I.Conjugative Transfer

Materials：9k-S⁰ plates, LB plates, Starkey-Na₂S₂0₃ solid medium,1.5 mL centrifuge tube, PBS buffer, 9k buffer

1.Collection of Bacterial Cells: Acidithiobacillus ferrooxidans ATCC 23270, serving as the recipient strain, was cultivated in 9K-S⁰ medium for three days until it reached the logarithmic growth phase. The culture contained 1% (w/v) elemental sulfur, and the cells were collected by centrifugation at 6000 rpm for 3 minutes when the OD₆₀₀ reached 0.4. E. coli SM10, carrying the recombinant plasmid and acting as the donor strain, was subcultured from an overnight culture into LB medium supplemented with 100 μg/mL streptomycin and grown to an OD₆₀₀ of 0.6 to 0.8. The cells were then collected by centrifugation at 7000 rpm for 3 minutes. 2.Washing of Bacterial Cells: The recipient cells were washed with a 2:2 wash solution and centrifuged at 2000 rpm for 1 minute to remove elemental sulfur. The supernatant was decanted as the cell suspension.
3.Conjugation: Both the recipient and donor cells were concentrated to an OD₆₀₀ of 1. They were mixed in a 1:2 ratio (recipient:donor), and 100 μL of the mixture was evenly spread onto a 2:2 solid conjugation plate containing a 0.45 μm filter membrane. The plates were incubated at 30℃ for 5 days in an upright position.
4.Membrane Washing: The 0.45μm membrane was removed from the plate and placed into a sterile 50 mL EP tube. The cells on the membrane were washed off with 5 mL of 2:2 wash solution, collected by centrifugation, and concentrated into a 1.5 mL EP tube. Serial dilutions were made at 10-fold, 50-fold, and 100-fold. An aliquot of 100 μL of the concentrated cell suspension was spread onto Starkey-Na₂S₂0₃ solid medium supplemented with 100 μg/mL streptomycin and incubated at 30℃ for 15-20 days, inverted, until colonies were observed.
5.Verification: Single colonies were first inoculated into 10 mL of 9K liquid medium containing 10% Fe²⁺ (49.64 g/L FeSO₄·7H₂O) without antibiotics and cultivated for 3 days until the culture turned red. The culture was then transferred into 30 mL of 9K-S⁰ liquid medium containing 200 μg/mL streptomycin for further cultivation for 6 to 10 days. The sulfur particles were observed to be fine and did not clump together, settling at the bottom with a turbid supernatant upon standing. A 20 mL aliquot of the culture was used to extract plasmid DNA, and PCR was performed for verification. If successful, subsequent experiments were conducted.

II. Determination of the Growth Curve of Engineered E. coli BL21

Materials：LB plates, 1.5 mL centrifuge tube, 96-well plate, Micro plate reader (produced by gene5^®)

1.Preparation of LB Liquid Medium: The engineered E.coli BL21 strain, cultured to the mid-log phase (OD₆₀₀ ~ 0.5), was used to inoculate fresh LB medium at a 1% inoculation ratio.
2.Cultivation: The culture was allowed to stand at 30 ℃ for 2 days and then transferred to a shaking incubator set at 180 rpm for a period of 24 hours. Samples were collected at 3, 10, 15, 21, and 25 hours post-inoculation to monitor the OD changes. Three parallels were set up for the experiment.
3.Sampling Method: Aliquots of 200 μL of the supernatant were taken to measure the OD₆₀₀. The growth curve was plotted based on the changes in OD₆₀₀. Each sample was tested in triplicate to ensure accuracy and consistency.

III. Determination of the Growth Curve of A. ferrooxidans Conjugants

Materials：9k-S⁰ plates, 9k-Fe²⁺ plates, 1.5 mL centrifuge tube, 96-well plate

1. Preparation of 9k-S⁰ Liquid Medium: The engineered A. ferrooxidans strain, cultured to the mid-log phase (OD₆₀₀ ~ 0.5), was used to inoculate fresh 9k-S⁰ medium at a 1% inoculation ratio. 2. Cultivation: The culture was allowed to stand at 30 ℃ for 2 days and then transferred to a shaking incubator set at 180 rpm for a period of 14 days. Samples were collected starting at the time of inoculation, with one sample taken every 24 hours to monitor OD changes. Three parallels were set up for the experiment.
3. Sampling Method: The samples were centrifuged at 2000 rpm for 30 seconds to precipitate elemental sulfur. Then, 200 μL of the supernatant was taken to measure the OD₆₀₀. The growth curve was plotted based on the changes in OD₆₀₀. Each sample was tested in triplicate to ensure accuracy and consistency.

