Our target gene DNA fragments are synthesized by the company or amplified through PCR.

1.Place the gel tray in the appropriate position in the gel cartridge and place the comb in the correct position.

2. Measure 0.5 g agarose, put it in a 250 mL Erlenmeyer flask, add 50 mL 1 × TAE buffer and mix, then put the Erlenmeyer flask in the oven and heat to boil until the agarose is completely dissolved.

3. Add Ultra GelRed (10,000 ×) to the solution.

4.Pour the solution into the gel casting tray.

5. After the gel has solidified, pull out the comb.

6. Place the gel in the electrophoresis chamber with enough 1x TAE buffer.

7. Transfer the mixture to the well on the gel with a pipette.

8. Power on, run at 130 V for 20 minutes.

1.Column equilibrium: Add 500 μL of equilibrium solution BL to the adsorption column CB2, centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Cut the single band of target DNA from the agarose gel, place it into a clean centrifuge tube, and weigh it.

3. Add moderate volume of solution PC to the glue block and place it in a water bath at 50°C for 10 min, during which the centrifuge tube is gently turned up and down to ensure that the glue block is fully dissolved.

4. Add the solution obtained in the previous step to an adsorption column CB2, centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

5. Add 600 μL of rinsing solution PW to the adsorption column CB2, centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

6. Repeat step 5.

7. The adsorption column CB2 is put into the collection tube and centrifuged at 12000 rpm for 2 min. The adsorption column is left at room temperature for several minutes and allowed to dry thoroughly.

8. Put the adsorption column CB2 into a clean centrifuge tube, add an appropriate amount of elution buffer EB to the middle position of the adsorption membrane by hanging drops, place it at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to collect the DNA solution.

Pure DNA fragments are obtained through agarose gel electrophoresis and DNA purification.

1.Prepare the system of Restriction Endonuclease cleaving DNA.

2. Restriction Endonucleases are incubated at 37°C for 1 h except for BamH I, which is incubated at 30°C. The mixture is further digested by reaction at 80°C for 5 min to obtain the linearized plasmidstored and store at 4°C.

1.Configuration of homologous recombination reaction system.

2. Use a pipettor to gently suck and mix (do not shake and mix violently), and briefly centrifugal the reaction solution to the bottom of the tube.

3. React at 37°C for 30 min, then drop to 4°C or immediately cool on ice.

1.Design of primers for single-base (or continuous multi-base) site-directed mutagenesis.

2.The target plasmid was amplified using Phanta Max Super-Fidelity DNA Polymerase.

3.Amplified product Dpn I digestion.

4.Digestion product concentration determination

5.If the digestion product has been purified by gel recovery using a high-quality kit and there is no obvious stray band or smear residue after electrophoresis, the concentration can be determined using an absorbance-based instrument such as Onedrop, but the concentration value is only credible when A260/A280 is between 1.8 and 2.0.

6.Calculate the amount of digestion product used

7.The optimal amount of Dpn I digestion product used = [0.02 × number of base pairs of fragment] ng (0.03 pmol).

8.One-step cloning, homologous recombination.

9.For single-base (or continuous multi-base) site-directed mutagenesis, set up the reaction system on ice.

10.Use a pipette to gently pipette and mix (do not shake to mix), and briefly centrifuge to collect the reaction solution to the bottom of the tube.

11.37°C, 30 min; cool to 4°C or immediately place on ice to cool.

12.Transformation of recombinant products.

13.Thaw the cloned competent cells on ice.

14.Take 10 μl of the recombinant product and add it to 100 μl of competent cells, gently tap the tube wall to mix (do not oscillate to mix), and let it stand on ice for 30 min.

15.After heat shock in a 42°C water bath for 90 sec, immediately place it on ice to cool for 2-3 min.

16.Add 1000 μl LB liquid culture medium (without antibiotics) and shake at 37°C for 1 h (speed 220 rpm).

17.Preheat the LB solid culture medium plate with the corresponding resistance in a 37°C incubator.

