. Experiments .

- AKTA pure™ chromatography system
- LB or TB liquid medium
- 50 mM Tris-HCl buffer (pH 7.5)
- Elution Solution A (50 mM Tris-HCl, 0.3 M NaCl, 20 mM imidazole, pH 7.5)
- Elution Solution B (50 mM Tris-HCl, 0.3 M NaCl, 200 mM imidazole, pH 7.5)
- HisTrap HP His tag protein purification columns
- Kanamycin (50 mg/mL)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm until the OD₆₀₀ of bacteria culture reached at 0.6~0.8.
3. Corresponding inducer is added into bacteria culture from step 2 and kept at Suitable culture conditions and last 16-20 h.
4. Bacteria culture form step 3 is centrifuged at 6500 rpm at 4 °C for 7 minutes to collect the cell and resuspended with 25-30 mL Tris-HCl buffer (pH 7.5)
5. Repeat step 4 twice.
6. The liquid from step 5 is treated with ultrasonication to lyse the cell.
7. The lysis is centrifuged at 9000 rpm at 4 °C for 20 minutes to remove the cell debris and then the supernatant is filter through a 0.22-μm membrane filter.
8. The target protein is purified from the supernatant using a HisTrap column with AKTA pure™ chromatography system.
9. The eluate containing the target proteins is dialyzed with Tris-HCl buffer (pH 7.5) overnight to remove imidazole for subsequent uses.

- Deli Ruiguan multifunctional office copy paper (A4)
- Blender
- Drying Oven
- Stirrer
- Water bath
- Gel imaging system
- 60 mm Büchner funnel
- i-Quip 7 cm fast quantitative filter paper
- Deink-enzymes
- Blotting papers

1. Papers printed identical content are shredded into small strips.
2. Strips from step 1 are weighed and added into appropriate amount of purified water to prepare 1% (w/v) pulp.
3. The complex from step 2 is grinded by a blender continuing 30 minutes.
4. 30 mL of the pulp is transferred into a flask and treated with 2 mL of 7.5 deink-enzyme solution and kept at the optimal temperature of the enzyme for 60 minutes while being stirred by a fixed-speed stirrer.
5. After step 4, the pulp is added 0.6 mL limonene and shaken at 25 °C, 200 rpm for 1 minute, and then transferred into a beaker and stirred by a fixed-speed magnetic stirrer for 1 minute.
6. After 1 minute, a blotting paper is placed on the surface of the pulp for 30 seconds then is removed.
7. The pulp is filtered by i-Quip 7 cm fast quantitative filter paper using a Büchner funnel to collect paper fibers.
8. Paper fibers from step 7 are complete dried at 65 °C in a drying oven to get a recycled paper.
9. The recycled paper from step 8 is imaged by the Coomassie Brilliant Blue mode of gel imaging system and then its average grayscale value is analyzed by Image Lab and our Image Gray Read Tool.

- Gel imaging system
- Fume hood
- 96-well plate
- 6-well plate
- Water-based fountain pen
- MUJI smooth oil-based blue ballpoint pen
- Deli Ruiguan multifunctional office copy paper (A4)
- Rhamnolipid samples
- rhamnolipid-limonene mixture
- SDS solution
- SDS-limonene mixture

1. The paper is cut into 2.5 cm × 2.5 cm squares.
2. Square papers are written with the identical content and dried at 65 °C for 10 minutes to ensure the ink is completely dry.
3. Each well of a 6-well plate is placed with a square paper from step 2 and added with 2.0 mL rhamnolipid-limonene mixture.
4. The square papers are taken out per 15 minutes and air-dried until 1 h.
Note: Take three parallel samples per treatment/time point to ensure experimental accuracy. 5. The square papers are imaged by the Coomassie Brilliant Blue mode of gel imaging system and analyzed by Image Lab.

