We conducted the design of plasmids involved in this project exclusively using Benchling, a platform for molecular biology research and design.

For this project, we acquired synthetic DNA as follows: All oligoDNA was purchased from Eurofins Scientific. Other synthetic gene fragments were sourced from gBlocks by Integrated Gene Technologies and Gene Fragments by TWIST BIOSCIENCE.

The following procedure was used to amplify DNA.

1. The sample was prepared as follows:

KOD ONE Blue was provided by Toyobo.

2. PCR was performed with the following thermal cycling conditions:

Gels were created for electrophoresis as follows:

1. Agarose was dissolved in 1x TAE buffer at a rate of 1% w/v and the mixture was heated until agarose dissolved.
2. The mixture was poured into a tray with a comb
3. EtBr was added at a 1:10 ratio to the gel, and gently mixed to ensure even distribution.
4. The gel was allowed to solidify at room temperature.

1x TAE buffer had 40 mM tris-acetate, and 1 mM EDTA. 10 mg/mL EtBr was purchased Takara Bio Inc.

This procedure was conducted to assess the length of DNA fragments which is amplified through techniques such as PCR.

1. The gel was placed in the electrophoresis chamber without removing it from the tray.
2. 1x TAE buffer was poured into the chamber until the gel was fully immersed.
3. An appropriate amount of size marker was added to the first lane.
4. The samples (3 µL each) were sequentially loaded in the lanes, starting from the second lane.
5. The chamber was turned on and electrophoresis was run at 100 V for 20 minutes.
6. After, the gel was carefully removed from the tray and transferred to another large, transparent tray.
7. The bands were visualized using a CCD camera.

Two kinds of size markers were used: λ styI, λ phage DNA fragments cleaved with restriction enzyme EcoT14I, and a mixture of TOYOBO's 100bp ladder and 6x loading dye. 10 µL of the former and 6 µL of the latter were used. The CCD camera was part of the ChemiDoc XRS+ System purchased from Bio-Rad Laboratories.

DpnI treatment was performed to cleave plasmids propagated in E. coli when they were used as PCR templates. This prevents any residual E. coli-derived plasmid from being carried over.

1. 1 µL of DpnI was added to 20 µL of the PCR product.
2. The mixture was incubated at 37°C for a minimum of 1 hour.

DpnI was purchased from Takara Bio Inc.

DNA purification was conducted to eliminate excess enzymes and residual substances after PCR.

1. 20 μl of the PCR product was mixed with 100 μl Buffer PB in a 1.5 mL tube.
2. This mixture was transferred to a silica membrane column and centrifuged at 14,000 rpm for 1 minute.
3. The supernatant was discarded.
4. 700 μl of Buffer PE was added to the column and centrifuged at 14,000 rpm for 1 minute at room temperature.
5. The supernatant was discarded.
6. The upper portion of the column was attached to a new 1.5 mL tube.
7. DNA was eluted with 10 μl of Milli-Q water.
8. The mixture was left for 1 minute and then centrifuged at 14,000 rpm for 1 minute.
9. Purified DNA was obtained in the 1.5 mL tube.

Silica membrane columns were purchased from Aji Bio-Pharma Oligos. Buffer PB and PE were purchased from QIAGEN.

To measure the concentration and purity of purified DNA, we used the NanoDrop One spectrophotometer from Thermo Fisher Scientific.

1. The instrument was turned on, and the arm was raised.
2. The platform was wiped with a Kimwipe which is wet with Milli-Q water, and dried by dabbing with a dry Kimwipe.
3. A blank measurement was taken using 1 µL of the same buffer used for the DNA solution.
4. The concentration and purity of the DNA were measured with 1 µL of the DNA solution.
5. After every measurement, the platform was again wiped with a Kimwipe wet with Milli-Q water and dried by dabbing.
6. After all measurements were taken, the arm was lowered, and the instrument was turned off.

We utilized the XE cocktail method (Liu et al., 2023), a low-cost PCR enzyme developed by former members of our team, to combine DNA fragments [1].

1. The thermal cycler was preheated to 37°C.
   
2. The samples were prepared as follows:
   

3. The cocktail was immediately transferred to the thermal cycler and the following program was run.

We introduced the plasmid into E. coli following this protocol.

1. Up to 2 μl of the assembly product or plasmid was added to 10 μl of competent cells.
2. The mixture was left on ice for 20 minutes.
3. The mixture was placed in a heat block at 42°C for 45 seconds.
4. The mixture was left on ice for 1 minute.
5. 100 μl of SOC medium was added.
6. The mixture was incubated at 37°C for 1 hour in the case of an assembly product, and 45 minutes for the plasmid.
7. The liquid was spread onto a plate following aseptic techniques.
8. The plates were labeled and incubated at 37°C overnight.

