L. plantarum L68 competent cell preparation and DNA transformation:

LB Medium

• 1.A single colony of L. plantarum L168 was inoculated in 5 mL of MRS media and cultured overnight at 37°C with shaking (200 rpm). The primary culture was diluted in a 1:50 (v/v) ratio in a 50 mL secondary culture composed of MRS media and 10% (w/v) glycine premixed in a 9:1 ratio.

• 2.The secondary culture was incubated at 37°C until OD600 reached 0.6~0.8, following which the cells were placed on ice for 15 minutes and then pelleted down by centrifuging at 4000 rpm (3363 g) for 10 min at 4°C.

• 3.Discard the supernatant and precipitate it with the same volume of 30% PEG 1500. Let it stand for 5min. Then centrifuge at 4500rpm and 4 ° C for 10min again. Discard the supernatant and add 30% PEG 1500 into precipitation at a ratio of 100:1 for resuspension. After resuspension,the competent cells were then dispensed in 50 μL aliquots for DNA transformation.

• 4.For all transformations, 500 ng of plasmid was added to the competent cells and then transferred to chilled 2 mm gap electroporation cuvettes (Bio-Rad Laboratories GmbH, Germany). Electroporation transformation was done with a single pulse at 2.2 kV, 4 ms, after which 500 μL of lukewarm MRS media was immediately added. The mixture was kept for incubation at 37°C for a recovery period of 2 h. Following the recovery phase, the cells were centrifuged at 5800 rpm (3363 g) for 2 min, 450 μL of the supernatant discarded, and 100 μL of the resuspended pellet was plated on MRS Agar supplemented with 10 μg/mL of Erythromycin. The plates were incubated at 37°C for 48 h to allow the growth of distinct single colonies.

Plasmid amplification:

• 1.Culture medium preparation:
  (1)Prepare 600ml LB liquid culture medium: Tryptone 6g, yeast extract 3g, NaCl 6g, water 600ml
  (2)Solid culture medium containing Erythromycin 60mL (final concentration of Erythromycin 250 μg/mL): [Note:the culture medium containing Erythromycin cannot be heated under high pressure, or Erythromycin will become invalid] Firstly, LB solid culture medium was prepared by supplementing LB liquid culture medium with 2% agar. Subsequently, a high voltage was applied, followed by the addition of erythromycin in the subsequent steps.

• 2.Take 100 μL DH5α Chemically Competent Cell, add 0.5 μL plasmid,and ice bath for 30min (this step needs to be done in the clean bench and on ice)

• 3.Heat shock at 42 ° C for 45seconds. Due to the occupation of the metal heat shock instrument, we used water bath instead. We need to turn on the hot water bath machine about 15 minutes in advance, and put it back on the ice right away. Then Ice bath for 2 minutes.

• 4.Add 400 μL Lysogeny broth, shake and culture at 37°C for 45-60min

• 5.Add Erythromycin into LB solid culture medium and coat with bacteria. The specific steps are as follows:

  • Ultraclean table sterilization;
  • Dissolve Erythromycin in absolute ethanol, and the concentration of Erythromycin absolute ethanol mixture is 50mg/ml. Ensure that the concentration of LB solid medium containing Erythromycin is 300-500 μg/mL.
  • High pressure LB solid culture medium has not been completely cooled (otherwise it is prone to solidification). If it has already solidified, it can be heated in a microwave oven first to ensure that it is in a liquid state. Then place it in the super clean table.
  • Inject alcohol into a small tube containing Erythromycin anhydrous ethanol mixture and put it into the clean bench.At the same time, prepare a number of 1.5 ml EP tubes, bacterial filters and syringes
  • use a syringe that retains needles to absorb all the Erythromycin anhydrous ethanol mixture in the small tube, then remove the needles, install the bacterial filter, and slowly push the syringe to filter the Erythromycin anhydrous ethanol mixture and inject it into a 1.5ml EP tube;
  • Use the gun head to absorb the Erythromycin anhydrous ethanol mixture in the EP tube with accurate dosage, and add it into the uncooled LB solid medium under high pressure.
  • Manually shake the glass bottle and mix well, then pour it into a flat plate (note that the lid of the plate should not be fully opened when pouring, it can be placed halfway below). After filling the plate, there is no need to cover it and wait for it to naturally cool and solidify.
  • If there is excess Erythromycin anhydrous ethanol mixture, write the substance, date, concentration and other information on the cover and side, and store at -80 ° C;
  • When the plate containing Erythromycin is solidified, take 50-100 μ Apply the shaken bacterial liquid onto the plate. First use 10 μ L's gun, suck 10 at a time μ L bacterial solution, point 6 on the plate.
  • Prepare a plastic scraper in advance. Spread the bacteria evenly on the plate, basically in a circular motion, Scrape and apply evenly;
  • Write the name, date, and other information on the back of the coated tablet, and place it in an oven for overnight cultivation. Due to the facultative anaerobic nature of Escherichia coli, sealing is not necessary. Pay attention to placing it upside down.