IV. Determination of Biofilm Formation by Engineered E. coli BL21

Materials：LB plates, 1.5 mL centrifuge tube, PBS buffer, Crystalline Violet Stain, 95% ethanol, 24-well plate, Micro plate reader (produced by gene5^®)

1. Activation and Cultivation: The engineered E. coli BL21 strain was streaked from a glycerol stock onto an LB agar plate and incubated to activate the culture. A single colony was then transferred to liquid LB medium and grown to the late logarithmic phase. The culture was centrifuged at 5000 rpm for 10 minutes to pellet the cells. The supernatant was discarded, and the cells were resuspended in an equal volume of sterile PBS buffer. This washing step was repeated.
2. Inoculation and Incubation: One hundred microliters of the resuspended cells were diluted in 900 μL of PBS to achieve a 10-fold dilution. This diluted cell suspension was mixed evenly and added to a 24-well plate at a 1% inoculation ratio, with 1 mL of the diluted cell suspension per well. Each strain was tested in quadruplicate, and the plate was incubated at 30℃ for 12h, 24h, 48h, and 72h to allow biofilm formation.
3. Cryo Violet Staining: After the specified incubation periods, the culture medium was carefully aspirated from each well using a 1 mL pipette, leaving the biofilm intact. One milliliter of crystal violet staining solution was added to each well, and the plate was incubated for 15 minutes to stain the biofilms. The staining solution was then aspirated, and each well was gently rinsed twice with 1 mL of sterile PBS buffer to remove unbound stain. Finally, add 1 mL of 95% ethanol solution to resuspend the biofilm, place it at 37 ℃ for 10 minutes to fully dissolve the biofilm, and use a microplate reader at 570 nm to measure the biofilm data.

V. Determination of Biofilm Formation by A. ferrooxidans Conjugants

Materials：9k-S⁰ plates, 1.5 mL centrifuge tube, PBS buffer, Crystalline Violet Stain, 95% ethanol, 24-well plate, Micro plate reader (produced by gene5^®)

1.Activation and Cultivation of Engineered Bacteria: The engineered A. ferrooxidans strain is streaked from a glycerol stock onto 9k-S⁰ medium for strain activation and subcultured to the late logarithmic phase. The culture is then centrifuged at 5000 rpm for 10 minutes to pellet the cells. The supernatant is discarded, and an equal volume of sterile PBS buffer is added to resuspend the cells. This washing step is repeated.
2. Preparation and Inoculation: One hundred microliters of the resuspended cell solution is mixed with 900 μL of PBS to create a 10-fold dilution. This diluted cell solution is mixed evenly and its OD₆₀₀ is measured using a UV-Vis spectrophotometer. The culture is diluted with 9K medium to adjust the cell density to an OD₆₀₀ of 0.05. The diluted cell solution is then added to a 24-well plate, with 1 mL of the cell solution per well. Each strain is tested in triplicate, with six sets in total, and the plate is incubated at 30℃ for 12h, 24h, 36h, 48h, 60h, and 72h to allow biofilm formation, after which the biofilms are stained with Crystal Violet.
3. Crystal Violet Staining Method: The staining procedure is performed as described previously.

VI. Determination of c-di-GMP Concentration

Materials：Enzyme-Linked Immunosorbent Assay (ELISA) Kit, BCA kit

Assay Kit

We employ an enzyme-linked immunosorbent assay (ELISA) kit specific for cyclic di-GMP (c-di-GMP) to measure its concentration. Since c-di-GMP is found within cellular proteins and the quantification of c-di-GMP requires data on the protein content of the cells, we utilize a bacterial protein extraction kit to isolate bacterial proteins and a BCA protein assay kit to determine protein concentrations.

