Materials

• 20 mL Escherichia coli (E. coli) BL21 overnight culture in Lysogeny Broth.
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 100 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 100 mM aqueous AgNO₃ solution.
• Lysogeny Broth.
• 50 mL Flasks.
• 1 mL plastic cuvettes.

Equipment

• Shaking incubator.
• UV-vis NIR spectrophotometer.

Protocol

1. Dilute the overnight culture down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
3. Add bacterial culture or Lysogeny Broth (final volume 10 mL) into intended flasks according to the layout below (e.g. 100µL).
4. Add 100 mM aqueous HAuCl₄ solution (e.g. 5 µL) into assigned flasks according to the layout below.
5. Add 100 mM aqueous AgNO₃ solution (e.g. 1 µL) into assigned flasks according to the layout below.
6. Scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.
7. Place flasks into a shaking incubator set to 37°C at 200 rpm for 72 hours.
8. Scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer every 12 hours.

50 mL Flasks

Flask #	Bacterial Culture	100 mM AgNO3	100 mM HAuCl4	LB
1: 1 mm AgNO3	9.9 mL	100 µL
2: 10 mm AgNO3	9 mL	1 mL
3: 1 mm HAuCl4	9.9 mL		100 µL
4: 10 mm HAuCl4	9 mL		1 mL
5: 1 mm AgNO3 3D	9.9 mL	100 µL
6: 10 mm AgNO3 3D	9 mL	1 mL
7: 1 mm HAuCl4 3D	9.9 mL		100 µL
8: 10 mm HAuCl4 3D	9 mL		1 mL
9: negative control			100 µL	9.9 mL
10: negative control			1 mL	9 mL
11: negative control		100 µL		9.9 mL
12: negative control		1 mL		9 mL
13: negative control				10 mL

Materials

• 20 mL Escherichia coli (E. coli) BL21 overnight culture in Lysogeny Broth.
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 100 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 100 mM aqueous AgNO₃ solution.
• Lysogeny Broth.
• 50 mL Flasks.
• 1 mL plastic cuvettes.

Equipment

• Shaking incubator.
• UV-vis NIR spectrophotometer.

Protocol

1. For the previous experiments called “Preliminary Flask Experiment”:
   1. Add 100 mM of aqueous HAuCl₄ solution (e.g. 5 µL) into assigned flasks according to the layouts below.
   2. Add 100 mM of aqueous AgNO₃ solution (e.g. 1 µL) into assigned flasks according to the layouts below.

For the 1mM of AgNO3 3D flask	Added 1 mM of HAuCl4
For the 10 mM of AgNO3 3D flask	Added 10 mM of HAuCl4
For the 1 mM of HAuCl4 3D flask	Added 1 mM of AgNO3
For the 10 mM of HAuCl4 3D flask	Added 10 mM of AgNO3

1. Dilute the overnight culture down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
3. Add bacterial culture or Lysogeny Broth (final volume 10 mL) into intended flasks according to the layout below (e.g. 100µL).
4. Add 100 mM aqueous HAuCl₄ solution (e.g. 5 µL) into assigned flasks according to the layout below.
5. Add 100 mM aqueous AgNO₃ solution (e.g. 1 µL) into assigned flasks according to the layout below.
6. Place flasks into a shaking incubator set to 37°C at 200 rpm for 72 hours.
7. Scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.

50 mL Flasks

Flask #	Bacterial Culture	100 mM AgNO3	100 mM HAuCl4	LB
1: 0.1 mM AgNO3	9.99 mL	10 µL
2: 0.5 mM AgNO3	9.95 mL	50 µL
3: 1 mM AgNO3	9.9 mL	100 µL
4: 10 mM AgNO3	9 mL	1 mL
5: 1 mM HAuCl4	9.99 mL		100 µL
6: 10 mM HAuCl4	9 mL		1 mL
7: 0.1 mM AgNO3 Control		10 µL		9.99 mL
8: 0.5 mM AgNO3 Control		50 µL		9.95 mL
9: 1 mM AgNO3 Control		100 µL		9.9 mL
10: 10 mM AgNO3 Control		1 mL		9 mL
11: 1 mM HAuCl4 Control			100 µL	9.99 mL
12: 10 mM HAuCl4 Control			1 mL	9 mL

Materials

• 2 x 20 mL Escherichia coli (E. coli) BL21 overnight culture grown in Lysogeny Broth.
• Sodium phosphate dibasic heptahydrate.
• Sodium phosphate monobasic monohydrate.
• 10 M HCl.
• 10 M NaOH.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 100 mM aqueous AgNO₃ solution.
• Lysogeny Broth.
• Sterile flat-bottom 96-well plate with lid.
• 50 mL Falcon tubes.
• 15 mL Falcon tubes.
• 2 mL Eppendorf tubes.
• 0.2 µm Syringe filter.
• 15 mL Syringe.
• 1 mL plastic cuvettes.