Bacterial amplification:

• 1.First amplification: after overnight culture in the medium containing Erythromycin. First, put the container, Lysogeny broth, etc. on the clean bench for ultraviolet irradiation for 15min, then add 0.8ml 50mg/ml Erythromycin in 80ml Lysogeny broth, shake and mix well. Then take 5ml into the EP tube. Pick up the colony (pick up the single colony on the Petri dish with a white

• 2.After completing the shaking of the shaking table for amplification again, take another 0.4ml of bacterial solution and add it to the final concentration of Erythromycin of 300-500 μ 40mL LB liquid culture medium with a concentration of g/mL, and then placed in a shaker for overnight cultivation.

Plasmid Extraction：

• 1.Add 500 to the adsorption column CP4 (the adsorption column is placed in the collection tube) μ Centrifuge the equilibrium solution BL at 12000 rpm for 1 minute, discard the waste liquid from the collection tube, and reposition the adsorption column in the recovery header.

• 2.Take 5-15ml of overnight cultured bacterial solution and add it to a centrifuge tube. Centrifuge at 12000 rpm for 1 minute, and extract the supernatant.

• 3.Add 500 to the EP tube with bacterial sediment μ L Solution P1, use a Pipette or vortex oscillator to completely suspend bacterial cell precipitation.

• 4. Add 500 to the EP tube μ Solution P2, gently flip up and down 6-8 times to fully lyse the bacterial body.

• 5.Add 500 to the EP tube μ Solution P4, immediately gently flip up and down 6-8 times, thoroughly mix, and white flocculent precipitates will appear. Place at room temperature for about 10 minutes, 12000 rpm, centrifuge for 10 minutes, and then,Sediment forms at the bottom of the EP tube.

• 6.Add the supernatant collected in the previous step to the filter column CS (with the adsorption column placed in the collection tube) in stages, centrifuge at 12000 rpm for 2 minutes, and collect the filtrate in a clean 2ml EP tube.

• 7.Add 0.3 times the volume of isopropanol to the filtrate, mix it upside down, and transfer it to the adsorption column CP4 (the adsorption column is placed in the collection tube).

• 8.At room temperature of 12000 rpm, centrifuge for 1 minute, remove the waste liquid from the collection tube, and place the adsorption column back into the collection tube.

• 9.Add 500 to the adsorption column CP4 μ l. Deprotein solution PD, 12000 rpm, centrifuge for 1 minute, pour out the waste liquid from the collection tube, and place the adsorption column CP4 in the recovery header.

• 10.Add 600 to the adsorption column CP4 μ Rinse solution PW at 12000 rpm, centrifuge for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column CP4 in the recovery header.

• 11.Add 600 to the adsorption column μ Rinse solution PW at 12000 rpm, centrifuge for 1 minute, and discard the waste liquid from the collection tube.

• 12.Place the adsorption column CP4 back into the recycling header, centrifuge at 12000 rpm for 2 minutes, and remove the residual rinsing solution from the adsorption column.

• 13.Place the adsorption column CP4 in a clean EP tube and add 100-300 suspended droplets to the middle of the adsorption membrane μ Elute buffer TB, place at room temperature for 2 minutes at 12000 rpm, and centrifuge for 1 minute to collect the plasmid solution into a centrifuge tube. Detect plasmid concentration, usually at 100ng/ μ Above l is sufficient.

Validation of Plasmids - Enzyme Digestion

Prepare a 20 μL System :

Regent	Dose
ddH2O	11.5 μL
Cutsmart	2 μL
Endonuclease 1	10.75 μL
Endonuclease 2	20.75 μL
Plasmid	5 μL

Table 1. Enzyme Digestion System

Add to the EP tube and put it into Metal bath at 37 ° C for 9 min

Agarose gel electrophoresis

• 1.Weigh 0.75g of Agarose gel, pour the corresponding volume of 1×TAE buffer into a 50 ml conical flask.

• 2.Place the Erlenmeyer flask in the microwave oven and dissolve it with maximum heat, take it out and mix it every 30s. When clarified, remove and cool toapproximately 60°C.