Three strains of competent cells were used in this experiment: DH5α, BL21, and BW25113. DH5α was purchased from TOYOBO, and BL21 (DE3) from COSMO BIO CO., LTD. The BW25113 strains were prepared in-house; see the Competent Cell Preparation section for the protocol.

We performed Direct Colony PCR to confirm the presence of the desired plasmid within the colonies grown on an agar plate.

1. The samples were prepared as follows:

2. A single, relatively isolated colony was picked using a pipette tip, and the tip was dipped into the liquid which is prepared as the above in a PCR tube.
3. The DNA was amplified using the following thermal cycling conditions:

To cultivate a significant quantity of E. coli that is believed to have incorporated the correct plasmid from direct colony PCR, we inoculated them in test tubes with LB medium.

1. 3 mL of LB medium with the appropriate antibiotic was prepared in a test tube.
   
2. A single colony was picked using a pipette tip and suspended in the prepared medium.
   
3. The test tube was incubated at 37°C and agitated 180 rpm for at least 16 hours in a shaker.
   

With this method, we extracted the desired plasmid from E. coli cells and purified it. For this procedure, we used the Promega Wizard Plus SV Minipreps DNA purification System.

1. 1.5 mL of the 3 mL culture from the test tube was decanted into a 1.5 mL tube.
2. The 1.5 mL tube was centrifuged at 13,000 rpm for 1 minute, and the supernatant was discarded
3. Steps 1 and 2 were repeated.
4. The 1.5 mL tube was centrifuged at 13,000 rpm for 1 minute.
5. The supernatant was removed with a pipette.
6. The precipitated cells were fully resuspended with 250 µL of CRA, vortexing, and pipetting.
7. 250 µL of CLA was added, and the solution was mixed with gentle inversion.
8. Once the solution became clear, 10 µL of Alkaline protease was added, and the solution was mixed with inversion. The solution was left for 5 minutes.
9. 350 µL of NSB was added and mixed well by vortexing.
10. The mixture was centrifuged at 13,000 rpm for 10 minutes.
11. An SV minicolumn was placed over a collection tube, and the supernatant was transferred into it.
12. The minicolumn and tube were centrifuged at 13,000 rpm for 1 minute.
13. The supernatant in the collection tube was discarded, and 750 µL of Column Wash Water was added to the tube.
14. The mixture was centrifuged at 13,000 rpm for 10 minutes.
15. The supernatant in the collection tube was discarded, and 250 µL of Column Wash Water was added to the tube.
16. The mixture was centrifuged at 13,000 rpm for 1 minute.
17. The supernatant in the collection tube was discarded, and the collection tube and column were again centrifuged at 13,000 rpm for 2 minutes.
18. The SV minicolumn was placed on the top of a 1.5 mL tube, and DNA was eluted with 50 µL of Milli-Q water.
19. The mixture was centrifuged at 13,000 rpm for 1 minute.
20. The DNA was obtained from the 1.5 mL tube.

We conducted sequence analysis using Sanger sequencing provided by Azenta Life Science. All functional regions of the plasmid used in this study were subjected to sequence analysis to verify their sequences.

Luria broth medium was used as the medium for the culture of E. coli.

1. The following compounds were mixed in a large glass jar.

2. This mixture was autoclaved at 121°C for 15 minutes to sterilize.
3. When it cooled to around 60°C, the appropriate antibiotics were added.

We prepared LB agar medium to create plates for culturing E. coli.

1. A mixture was prepared in a large glass jar as follows:

2. The mixture was autoclaved at 121°C for 15 minutes to sterilize.
3. When the mixture cooled to around 60°C, it was mixed thoroughly and the appropriate antibiotics were added to it.
4. ~20 mL of the agar medium was poured into each plate and allowed to solidify.

This type of agar plate was used to test the expression of proteases. This protocol was adapted from a video [2].

1. Solution A was prepared as follows:

2. Solution B was prepared as follows:

3. The two solutions were autoclaved at 121°C for 15 minutes.
4. When the solutions cooled to around 60°C, they were mixed before addition of the appropriate antibiotics.
5. ~20 mL of this solution was poured into each plate and allowed to solidify.

The skim milk was Hokkaido skim milk purchased from MEGMILK SNOW BRAND Co., Ltd

Transformed BL21 strains with the target plasmid were cultured in 1 L of LB medium to produce a high volume of protein.