Equipment

• Tabletop centrifuge.
• Rod sonicator (MS-72 probe).
• Tecan spark microplate reader.
• Shaking incubator.
• UV-vis NIR spectrophotometer.

Protocol

First Iteration (Plate A +B)

1. For 100 mM phosphate buffer pH 7:
   1. In a suitable container, add 800 mL ddH₂O.
   2. Add 15.487 grams of sodium phosphate dibasic heptahydrate to the solution.
   3. Add 5.827 grams of sodium phosphate monobasic monohydrate to the solution.
   4. Adjust pH with 10 M HCl or 10 M NaOH to pH 7.
   5. Add ddH₂O to a final volume of 1 L.
2. Dilute the overnight culture down to an OD600 of 0.2.
3. Grow diluted culture in a shaking incubator set to 37°C at 200 rpm until it reaches an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
4. Once the cultures have reached this phase: Transfer both of the cultures into separate 50 mL falcon tubes. Falcon tube 1 will be used directly in the 96-well plates and be referred to as the “cell system”.
5. Centrifuge falcon tube 2 at 4000 rpm for 10 minutes.
   1. Take supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into new 50 mL falcon tubes. This will be referred to as supernatant from now on.
   2. Resuspend the pellet in 6 mL of lysogeny broth. Split the resuspended pellet into 3 Eppendorf tubes.
   3. Sonicate one of the samples with a rod sonicator (MS72, 10% amplitude) for 20 seconds.
   4. Sonicate one of the samples with a rod sonicator (MS72, 10% amplitude) for 30 seconds.
   5. Sonicate one of the samples with a rod sonicator (MS72, 10% amplitude) for 45 seconds.
   6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
   7. The supernatant of these sonicated samples will now be referred to as “cell-free extract”.
   8. The pellet of these sonicated samples will now be referred to as “debris”.
6. Add cell-free extract into the intended wells of the 96-well plates according to the layout below.
   1. Plate A
      1. Row 1-4 uses 20-second sonication cell-free extracts.
      2. Row 5-8 uses 30-second sonication cell-free extracts.
   2. Plate B
      1. Row 1-4 uses 45-second sonication cell-free extracts.
7. Add supernatant, or cell-free extract into intended wells of the 96-well plates according to the layout below.
8. Resuspend the debris in 1 mL phosphate buffer and add into the intended wells of the 96-well plates according to the layout below.
9. Add culture (cell system), or Lysogeny Broth into the intended wells of the 96-well plates according to the layout below.
10. Add phosphate buffer into the intended wells of the 96-well plates according to the layout below.
11. Add 100 mM aqueous AgNO₃ solution into the intended wells of the 96-well plates according to the layout below.
12. Place 96-well plates in a shaking incubator set to 37°C at 200 rpm for 72 hours.
13. Scan absorbance from 350 nm to 1000 nm with a microplate reader.

Second Iteration (Plate C +D)

1. For the overnight cultures, measure OD600 and dilute down to an OD600 of 0.2 if needed.
2. Grow diluted culture in a shaking incubator set to 37°C at 200 rpm until it reaches an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth so samples are taken during this phase.)
3. Once the cultures have reached this phase: Transfer both of the cultures into separate 50 mL falcon tubes. Falcon tube 1 will be used directly in the 96-well plates and be referred to as the “cell system”.
4. Centrifuge falcon tube 2 at 4000 rpm for 10 minutes. Discard supernatant
   1. Resuspend the pellet in 1 mL of Lysogeny Broth.
   2. Sonicate one of the samples with a rod sonicator (MS72, 10% amplitude) for 45 seconds.
   3. The supernatant of this sonicated sample will now be referred to as “cell-free extract”.
5. Add cell-free extract into intended wells of 96-well plates according to the layout below.
6. Add culture (cell system), or Lysogeny Broth into the intended wells of 96-well plates according to the layout below.
7. Add 100 mM aqueous AgNO₃ solution into intended wells of 96-well plates according to the layout below.
8. Place 96-well plates in a shaking incubator set to 37°C at 200 rpm for 72 hours.