- Chitosan powder
- 2.0% (w/w) acetic acid (HAc)
- 0.1 M NaOH
- 2.5% (w/w) glutaraldehyde
- 0.1 M HCl
- Diluted ink solution (diluted 2000 times)
- Water bath
- Tecan Infinite M200 microplate reader

1. 0.45 g of chitosan is dissolved by 15 mL of 2.0 % (w/w) HAc.40 mL of 0.1 M NaOH is added into the chitosan solution.
2. The solution from step 2 is centrifuged at 3600 rpm for 5 minutes to collect the precipitation.
3. The precipitation is resuspended by 50 mL of purified water to repeat step 3 until the pH of the supernatant is neutral.
4. The precipitation is transferred to a petri dish, flatten with a spatula, dried at 80 °C to form a film and then weighed.
5. The dry film is treated with 2.5% (w/w) glutaraldehyde at 60 °C for 24 h to form crosslinked Chitosan.
Note: The ratio of glutaraldehyde to chitosan is 25 mL/g. 6. The cross-linked Chitosan is separated from the complex from step 6 and then dried at 80 °C.
7. Discard the glutaraldehyde, and dry the sample again in the oven at 80 °C.

1. 0.10 g of crosslinked chitosan is added to a 1.5-mL centrifuge tube.
2. The crosslinked chitosan is soaked by 1.0 mL 0.1 M HCl for 90 minutes.
3. The crosslinked chitosan is wished by water until the water is neutral and air-dried.
4. 1.0 mL of diluted ink is mixed with the crosslinked chitosan from step 3.
5. OD₄₀₀ of the supernatant is measured by Tecan Infinite M200 microplate reader immediately and then measured per 1 h until 4 h.
6. Calculate the adsorption rate based on the standard curve.

- 2.5% (w/w) rhamnolipid solution
- 2.5% (w/w) SDS solution
- M&G MG6128 black ink refill
- 75% (v/v) alcohol
- 1 cm × 1 cm square papers- Metal bath
- Tecan Infinite M200 microplate reader

1. The ink is transferred into a 2-mL centrifuge tube and diluted by 75% (v/v) alcohol 10-20 times approximately.
2. 15 µL diluted ink is added on a square paper and spread evenly by a pipette tip.
3. The square paper is air-dried for 3 minutes and then placed into a 2-mL centrifuge tube.
Note: Take five parallel samples per experiment to ensure experimental accuracy. 4. 1 mL of the reaction liquid (SDS solution, rhamnolipid solution, or purified water) is added into the centrifuge tube from step 4.
5. The centrifuge tube is shaken at 25 °C, 1000 rpm for 10 minutes by metal bath.
6. OD₄₀₀ of the supernatant in the centrifuge tube is measured.
7. The ink concentration of the supernatant is calculated by using the standard curve.

- Tecan Infinite M200 microplate reader
- Centrifuge
- Chloramphenicol (34 mg/mL)
- Kanamycin (50 mg/mL)
- LB liquid medium
- IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
- double sterile water
- L-arabinose stock solution (20% (w/v))

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm overnight.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm until the OD₆₀₀ of bacteria culture reached at 0.6~0.8.
3. 0.5 mM IPTG is added into bacteria culture to induce the expression of sfGFP at 18 °C for 20 h.
4. After 10 h, 0.25% arabinose is added into bacteria culture to initiate autolysis system expression.
5. The fluorescence intensity of the bacteria culture is measured immediately by Tecan Infinite M200 microplate reader.
6. The bacteria culture is centrifuge at 6500 rpm for 10 minutes and then measured the fluorescence intensity of the supernatant by Tecan Infinite M200 microplate reader.
7. After 10 h, the fluorescence intensity of the bacteria culture and the supernatant are measured again.
8. The lysis efficiency is calculated by dividing the fluorescence intensity of the supernatant by the fluorescence intensity of the bacteria culture.
Note:
To further improve the autolysis efficiency of the E. coli FLSA system, the following two methods can be applied:
1. When inducing the Fhud-T7Lysozyme-SsrA module with 0.25% arabinose, add 0.5% Triton X-100 to the culture (add Triton X-100 along with arabinose).
2. Adjust the pH of the induced culture medium to 8.0 using 2 M NaOH ( before adding arabinose), and then shake at 25°C for 30 minutes. Analyze the sfGFP distribution with a microplate reader and 10% SDS-PAGE. Quantify the band intensity using ImageJ. Define the lysis or release efficiency as the percentage of the target protein in the supernatant divided by the total target protein in the culture.