• 3. Add nucleic acid dye 3 μL.

• 4.Pour into the mold, wait for solidification at room temperature, and remove.

• 5.Add enough 1×TAE buffer to the electrophoresis tank until the gel surface is covered.

• 6.Remove the slot cover, slowly add the DNA sample, cover the slot cover, and remove after 30 minutes of electrophoresis.

Polymerase Chain Reaction

1.Add reagents according to the table below:
Prepare a 20 μL System :

Regent	Dose
GXL Buffer	10μL
dNTP mix	4μL
Gxl	1μL
Pre primer	2.5μL
Back primer	2.5μL
ddH20	29μL
Template sequence	1μL

Table 2. Polymerase Chain Reaction System

2.Steps of reaction

Temperature	Time
95℃	30s
95℃	15s
60℃	40s
72℃	5min
8℃	infinite

Table 3. Steps of reaction

Step2 to Step4 30cycles

Extraction of total bacterial RNA

• After verifying the correctness of L168, L168-branding iron, and L168-protoporphyrin, single colonies of each were selected and inoculated in 8 ml of MRS medium for overnight incubation. Sterilized 15ml clear glass tubes and stoppers were prepared in advance. On the following day, bacterial inoculation was performed at a dilution ratio of 1:100. After approximately 4 hours of incubation, the culture was centrifuged at 4 degrees Celsius and 4000 rpm for 5 minutes when the optical density (OD) reached around 0.7. The supernatant and bacterial cells were separated, with the bacterial cells intended for RNA extraction and the supernatant reserved for metabolite measurement.
• After culturing the bacteria, the extraction of total bacterial RNA was initiated:

• 1.The culture was centrifuged at 400 rpm and 4 °C for 5 minutes, and the supernatant was carefully removed. The bacterial pellet was then washed and resuspended in 1 mL of PBS. The bacterial suspension was transferred to a RNAse-free 1.5 mL EP tube. Subsequently, centrifugation was performed at 12,000 rpm for 2 minutes, and the supernatant was discarded.; 

• 2.The bacterial pellet was resuspended by adding 500 μL of lysozyme solution (20mg/mL, prepared by dissolving 20mg of lysozyme in 1mL TE buffer). The suspension was vigorously mixed and then incubated in a 37 °C metal bath for 30 minutes.

• 3.Following centrifugation at 12,000 rpm for 2 minutes, the supernatant was carefully removed.

• 4. Add 1 mL of Trizol to the bacterial pellet, resuspend thoroughly, and place the mixture on ice for 5 minutes. Note: When treating L168 with lysozyme, clumping may occur, and optimization of the cell lysis method is recommended to improve wall breaking efficiency.

• 5.Add 0.2 mL of chloroform to the Trizol-bacterial mixture, vortex vigorously for 15 seconds, and allow it to stand at room temperature for 5 minutes.

• 6.Centrifuge the mixture for 15 minutes at 12,000 rpm and 4°C. After centrifugation, the sample will separate into three distinct layers: the upper layer is a colorless aqueous phase containing dissolved RNA, the middle layer is a white interphase (avoid aspirating this layer), and the bottom layer is a red organic phase. The volume of the colorless aqueous phase is approximately 60% of the initial Trizol volume.

• 7.Transfer the colorless aqueous phase into a new RNAse-free 1.5 mL EP tube. Add 0.5 mL of isopropanol (0.5 mL per 1 mL of Trizol used initially), gently invert the tube to mix, and let it stand at room temperature for 10 minutes. Optionally, you can add 2 μL of glycogen to enhance the visibility of RNA precipitation.

• 8.Centrifuge the mixture for 10 minutes at 12,000 rpm and 4°C. Following centrifugation, sedimentation may appear on the walls of the tube.

• 9.Carefully remove the supernatant without disturbing the RNA pellet. Slowly add 75% ice-cold ethanol (prepared in advance with DEPC-treated water and anhydrous ethanol) to wash the RNA. It is recommended to add at least 1 mL of 75% ice-cold ethanol for every 1 mL of Trizol used initially. Gently pipette or blow on the tube to precipitate and resuspend the RNA pellet. Centrifuge the tube for 8 minutes at 7,500 rpm and 4°C.

• Repeat 9.

• 10.Remove the supernatant carefully, then perform a short spin and use a pipette to remove any excess ethanol. After air-drying the RNA pellet for 10 minutes in a Laminar Flow Cabinet, add 20-35 μL of DEPC-treated water to dissolve the RNA.

• 11.After extraction, the concentration of RNA was measured using an ultramicrosample spectrophotometer.