Experiments

LB Agar Plate Preparation

1. Take one bottle of BeyoPure™ LB Broth with Agar (Catalog No. ST158), open the package, and add it entirely into a 1L blue-capped bottle.
2. Add ultrapure water from the Milli-Q® Type 1 Ultrapure Water System until it reaches 500 ml, shake well, apply autoclave tape, and slightly loosen the bottle cap.
3. Sterilize at 121°C for 30 minutes.
4. Allow it to cool to around 60°C, then add the corresponding antibiotic.

Antibiotic Stock Solution Preparation

1. Kanamycin Stock Reagent

1. Weigh 1 g of kanamycin powder (Beyotime: ST101) and add it into a 50 ml centrifuge tube.
2. Add 20 ml of sterilized ddH2O (Sangon Biotech: B541017), vortex to mix, let it stand for 3 minutes, and filter sterilize using a sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE, 0.22 μm).
3. 50 mg/ml kanamycin stock solution was prepared as described above and subsequently aliquoted into 2 ml EP tubes for storage.

2. Chloramphenicol Stock Reagent

1. Weigh 600 mg of chloramphenicol powder (Beyotime: ST2722-1g) and add it into a 50 ml centrifuge tube.
2. Add 20 ml of sterilized ddH2O (Sangon Biotech: B541017), vortex to mix, let it stand for 3 minutes, and filter sterilize using a sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE, 0.22 μm).
3. 30 mg/ml Chloramphenicol stock solution was prepared as described above and subsequently aliquoted into 2 ml EP tubes for storage.

Preparation of Different Biomarker Stock Solutions

1.Uric Acid

• 50 ml of 1X PBS (Beyotime C0221A) was added to a 50 ml centrifuge tube, followed by the addition of 0.0168 g of uric acid. The mixture was vortexed, allowed to stand for 3 minutes, and then sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting solution was aliquoted into 2 ml EP tubes for storage.

2.Lactic Acid

• 0.2 g quantity of 90% lactic acid solution was pipetted into a 50 ml centrifuge tube, and ultrapure water from the Milli-Q® Type 1 Ultrapure Water System was added to a final volume of 50 ml. The solution was vortexed, allowed to stand for 3 minutes, and then sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting solution was aliquoted into 2 ml EP tubes for storage.

3.Tryptophan

• 0.1 g quantity of tryptophan was weighed and added to a 50 ml centrifuge tube, and ultrapure water from the Milli-Q® Type 1 Ultrapure Water System was added to a final volume of 50 ml. The mixture was vortexed, allowed to stand for 3 minutes, and then sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). A 1 mM tryptophan stock solution was thus prepared and aliquoted into 2 ml EP tubes for storage.

4.Glucose

• 1.8 g quantity of glucose was weighed and added to a 50 ml centrifuge tube, and ultrapure water from the Milli-Q® Type 1 Ultrapure Water System was added to a final volume of 50 ml. The solution was vortexed, allowed to stand for 3 minutes, and then sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). A 200 mM glucose stock solution was prepared and aliquoted into 2 ml EP tubes for storage.

Preparation of Biomarker Induction Solutions at Different Concentrations

Bacterial Transformation

1. Trans 5a Chemically Competent Cells (TransGene CD201-01) were retrieved from the -80°C freezer and allowed to thaw on ice for 10 minutes.
2. 10 ng of the target plasmid was added to the thawed competent cells, gently mixed, and incubated on ice for 30 minutes. During this period, a water bath was prepared and set to 42°C.
3. The competent cell-plasmid mixture was subjected to heat shock at 42°C for 45 seconds, followed by an immediate transfer to ice for 2 minutes without agitation of the centrifuge tube.
4. Subsequently, 500 µl of sterile LB medium (without antibiotics) was added to each tube, mixed thoroughly, and incubated at 37°C with shaking at 200 rpm for 1 hour to facilitate bacterial recovery. Meanwhile, LB plates with the appropriate antibiotic were prepared and placed in the 37°C incubator.
5. Transformed competent cells were plated onto LB agar plates containing the appropriate antibiotic and spread evenly. The plates were incubated at 37°C until the liquid was absorbed, then inverted and incubated overnight at 37°C.

Bacterial Seed Culture Preparation, Induction, and Fluorescence Assay Testing

1. Add 3 ml of appropriate antibiotic LB into a 12 ml culture tube.
2. Pick 3 single colonies from the plate into the 3 ml LB with the appropriate antibiotic.
3. Place in a shaker at 220 rpm, 37°C, and incubate overnight.
4. Take 60 µl of the overnight culture and add it to 6 ml of LB with the appropriate antibiotic, and incubate at 220 rpm until the OD reaches 0.2-0.3, allowing the culture to reach the log phase.
5. Mix the log-phase culture well using a pipette, and transfer 900 µl into a 96-well deep-well plate.
6. Add 100 µl of the prepared biomarker inducer (uric acid/lactic acid/glucose/tryptophan) into the 900 µl log-phase culture.
7. Place the deep-well plate into a shaker at 1000 rpm, 37°C, and incubate overnight.
8. Transfer 150 µl of the overnight culture from the deep-well plate into a black microplate.
9. Place it into a TECAN Spark to measure the corresponding OD and fluorescence values.

Fluorescent Protein Standard Expression and Purification

1. Pick 3 single colonies from the plate into 50 ml of LB with the appropriate antibiotic.
2. Place in a shaker at 220 rpm, 37°C, and incubate overnight.
3. Centrifuge at 10000 rpm for 5 minutes and discard the supernatant.
4. Add 8 ml of lysis buffer, and mix thoroughly.
5. Place in an ultrasonic cell disruptor for 5 minutes:
   • Power = 600W
   • Sonication on-time: 5s
   • Sonication off-time: 5s
6. Centrifuge and collect the supernatant.
   • The subsequent protein purification will use the Beyotime His-tag Protein Purification Kit (Denaturant-resistant): P2229S for purification.
   • Load BeyoGold™ His-tag Purification Resin (denaturant-resistant) into the column, equilibrate the purification column twice with 1 ml lysis buffer.
   • Load the supernatant onto the column. To improve yield, collect the flow-through and reload it 3-5 times to fully bind the His-tag protein.
   • Wash the column 5 times with 1 ml of denaturing lysis buffer.
   • Wash the column 5 times with 1 ml of washing buffer.
   • Elute the protein 6-10 times with 0.5 ml of elution buffer, collecting the final elution.

SDS-PAGE

1. Take 20 µl of each sample and add 5 µl of SDS-PAGE protein loading buffer (Boyetime P0015), vortex to mix, and incubate at 98°C in a water bath for 10 minutes to prepare loading samples.
2. Centrifuge at 10000 rpm for 1 minute.
3. Prepare 1X MOPS-SDS running buffer by diluting 50 ml of 20X MOPS-SDS running buffer in 950 ml of ddH2O.
4. Set up YoungPAGE™ (Genscript: M00928) pre-cast gels in a Biorad Mini-PROTEAN Tetra Vertical Electrophoresis Cell according to the manufacturer's instructions, and add the 1X MOPS-SDS running buffer.
5. Load 10 µl of the prepared samples into the gel wells, and add Broad Multi Color Pre-Stained Protein Standard (Genscript: M00624) as the protein molecular weight marker into any empty wells.
6. Run the electrophoresis at 200 mV for 20 minutes, adjusting the electrophoresis time based on the actual results.
7. After electrophoresis, remove the SDS gel and proceed with staining and destaining according to the manufacturer's instructions.
8. After destaining, place the gel in a ChemiDoc Imaging System (Biorad: 12003153) for gel imaging and upload the images.

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