1. 1 L of LB medium solution containing the appropriate antibiotics was prepared in a 2 L flask.
2. A reference test tube with 3 mL of the medium which was the same as the above was prepared.
3. The colonies on the plate were dissolved with repeated pipetting with 3 mL of medium from the flask.
4. The resulting suspension was added to the medium in the flask and mixed well.
5. A test tube with 3 mL of the bacterial solution from the flask was prepared to measure OD600.
6. The test tubes and flask were incubated in appropriate culture chambers at 37°C with shaking at 180 rpm.

In this experiment, we considered the OD600 of the culture in the test tube and the culture in the flask to be approximately equal.

We induced the expression of our lactose promoter-controlled target protein using IPTG (isopropyl β-D-thiogalactopyranoside) following the steps described below:

1. 1 M IPTG solution was prepared with Milli-Q water.
2. Periodic OD600 measurements (~every 20 minutes) of the culture in the test tube were started approximately 1 hour after the start of large-scale cultivation.
3. When the OD600 fell within the range of 0.3 to 0.6, 0.25 mL of the IPTG solution was added to the culture.
4. Cultivation was continued for approximately 3.5 hours before additional processing.

IPTG was purchased from Nacalai Tesque.

E. coli cultured in large quantities were collected for further downstream applications.

1. The E. coli culture was decanted from the flask into three separate 200 mL bottles.
2. The volume in each bottle was adjusted to be the same.
3. The bottles were centrifuged at 5,000 rpm for 10 minutes, and the supernatant was discarded.
4. Steps 1 to 3 were repeated.
5. The E. coli from the bottles were collected into three 50 mL tubes using a spoon.
6. The tubes were centrifuged at 3,500 rpm for 2 minutes.
7. The supernatant was discarded, if present.
8. These cells were immediately used; otherwise, these were stored at -80°C.

This method was used to disrupt the E. coli cell membranes to check the intracellular presence of proteins.

1. Buffer C was chilled on ice.
2. 10 mL of Buffer C was added to the E. coli pellet obtained from the Retrieval of E. coli before, and the cells were suspended using pipetting or vortexing.
3. This suspension was transferred to a 50 mL tube.
4. The sonication machine was set to a Duty cycle of 90%.
5. The E. coli solution was ultrasonicated using the sonication machine for 10 seconds.
6. The solution was allowed to stand for 50 seconds.
7. Steps 5 and 6 were repeated four more times.

The composition of Buffer C was as follows:

The sonication machine used was the Branson Sonifier 450.

We extracted the protein from E. coli lysate after sonication following the procedure described below.

1. The ultrasonicated sample was decanted into a 15 mL tube.
2. The tube was centrifuged at 9,000 rpm for 10 minutes at 4°C.
3. The supernatant was decanted into another 15 mL tube.
4. 1 mL of nickel beads solution was added into a 1.5 mL.
5. The 1.5 mL tube was centrifuged with a tabletop centrifuge for 10 seconds.
6. The supernatant was removed with a pipette.
7. 1 mL of Buffer C was added into the 1.5 mL tube.
8. The 1.5 mL tube was centrifuged with a tabletop centrifuge for 10 seconds.
9. The supernatant was removed with a pipette.
10. Steps 7 to 9 were repeated twice more.
11. The nickel beads were suspended with 1 mL of Buffer C, and the resulting mixture was transferred to the 15 mL tube from step 3.
12. The tube was set on a rotator and agitated for 1 hour.
13. The 15 mL tube was centrifuged at 9,000 rpm for 1 minute at 4°C.
14. The supernatant was discarded.
15. The nickel beads were resuspended with 1 mL of Buffer C in the 15 mL tube and the resuspension was loaded into a column.
16. A total of an additional 10 mL Buffer C was passed through the column.
17. The column was placed over a newly prepared 1.5 mL tube and the protein was eluted with 500 µL of 250 mM aqueous Imidazole solution.

The nickel beads solution we used was Ni Sepharose 6 Fast Flow. The collection columns we used were BIO RAD Micro Bio-Spin Columns. Imidazole was purchased from Nacalai Tesque.

TCA precipitation was carried out to concentrate proteins.

1. The protein solution was placed in a 1.5 mL tube.
2. 100% w/v TCA stock was mixed with the solution in a 4:1 ratio.
3. The solution was left on ice for 10 minutes.
4. The chilled solution was centrifuged at 14,000 rpm for 5 minutes.
5. The supernatant was discarded, and 200 µL of 100% cold acetone stock was added to resuspend the pellet.
6. Steps 4 and 5 were repeated twice more.
7. The pellet was air-dried at 95°C for 5 minutes using a heat block.
8. The pellet was resuspended in 2X SDS buffer.