Collect the bacterial culture in the late logarithmic phase and measure the OD₆₀₀. Concentrate and resuspend the culture in PBS to achieve a 1 mL suspension with an OD₆₀₀ of 1.8.
Bacterial Protein Extraction Kit Procedure:
① Preparation of Extraction Solution: Mix Reagent A1 and Reagent A2 in a 9:1 ratio to create Protein Extraction Solution A. Thoroughly mix and keep the solution on ice until use.
② Centrifugation of Bacterial Suspension: Centrifuge the bacterial suspension at 4℃ at 10,000×g for 5 minutes. Discard the supernatant and remove any remaining liquid as much as possible to collect the bacterial cells.
③ Washing of Bacterial Cells: Wash the bacterial cells twice with PBS.
④ Protein Extraction: Add 500 uL of protein extraction solution per 50-100 mg wet weight of bacterial sample. Vortex to mix well and incubate at 2-8℃ with shaking for 20-30 minutes.
⑤ Sonication: Sonicate the suspension on ice under conditions of 150-300 watts, 10 seconds sonication/10 seconds interval, until the bacterial suspension becomes clear.
⑥ Centrifugation and Collection of Supernatant: Centrifuge the bacterial suspension at 4℃ at 12,000×g for 10 minutes.
Transfer the supernatant into a pre-chilled clean centrifuge tube. The supernatant now contains the total bacterial protein extract.
Determination of c-di-GMP Concentration Using a c-di-GMP Enzyme-Linked Immunosorbent Assay (ELISA) Kit Procedure:
1.Dilution of Standards: The kit provides a concentrated standard. Prepare dilutions in small test tubes as per the chart below:

40 pg/mL	Standard number 5	Add 150 μL of the original double standard to 150 μL of standard diluent
20 pg/mL	Standard number 4	Add 150 μL of standard 5 to 150 μL of standard diluent
10 pg/mL	Standard number 3	Add 150 μl of standard 4 to 150 μL of standard diluent
5 pg/mL	Standard number 2	Add 150 μL of standard 3 to 150 μL of standard diluent
2.5 pg/mL	Standard number 1	Add 150 μL of standard 3 to 150 μL of standard diluent

2.Sample Application: Prepare blank wells (no samples or enzyme conjugates added, but follow the same procedure for all other steps), standard wells, and sample wells. Add 50 μL of each standard to the appropriate wells on the ELISA plate. For sample wells, first add 40 μL of sample diluent, then add 10 μL of the sample (resulting in a final dilution of the sample of 5 times). Apply samples to the bottom of the wells, avoiding contact with the walls, and gently mix by tapping.
3.Incubation: Seal the plate with adhesive film and incubate at 37 ℃ for 30 minutes.
4.Dilution of Wash Solution: Dilute the concentrate wash solution 30-fold with distilled water and keep it ready for use.
5.Washing: Carefully remove the adhesive film, discard the liquid, and dry by tapping. Fill each well with the diluted wash solution, let it sit for 30 seconds, then discard. Repeat this process five times and dry by tapping.
6.Addition of Enzyme Conjugate: Add 50 μL of enzyme conjugate to each well, excluding the blank wells.
7.Incubation: Follow the same procedure as step 3.
8.Washing: Follow the same procedure as step 5.
9.Color Development: To each well, first add 50 μL of substrate A, then 50μl of substrate B. Gently mix and incubate at 37℃ in the dark for 10 minutes to develop the color.
10. Termination: Add 50 μL of stop solution to each well to stop the reaction (the color will change from blue to yellow immediately).
11.Measurement: Zero the ELISA reader with the blank well and measure the absorbance (OD value) at 450 nm for each well in sequence. The measurement should be conducted within 15 minutes after the addition of the stop solution.
Determination of Protein Concentration Using the BCA Assay Kit Procedure:
1.Dilution of Standards: The kit provides a concentrated bovine serum albumin (BSA) standard. Dilute the standard in small test tubes according to the chart below:

Name of standard sample	Diluent volume（μL）	Standard liquid volume（μL）	Final concentration（μg/mL）
A	0	300	2000
B	125	375	1500
C	375	325	1000
D	175	B tube take 175	750
E	325	C tube take 325	500
F	325	E tube take 325	250
G	325	F tube take 325	125
H	400	G tube take 325	25
I	400	0	0

2.Preparation of BCA Working Solution: Based on the number of standard samples and sample wells, prepare a sufficient amount of BCA working solution with a volume ratio of 50 mL of Reagent A to 1 mL of Reagent B. Mix thoroughly.
3. Application of Standards and BCA Working Solution: Add the appropriate amount of standard samples to a 96-well plate. Add 200 μL of BCA working solution to each well. Measure the absorbance at 562 nm using a microplate reader. Plot a standard curve based on the results of the standards.
4. Sample Analysis: Take 20 μL of the prepared protein sample and add it to 200 μL of BCA working solution in the microplate wells. Set the microplate reader to 562 nm and measure the sample absorbance. Calculate the protein concentration of the sample based on the standard curve and the solution concentration. Perform each experiment in triplicate.

