Experiments

PCR

The following procedure was used to amplify DNA.

Gel Preparation

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 from Takara Bio Inc.

Gel Electrophoresis

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.

Dpnl Treatment

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

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.

DNA Concentration Measurement

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.

DNA Assembly

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:

Component	μL
Inserts (diluted to 10~30 ng/μL)	1 each
Vector	1
2x XE cocktail	total volume of other components

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

Step	Temperature (°C)	Time(min)
1	37	pause
2	37	5
3	65	5
4	4	pause

4. [1]Liu, A.Y., Koga, H., Goya, C., Kitabatake, M. (2023). Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors. Genes to Cells, 28(8), 553-562. https://doi.org/10.1111/gtc.13034

Transformation into E.coil

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.

Direct Colony PCR

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:

Component	Amount(μL)
Primers (diluted to 10 mM)	0.3
2X KOD ONE Blue	5
Milli-Q water	To 10
Total	10

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:

Step	Temperature (°C)	Time(s)	Repetitions
Pre-heating	98	30	1
Denaturation	98	10	29
Annealing	55	5	29
Extension	68	≤1 kb: 1 1~10 kb: 5 / kb	29
Hold	4	pause	--

Inocuration

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.

Miniprep

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.

Sequencing

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.