1. The samples are prepared with 20 µL each using 2x SDS buffer.
2. 20 µL of each sample was loaded in the lanes of the polyacrylamide gel.
3. Electrophoresis was conducted at 20 mA and 10 W for 70 minutes.

The 2x SDS buffer had the following composition [3]:

The polyacrylamide gel we used was Extra PAGE One 5-20% from Nacalai Tesque.

1. The Anti-His-tag mAb 2000-fold was diluted with 5 mL of the skim milk solution used for sheet blocking.
2. Excess portions at the edges of the sheet were trimmed off.
3. The sheet was sandwiched into a hybrid bag and sealed on three sides using a sealer.
4. The Anti-His tag solution was added to the bag, and the remaining side was sealed with a sealer.
5. The bag was shaken for 1 hour on a shaker.
6. One side of the bag was cut, and the liquid inside was discarded.
7. Two other sides were cut open to carefully remove the sheet.
8. The sheet with the protein-binding side was placed facing up in a tray, immersed with TBST, and shaken for 5 minutes on a shaker. TBST was then discarded.
9. The above washing step was repeated twice more.
10. 10 mL of TBST with HRP was used to immerse the sheet and HRP was added to make it 10,000-fold.
11. The tray was shaken for 45 minutes on a shaker.
12. 2 mL of ECL plus Solution A was mixed with 50 µL of Solution B.
13. The sheet was placed on a plastic wrap, immersed in the HRP solution, and left for 5 minutes.
14. The sheet was placed in a new bag and the bag was sealed on three sides with a sealer.
15. The remaining liquid inside the bag was soaked up with a towel before sealing the remaining side.
16. The bands on the sheet were visualized with a CCD camera.

The composition of TBST was as follows:

This solution had a pH of 7.4 at room temperature.

The Anti-His-tag mAb from mouse was purchased from MBL Life Science, and the ECL plus was purchased from Thermo Fisher Scientific

The OD600 was measured using CO8000 Biowave from Funakoshi.

1. A blank measurement using LB medium without E. coli was taken.
2. The samples' OD600 were measured.
3. If the OD exceeded 2.0, the sample was diluted 11-fold by mixing 300 µL of the culture with 3 mL of LB, and the OD of that diluted sample was measured and multiplied by 11 to get the original sample.

We confirmed in our preliminary experiments that the dilution factor and OD600 are linearly correlated when the OD600 exceeds 2.0 using the above method.

We prepared competent cells of BW25113 using the following method.

1. The desired E. coli strain was cultivated in 3 mL of medium until the OD600 reaches 0.35.
   
2. Once the OD600 reaches 0.35, the culture was cooled on ice.
   
3. The culture was transferred to a 1.5 mL tube and centrifuged at 13,000 rpm for 1 minute
   
4. The supernatant was discarded.
   
5. Steps 3 to 4 were repeated to collect the pellet.
   
6. The pellet was resuspended in 500 µL of 50 mM CaCl₂ and centrifuged at 13,000 rpm for 1 minute.
   
7. The supernatant was discarded, and the pellet resuspended in 250 µL of 50 mM CaCl₂, and centrifuged at 13,000 rpm for 1 minute.
   
8. The supernatant was discarded, and the pellet was resuspended in 50 mM CaCl₂ solution containing 10% glycerol.
   
9. The competent cells were stored at -80°C.
   

BW25113 cells were provided by the National Institute of Genetics, Japan.

AHL Stock

1. 5 mg of AHL (IUPAC name: N-(β-ketocaproyl)-L-homoserine lactone, CAS Registry No. 143537-62-6) was dissolved in 10 mL of DMSO and stored at -80°C.
   
2. Aliquots of 10 µL were taken, diluted to 0.5 µM in H₂O, and stored them at -20°C for experimental use.
   

AHL was purchased from Sigma-Aidrich Japan.

Arabinose Stock

1. One (1) mg of arabinose was dissolved in 10 mL of H₂O and stored at room temperature [4].

Arabinose was purchased from Nacalai Tesque.

This procedure was performed to quantify the target plasmids present in the lysate.

1. The sample was prepare as follows:

2. Real-time PCR was performed with the following thermal cycling conditions:

The kit used was Luna Universal One-Step RT-qPCR Kit from Bio Labs.

Fluorescence measurements were conducted using the Qubit® 3.0 Fluorometer, from Life Technologies Japan Ltd. Before the measurements, the range for obtaining a linear calibration curve was determined using mixtures 180 µL of PBS and 20 µL of the sample . The final fluorescence values reported in the results are the fluorescence of the samples minus the background fluorescence from LB.