LB Preparation

1. Take one bottle of BeyoPure™ LB Broth (Catalog No. ST156), 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.

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.

Stock Solution Preparation

50 mg/ml Kanamycin Stock Solution

1. Weigh 1 g of kanamycin powder (Beyotime: ST101) and add it into a 50 ml centrifuge tube, 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) and aliquot it into 2 ml EP tubes for storage.

30 mg/ml Chloramphenicol Stock Solution

1. Weigh 600 mg of chloramphenicol powder (Beyotime: ST2722-1g) and add it into a 50 ml centrifuge tube, 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), and aliquot it into 2 ml EP tubes for storage.

AHL Solution Preparation (N-[(3S)-Tetrahydro-2-oxo-3-furanyl]hexanamide, 3O-C6-HSL, HHL CAS: 143537-62-6)

1. Weigh 4.15 mg of AHL into a 50 ml centrifuge tube.
2. Add ultrapure water from the Milli-Q® Type 1 Ultrapure Water System until it reaches 12.5 ml, vortex to mix, and let it stand for 3 minutes.
3. Filter sterilize using a sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE, 0.22 μm) to prepare a 1.56 mM AHL stock solution, and aliquot it into 2 ml EP tubes for storage.

Plasmid Extraction from Single Colonies

1. All plasmid extraction reagents are used with the Tiangen Plasmid Mini Kit (DP103).
2. Retrieve the glycerol stock of the target plasmid from the -80°C freezer, streak it onto an appropriate antibiotic plate, and incubate overnight at 37°C.
3. Pick a single colony from the plate and inoculate it into 5 ml of LB with the appropriate antibiotic, incubating overnight at 220 rpm.
4. Extract the plasmid using the Tiangen Plasmid Mini Kit.
5. Measure the plasmid concentration using NanoDrop One.

Plasmid Extraction from a Library

1. Retrieve the glycerol stock of the target library from the -80°C freezer, streak it onto an appropriate antibiotic plate, and incubate overnight at 37°C. Thaw the remaining bacterial suspension and transfer it into 100 ml of LB with the appropriate antibiotic, incubating for 6 hours.
2. Extract the plasmid library according to the instructions in the Tiangen Plasmid Mini Kit (DP103), and measure the library concentration using NanoDrop One.
3. Pick 10 single colonies from the plate and perform Sanger sequencing for sampling to verify the accuracy of the library.

Bacterial Transformation

1. Retrieve Trans 5a Chemically Competent Cells (TransGene CD201-01) from the -80°C freezer and place them on ice to thaw for 10 minutes.
2. Add 10 ng of the target plasmid to the thawed competent cells, gently mix, and incubate on ice for 30 minutes. Meanwhile, prepare a water bath at 42°C.
3. Heat shock the mixture of competent cells and plasmid at 42°C for 45 seconds, then immediately transfer the tube to ice for 2 minutes without shaking the centrifuge tube.
4. Add 500 µl of sterile LB medium (without antibiotics) to each tube, mix well, and incubate at 37°C with shaking at 200 rpm for 1 hour to allow bacterial recovery. Meanwhile, prepare the appropriate antibiotic LB plates and place them in the 37°C incubator.
5. Based on the experimental requirements (plasmid or recombinant ligation product transformation), pipette different volumes of transformed competent cells onto LB agar plates containing the appropriate antibiotic and spread evenly. Place the plates in the incubator at 37°C until the liquid is absorbed, then invert the plates and incubate overnight at 37°C.
6. Store the plates at 4°C after the colonies have grown.

Fluorescent Kinetic Assay

1. 3 single colonies were inoculated into 0.9 ml LB medium with specific antibiotics in a 2-ml 96-well deep-well plate (NEST). The plates were sealed with breathable Nunc Seals (Thermo Scientific) and incubated at 37 °C and 1000 r.p.m. overnight.
2. The overnight cultures were diluted 1:300 into 900 µl LB medium with antibiotics in 2-ml 96-well deep-well plates (NEST); the plates were sealed with Nunc Seals and incubated at 37 °C and 1,000 r.p.m. AHL solutions were then added to achieve the desired ion concentrations in the final system for our experimental conditions.
3. After mixing by pipetting, 135 µl cultures of each were transferred into 3 black 96-Well Cell Culture Plates with Clear Bottom (In Vitro Scientific) respectively to set up replicates, another 15 ul mineral oil were added in case of evaporation.
4. The kinetic of fluorescence readings (515 nm excitation wavelength and 555nm emission wavelength) were measured overnight in every 10 minutes on Spark® Multimode microplate Reader.

Library Construction

The resulting products were cleaned up using TIANquick Midi Purification Kit (TIANGEN) and then transformed to 100 µl DH5a Chemically Competent Cell (TransGen) and then plated on plates containing LB + 2% agar and grown overnight at 37 °C. Plates were stored at 4 °C after growth.

Item	Volume (µl)
Promoter library	3
Vector Plasmid	3
T4 Ligase buffer	2
rCutsmart	2
BsaI-HF®v2 (NEB)	1.5
T4 DNA Ligase (NEB)	1.5
ddH2O	7
Total	20

Plasmid library was assembled in one step. Promoter library were inserted into Vector plasmid sequentially with BsaI-HF®v2(NEB), rCutsmart, and T4 DNA Ligase (NEB) using the program: 37 °C for 5 min, 16 °C for 5 min, 60 cycles.

Fine-Tuning Circuit Screening Assay

1. 96 single colonies from Library construction were inoculated into 0.9 ml LB medium with specific antibiotics in a 2-ml 96-well deep-well plate (NEST). The plates were sealed with breathable Nunc Seals (Thermo Scientific) and incubated at 37 °C and 1000 r.p.m. overnight.
2. The overnight cultures were diluted 1:300 into 900 µl LB medium with antibiotics in 2-ml 96-well deep-well plates (NEST); the plates were sealed with Nunc Seals and incubated at 37 °C and 1,000 r.p.m. After 3 h, Cultures were diluted 1:100 into 900 µl LB medium with antibiotics in 2-ml 96-well deep-well plates (NEST), and ion solutions were then added to achieve the corresponding Cu(II) concentrations.
3. After mixing by pipetting, 135 µl cultures of each were transferred into 3 black 96-Well Cell Culture Plates with Clear Bottom (In Vitro Scientific) respectively to set up replicates, and another 15 ul mineral oil was added in case of evaporation.
4. The kinetic of optical density at 600 nm (OD600) and fluorescence readings (515 nm excitation wavelength and 555nm emission wavelength) were measured overnight every 30 minutes on Spark® Multimode microplate Reader. We inscribed the maximum fluorescence values at each of the two concentrations and calculated the corresponding fluorescence multiplicity.