert comb; allow to polymerize.
2. Mix protein samples with 5× loading buffer; boil at 96 °C for 10 min.
3. Load samples and ladder; run at 120 V until dye front reaches bottom.

Coomassie Brilliant Blue Staining

Staining solution

1. After SDS-PAGE electrophoresis, carefully disassemble the unit and remove the gel. Rinse gently with deionized water to remove excess buffer.
2. Submerge the gel in staining solution; incubate with gentle agitation for 30 min.

Destaining solution

1. Transfer gel to destaining solution, agitate gently, and replace solution periodically until background is clear.
2. Document the gel using a gel imaging system.

Tyrosinase Activity Assay (Spectrophotometric)

1. Prepare the reaction mixture containing 2 mM L-tyrosine and 0.2 mM CuSO₄ in Tris-HCl buffer (pH 7.4). Vortex for at least 1 min to oxygenate.
2. For each well of a 96-well plate, add 290 μL of the reaction mixture and 10 μL of the enzyme sample.
3. Incubate the plate at 50 °C. Measure the absorbance at 475 nm at 0, 5, and 10 min.
4. Calculate the slope of A₄₇₅ increase over time (0–10 min). This slope represents average enzyme activity.
5. One unit of enzyme activity (U) is defined as the amount of enzyme that produces 1 μmol of dopachrome per minute, corresponding to an increase in A₄₇₅ of 0.001 per minute.

Tyrosinase Activity Assay (Plate)

Spread recombinant colonies onto LB screening plates containing the appropriate antibiotic. Incubate at 37 °C for 40 hours. The development of a black colour indicates tyrosinase activity under the given culture conditions.

Glycosyltransferase Activity Assay

Prepare the reaction system according to the following table. Use the supernatant or pellet fraction from the sonicated E. coli cell lysate as the enzyme source. For the blank control, replace the enzyme solution with the Tris-HCl buffer used for cell collection.

1. Initiate the reaction by adding 10 μL of 10 μM 1-naphthol (anhydrous ethanol) to reach a final 1 mL volume. Mix gently and incubate at 37 °C for 1 min.
2. Quickly transfer 200 μL of the reaction mixture into each well of a black 96-well plate; perform measurements in triplicate.
3. Place the plate in a preheated microplate reader (37 °C). Set excitation to 287 nm, emission to 335 nm, slit width 5 nm.
4. Record fluorescence and analyse to determine enzyme activity.