- LB liquid medium chloramphenicol
- Chloramphenicol (34 mg/mL)
- PBS buffer (pH 7.5)
- L-arabinose stock solution (20% (w/v))
- Tecan Infinite M200 microplate reader
- Centrifuge
- HisTag-Spytag-GFP

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm overnight.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm until the OD₆₀₀ of bacteria culture reached at 0.6~0.8.
3. 0.2% arabinose is added into bacteria culture to induce the expression of INPNC-HisTag-SpyCatcher in E. coli BL21 (DE3) at 37 °C for 12 h.
4. 0.2 mg/mL HisTag-Spytag-GFP is added into bacteria culture and kept at 37 °C, 200 rpm for 12 h.
5. 1 mL of bacteria culture is centrifuged at 6500 rpm for 5 minutes, then 1 mL fresh LB liquid medium was used to resuspend the precipitate, repeat 2 times for centrifugation and suspension under the same conditions.
6. Then the fluorescence intensity of the precipitate is measured by Tecan Infinite M200 microplate reader.

- LB liquid medium
- Ampicillin (100 mg/mL)
- LB solid plates
- L-arabinose stock solution (20% (w/v))
- D-glucose stock solution (50% (w/v))

1. Bacteria culture is inoculated into LB liquid medium at 2% (v/v) and kept at 37 °C, 200 rpm overnight (12-16 h).
2. The overnight culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm. Two groups are set: one group (L-arabinose) is added L-arabinose stock solution to reach the concentration of 0.02% and the other group (D-glucose) is added D-glucose stock solution to reach the concentration of 0.05%.
Note: Each group should have three parallel cultures. 3. After culturing for 6 h, 20 μL of the bacteria culture is taken and mixed thoroughly with 180 μL of fresh LB medium containing the same antibiotic concentration.
4. 20 μL of the diluted culture is taken and mixed with another 180 μL of fresh LB medium with the same antibiotic concentration. Repeat this dilution process 7 times, resulting in 8 dilution levels.
5. 5 μL of each diluted culture is taken and spotted onto pre-prepared solid plates containing the same antibiotic concentration. Then the plates are incubated at 37 °C for 12 h.
6. The survival ratio is calculated. First, the colony-forming units (CFU)/mL is calculated: CFU/mL = colony count × dilution factor / 0.005 mL (a single dilution has a dilution factor of 10, two dilutions would be 10², and so on). The survival ratio (log₁₀) is calculated as: log10[(CFU/mL of the L-arabinose group)/(CFU/mL of the D-glucose group)].

- LB liquid medium
- Ampicillin (100 mg/mL)
- Self-made blue-light illumination device

1. Bacteria culture is inoculated into LB liquid medium at 2% (v/v) and kept at 37 °C, 200 rpm overnight (12-16 h).
2. The overnight culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm. Two groups are set: one group (ILLUMINATION) is exposed to blue light (with a relative light intensity of 250) and the other group (DARKNESS) is kept in darkness.
Note: Each group should have three parallel cultures. 3. After culturing for 6 h, 20 µL of the bacteria culture is taken and mixed thoroughly with 180 µL of fresh LB medium containing the same antibiotic concentration.
4. 20 μL of the diluted culture is taken and mixed with another 180 μL of fresh LB medium with the same antibiotic concentration. Repeat this dilution process 6 times, resulting in 7 dilution levels.
5. 5 μL of each diluted culture is taken and spotted onto pre-prepared solid plates containing the same antibiotic concentration. Then the plates are incubated at 37 °C for 12 h.
6. The survival ratio is calculated. First, the colony-forming units (CFU)/mL is calculated: CFU/mL = colony count × dilution factor / 0.005 mL (a single dilution has a dilution factor of 10, two dilutions would be 10², and so on). The survival ratio (log10) is calculated as: log10[(CFU/mL of the ILLUMINATION group)/(CFU/mL of the DARKNESS group)].