18.Centrifuge at 6000 rpm for 5 min and discard 900ul of supernatant. Resuspend the bacteria with the remaining culture medium and gently spread it on the plate containing the correct resistance with a sterile spreader.

19.Incubate inverted in a 37°C incubator for 12-16 h.

20.Identification of recombination products.

21.After overnight culture, if the number of clones on the recombination reaction transformation plate is significantly higher than the negative control, several single clones can be picked and inoculated into LB liquid culture medium containing appropriate antibiotics for overnight culture, and the plasmid can be extracted for first-generation sequencing.

1.Prepare the PCR reaction solution according to the specified system.

2.Select multiple single colonies from the transformed petri dishes incubated overnight and transfer them into thereaction system using a sterile tip.

3.Complete the reaction and verify it using nucleic acid gel electrophoresis. lf fragments of the appropriate size arepresent, streak the colony plates and incubate them before transferring to liguid medium. Store the strain in a 1:solution of alycerol and bacteria, and send the remaining samples for sequencing.

1. Column equilibrium: Add 500 μL of equilibrium solution BL to the adsorption column CP3, centrifuge at 12000 rpm for 1min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Take 1 to 5 mL of the bacterial solution cultivated overnight, add it to the centrifuge tube, centrifuge at 12000 rpm for 1 min, and remove the supernatant as much as possible.

3. Add 250 μL of solution P1 (with added RNaseA) to the centrifuge tube containing the bacterial precipitate to completely suspend the bacterial precipitate.

4. Add 250 μL solution P2 to the centrifuge tube and gently turn it up and down for 6 to 8 times to fully lyse the thallus.

5. Add 350 μL of solution P3 to the centrifuge tube and immediately turn it gently up and down 6 to 8 times, at which point a white flocculent precipitate appears. The samples are centrifuged at 12000 rpm for 10 min.

6. Transfer the supernatant collected in the previous step to the adsorption column CP3 using a pipetting gun. After centrifugation at 12000 rpm for 30~ 60 sec, the waste liquid in the collection tube is poured out and the adsorption column CP3 is placed into the collection tube.

7. Add 600 μL of rinsing solution PW to the adsorption column CP3 (absolute ethanol has been added), centrifuge at 12000rpm for 30~60 sec, pour out the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

8. Repeat steps.

9. The adsorption column CP3 is put into the collection tube and centrifuged at 12000 rpm for 2 min.

10. Place the adsorption column CP3 in a clean centrifuge tube, add 50 to 100 μL of elution buffer EB to the middle part of the adsorption membrane, place it at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to collect the plasmid solution into the centrifuge tube.

1.Add 1 mL of overnight culture seed medium to a shaker flask containing 100 mL of LB medium and culture in a shaker at 37°C to about OD₆₀₀=0.5.

2. Centrifugal harvesting, 6000 rpm, 5 min.

3. Add 10 mL of pre-cooled 100 mM CaCl₂ for resuspension.

4. Apply cold compress on ice for 20 min.

5. Centrifugal harvesting, 4000 rpm, 10 min.

6. Add 4 mL of pre-cooled 100 mM CaCl₂ and resuspend again.

7. Apply cold compress on ice for 20 min.

8. Centrifugation, 4000 rpm, 10 min.

9. Add 1mL of pre-cooled 100 mM CaCl₂ and 15% glycerol mixed solution.

10. Aliquot in pre-cooled 1.5 mL centrifuge tubes and store at -80°C.

1.Take competent cells from -80°C refrigerator and put it on ice.

2. When the competent cells thaw, add 10 μL DNA ligation product or 2 μL plasmid per tube, place the mixture on ice for 30 min.

3. Heat shock at 42°C for exactly 45 seconds.

4.Put the 1.5 mL tubes back on ice for 3 min.

5. Add 800 μL LB fluid medium without antibiotics into the 1.5 mL tubes and then culture in the shaker incubator at 37°C for an hour.

6. Extract 100-200 μL bacteria liquid, transferred to LB solid medium with antibiotics and spread plates.

7.Place plates upside down and incubate at 37°C overnight.