VII. Gold Transcription Factor Specificity Verification Experiment

Materials：LB medium, Colony template, PBS buffer, 96-well plate, Micro plate reader (produced by gene5^®)

1.Activation and Inoculation of E. coli BL21 Cells Containing the Recombinant Plasmid: Activate E. coli BL21 cells harboring the recombinant plasmid and inoculate them into LB medium at a ratio of 1%. Add 0.5 mM IPTG along with varying concentrations of HAuCl4 (0 µM, 0.25 µM, 1 µM, 5 µM, 10 µM, and 20 µM) for induction.
2.Sampling and Measurement of Fluorescence: Collect cells at 0, 6, 12, 18, and 24 hours of cultivation by centrifugation at 12,000 rpm for 2 minutes. Harvest the bacterial cells and wash with PBS buffer solution. Take 200 μL of the bacterial suspension and measure the fluorescence data versus OD₆₀₀ using a microplate reader in a 96-well plate, with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
3.Overnight Cultivation and Fluorescence Measurement: Cultivate the engineered E. coli BL21 strain in LB medium containing 20 µM HAuCl₄ (Au³⁺), 20 µM CuSO₄ (Cu²⁺), 20 µM CdCl₂ (Cd²⁺), 20 µM ZnCl₂ (Zn²⁺), or 20 µM NiCl₂ (Ni²⁺), with a control group without metal ion induction. Measure the ratio of fluorescence to OD₆₀₀.

VIII. Suicide Module Verification Experiment

Materials：LB medium, Colony template, 96-well plate, Micro plate reader (produced by gene5^®)

1.Cultivation of E. coli BL21 Cells Transformed with the Plasmid Expression Vector: Cultivate E. coli BL21 cells harboring the plasmid expression vector for 12 hours until they reach the late logarithmic phase.
2.Addition of Inducers and Measurement of OD₆₀₀: Measure the OD₆₀₀ of the culture before adding the inducers. Set up four control groups: one without IPTG and Au³⁺, one with 0.5 mM IPTG but without Au³⁺, one with 20 μM Au³⁺ but without IPTG, and one with both 0.5 mM IPTG and 20 μM Au³⁺.
3.Measurement of OD₆₀₀ After Inducer Addition: After the addition of the inducers IPTG/Au³⁺, measure the OD₆₀₀ every 3 hours. Subtract the OD₆₀₀ value at the 12th hour from the subsequent OD₆₀₀ measurements to obtain the growth curve data. Plot the difference in OD₆₀₀ values over time to create a growth curve.

IX.9k liquid culturte medium

Materials for 1 L: (NH₄)₂SO₄ 30 g
KCl 1 g
K₂HPO₄ 5 g
MgSO₄·7H₂O 5 g
Ca(NO₃)₂ 0.1 g

Protocol:

1. Measure materials to scale that in need
2.Add the required proportion of water, shake the bottle until the solids completely dissolve.
3. Adjust pH to 2.0 using 30% concentrated sulphuric acid.
4.Place the bottles into the autoclave for sterilization, under 121°C for 30 minutes.
5.Let the bottles cool down under room temperature. The solution would preserve under room temperature for a maximum of 7 days.
6. Add 1% sulphur monomer or 44.22 g/L ferrous sulphate heptahydrate depending on culture conditions

X.Starkey-Na₂S₂O₃ solid culturte medium

Materials for 1 L: LiquidA： (NH₄)₂SO₄ 6 g
KH₂PO₄ 6 g
MgSO₄·7H₂O 0.1 g
CaCl₂ 0.37 g
LiquidB:
agar 20 g
LiquidC：
Na₂S₂O₃·5H₂O 3 g
FeSO₄·7H₂O 0.012 g
20 mL H₂O

Protocol:

1.Measure materials to scale that in need
2.Add the required proportion of water, shake the bottle until the liquid A, B, C solids completely dissolve.
3.Place the liquid A, B bottles into the autoclave for sterilization, under 121°C for 30 minutes.Liquid C filtrate for sterilization
4. Mix equal volumes of liquid A and liquid B, then add 5% of liquid C of the total volume of liquid A and B, mix well and prepare the medium plate.