-1 M MgSO₄ solution
-1 M CaCl₂ solution
-40% Glycerin solution
-5×M9 Salt Solution

- LB liquid mediums
- L-arabinose stock solution (0.2 g/mL)
- Potassium dichromate solution (1 g/L)
- 50% H₂SO₄
- 50% H₃PO₄
- Chloramphenicol (34 mg/mL)
- Camera
- Potassium dichromate solids
- Chromogenic Agent

1. The 5×M9 Salt solution, 1 M CaCl₂ solution and 1 M MgSO₄ solution are sterilized at 115 °C for 30 minutes.
2. The 40% Glycerin is filtered by using a 0.22-μm syringe filter.
3. These solutions are mixed according to the following formula to prepare the M9 medium:

1. K₂Cr₂O₇ solution is prepared at concentrations of 1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L in a 10 mL centrifuge tube.
2. 100 μL of 50% H₂SO₄ and 100 μL of 50% H₃PO₄ are added to tubes from step 1, mixed well, and then add 400 μL of the chromogen agent, react for 10 minutes.
3. After reaction, the pictures of tubes from step 2 are taken by using the high-saturation mode of the camera.
4. RGB values of three points in each tube are measured, and take the average G values to draw the standard curve.

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm overnight.
2. 1 mL of bacteria culture is centrifuged at 6500 rpm for 3 minutes, then 1 mL fresh M9 medium was used to resuspend the precipitate, repeat 2 times for centrifugation and suspension under the same conditions.
3. The bacteria culture from step 2 is inoculated into 20 mL of M9 medium at a concentration of 2% and kept at 37 °C, 200 rpm for 12 h.
4. Bacteria culture from step 2 is inoculated into M9 mediums at 4% (v/v) and kept at 37 °C, 200 rpm until the OD₆₀₀ of bacteria culture reached at 0.7~0.9.
5. 0.2% arabinose is added into bacteria culture to initiate the expression of INPNC-linker-MT3-linker-MT2A at 37 °C, 200 rpm.
6. After 4 h, 1.25 mL 1 g/L K₂Cr₂O₇ solution is added into bacteria culture and then incubate at 200 rpm at 37°C for 24 h.
7. 4.5 mL of the bacterial cultures is collected at 0 h and 24 h.
8. 1.5 mL of the supernatant is placed into a 2.0 mL centrifuge tube and centrifuge at 6500 rpm for 20 minutes at room temperature.
9. The supernatant is filtered by using a 0.22-μm syringe filter.
10. 15 μL of 50% H₂SO₄ and 15 μL of 50% H₃PO₄ are added to the filtrate, and then 60 μL of chromogen agent is added after mixing, and the reaction is carried out for 10 minutes.
11. After reaction, the pictures of tubes from step 2 are taken by using the high-saturation mode of the camera.
12. RGB values of three points in each tube are measured, take the average G values and substitute it into the standard curve to calculate the chromium concentration in the original samples.

- Tecan Infinite M200 microplate reader
- Centrifuge
- Kanamycin (50 mg/mL)
- LB liquid medium
- IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
- double sterile water

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm overnight.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm until the OD₆₀₀ of bacteria culture reached at 0.6~0.8.
3. 0.1 mM IPTG is added into bacteria culture to induce the expression of signal peptide-GFP at 37 °C for 10 h.
4. 1 mL of the bacterial cultures is collected per hour until 10 h.
5. The fluorescence intensity of the bacteria culture is measured immediately by Tecan Infinite M200 microplate reader.
6. The bacteria culture is centrifuged at 6500 rpm for 10 minutes and then measured the fluorescence intensity of the supernatant by Tecan Infinite M200 microplate reader.
7. The secretion efficiency is calculated by dividing the fluorescence intensity of the supernatant by the fluorescence intensity of the bacteria culture.

- Tecan Infinite M200 microplate reader
- Centrifuge
- Kanamycin (50 mg/mL)
- LB liquid medium
- IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
- double sterile water
- δ-aminolevulinic acid hydrochloride (0.5 mol/L)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm overnight.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 25 °C, 150 rpm until the OD₆₀₀ of bacteria culture reached at 0.6.
3. 0.2 mM IPTG and 0.5mM δ-aminolevulinic acid hydrochloride are added into bacteria culture to induce the expression of signal peptide-GFP at 37 °C for 36 h.
4. 1 mL of the bacterial cultures is collected per 6 h until 36 h.
5. The bacteria culture is centrifuged at 6500 rpm for 10 minutes and take the supernatant for SDS-PAGE (Gray scale value analysis was performed on the bands).
6. The supernatant from the 36th h is taken for deinking pulp experiment.