A colony obtained from a plate streak was inoculated into 5 mL of the seed medium with appropriate concentration of antibiotics and cultivated at 37°C and 200 rpm for 8-10 hours. Subsequently, a 1% inoculum was transferred into 10 mL of LB medium (supplemented with sodium pyruvate, methionine, and threonine if the host cell was NT1003) for continued cultivation. When the OD₆₀₀ of the culture reached 0.6-0.8, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mmol/L to induce the expression of lysine decarboxylase. Induction continued overnight at 37°C. The cells were harvested by centrifugation at 5000 rpm for 10 minutes. The supernatant was discarded, and the cell pellet was resuspended in PBS buffer (pH 7.0). The OD₆₀₀ was adjusted to 10, and the cell suspension was stored at 4°C for later use.

The catalytic reaction using the prepared cells above was performed at 37°C and 200 rpm. The reaction system for whole-cell catalysis to produce cadaverine was composed as follows:

The seed medium for E. coli NT1003 and its recombinant strains included 10 g/L ammonium sulfate, 10 g/L yeast extract, 5 g/L peptone, 5 g/L sodium glutamate, 5 g/L sucrose, 0.3 g/L methionine, 0.3 g/L threonine, 77 mg/L MnSO₄·H₂O, 1 g/L MgSO₄·7H₂O,0.032 g/L FeSO₄·7H₂O, 0.5 g/L K₂HPO₄, 0.55 g/L sodium pyruvate, along with an appropriate concentration of antibiotics.

The fermentation medium consisted of 20 g/L glucose, 10 g/L (NH₄)₂SO₄, 5 g/L yeast extract, 5 g/L peptone, 0.5 g/L KCl,1.6 g/L MgSO₄·7H₂O, 32 mg/L FeSO₄·7H₂O, 32 mg/L MnSO₄·H₂O, 86 mg/L ZnSO₄·7H₂O, 77 mg/L CuSO₄,100 mM sodium molybdate (Na₂MoO₄), 10 g/L CaCO₃, 60 mg/L vitamin B₁, 10 mg/L nicotinamide, 30 μg/L biotin, 0.3 g/L threonine,0.3 g/L methionine.

The antibiotics used in this study included 50 μg/mL of kanamycin, 100 μg/mL ampicillin, or/and 50 μg/mL streptomycin.

IPTG-induced cultivation was conducted in 500 mL conical flasks containing 30 mL of fermentation medium. The bacterial culture from an overnight pre-culture in seed medium was transferred to the aforementioned fermentation medium at an initial inoculum of OD₆₀₀=0.2, followed by shaking incubation at 37°C and 220 rpm. When the cell density reached OD₆₀₀=0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM to induce the expression of pathway enzymes. Simultaneously, the shaking speed was reduced to 220 rpm while maintaining the cultivation temperature at 37°C for continued incubation.

For temperature-regulated cultivation, the procedures for strain activation and seed culture were identical to those used in IPTG-induced cultivation. After transferring the seed culture to the fermentation medium, the cells were initially cultured at 37°C and 220 rpm to promote growth. Once the cell density reached OD₆₀₀=0.6, the temperature was rapidly shifted to 42°C while maintaining the shaking speed at 220 rpm for an additional 3 hours. This process activated the temperature-sensitive promoter-based expression system, thereby achieving high-efficiency expression of the target gene.

Cell density was monitored by measuring the absorbance at 600 nm using a UV spectrophotometer.

Nucleic acid concentration was determined by measuring the A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios using Nanodrop.

The concentration of the fluorescent substance was determined by measuring the fluorescence intensity at 515/526nm of a 200µL sample using a fluorescence spectrometer.

Glucose and lysine contents were detected using a SBA (Biosensor Analyzer). After calibration with 100 g/L glucose and lysine standard solutions, the glucose and lysine contents in the samples were measured.

Cadaverine was determined via dansyl chloride derivatization. The method involves pre-column derivatization with dansyl chloride, separation of the products on an HC-C18 column, and a mobile phase composed of an acetonitrile-water mixture.