96-Well Plates

Plate A

	1	2	3	4	5	6
A	Cell free extract 5ul 145ul of buffer	Cell free extract 5ul 145ul of buffer	Cell free extract 5ul 145ul of buffer	Cell free extract 10ul 140ul of buffer	Cell free extract 10ul 140ul of buffer	Cell free extract 10ul 140ul of buffer
B	Cell-free extract 5uL 1.5uL 100 mM AgNO3 143.5uL buffer	Cell-free extract 5uL 1.5uL 100 mM AgNO3 143.5uL buffer	Cell-free extract 5uL 1.5uL 100 mM AgNO3 143.5uL buffer	Cell-free extract 10uL 1.5uL 100 mM AgNO3 138.5uL buffer	Cell-free extract 10uL 1.5uL 100 mM AgNO3 138.5uL buffer	Cell-free extract 10uL 1.5uL 100 mM AgNO3 138.5uL buffer
C	Cell-free extract 5uL 75uL 10mM AgNO3 70uL buffer	Cell-free extract 5uL 75uL 10mM AgNO3 70uL buffer	Cell-free extract 5uL 75uL 10mM AgNO3 70uL buffer	Cell-free extract 10uL 75uL 10mM AgNO3 65uL buffer	Cell-free extract 10uL 75uL 10mM AgNO3 65uL buffer	Cell-free extract 10uL 75uL 10mM AgNO3 65uL buffer
D	Cell-free extract 5uL 15uL 100 mM AgNO3 130uL buffer	Cell-free extract 5uL 15uL 100 mM AgNO3 130uL buffer	Cell-free extract 5uL 15uL 100 mM AgNO3 130uL buffer	Cell-free extract 10uL 15uL 100 mM AgNO3 125uL buffer	Cell-free extract 10uL 15uL 100 mM AgNO3 125uL buffer	Cell-free extract 10uL 15uL 100 mM AgNO3 125uL buffer
E	Cell free extract 5ul 145ul of buffer	Cell free extract 5ul 145ul of buffer	Cell free extract 5ul 145ul of buffer	Cell free extract 10ul 140ul of buffer	Cell free extract 10ul 140ul of buffer	Cell free extract 10ul 140ul of buffer
F	Cell-free extract 5uL 1.5uL 100 mM AgNO3 143.5uL buffer	Cell-free extract 5uL 1.5uL 100 mM AgNO3 143.5uL buffer	Cell-free extract 5uL 1.5uL 100 mM AgNO3 143.5uL buffer	Cell-free extract 10uL 1.5uL 100 mM AgNO3 138.5uL buffer	Cell-free extract 10uL 1.5uL 100 mM AgNO3 138.5uL buffer	Cell-free extract 10uL 1.5uL 100 mM AgNO3 138.5uL buffer
G	Cell-free extract 5uL 75uL 10mM AgNO3 70uL buffer	Cell-free extract 5uL 75uL 10mM AgNO3 70uL buffer	Cell-free extract 5uL 75uL 10mM AgNO3 70uL buffer	Cell-free extract 10uL 75uL 10mM AgNO3 65uL buffer	Cell-free extract 10uL 75uL 10mM AgNO3 65uL buffer	Cell-free extract 10uL 75uL 10mM AgNO3 65uL buffer
H	Cell-free extract 5uL 15uL 100 mM AgNO3 130uL buffer	Cell-free extract 5uL 15uL 100 mM AgNO3 130uL buffer	Cell-free extract 5uL 15uL 100 mM AgNO3 130uL buffer	Cell-free extract 10uL 15uL 100 mM AgNO3 125uL buffer	Cell-free extract 10uL 15uL 100 mM AgNO3 125uL buffer	Cell-free extract 10uL 15uL 100 mM AgNO3 125uL buffer

Plate A continued

	7	8	9	10	11	12
A	Cell free extract 20ul 130ul of buffer	Cell free extract 20ul 130ul of buffer	Cell free extract 20ul 130ul of buffer	Cell free extract 50ul 100ul of buffer	Cell free extract 50ul 100ul of buffer	Cell free extract 50ul 100ul of buffer
B	Cell-free extract 20uL 1.5uL 100 mM AgNO3 128.5uL buffer	Cell-free extract 20uL 1.5uL 100 mM AgNO3 128.5uL buffer	Cell-free extract 20uL 1.5uL 100 mM AgNO3 128.5uL buffer	Cell-free extract 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Cell-free extract 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Cell-free extract 50uL 1.5uL 100 mM AgNO3 98.5uL buffer
C	Cell-free extract 20uL 75uL 10mM AgNO3 55uL buffer	Cell-free extract 20uL 75uL 10mM AgNO3 55uL buffer	Cell-free extract 20uL 75uL 10mM AgNO3 55uL buffer	Cell-free extract 50uL 75uL 10mM AgNO3 25uL buffer	Cell-free extract 50uL 75uL 10mM AgNO3 25uL buffer	Cell-free extract 50uL 75uL 10mM AgNO3 25uL buffer
D	Cell-free extract 20uL 15uL 100 mM AgNO3 115uL buffer	Cell-free extract 20uL 15uL 100 mM AgNO3 115uL buffer	Cell-free extract 20uL 15uL 100 mM AgNO3 115uL buffer	Cell-free extract 50uL 15uL 100 mM AgNO3 85uL buffer	Cell-free extract 50uL 15uL 100 mM AgNO3 85uL buffer	Cell-free extract 50uL 15uL 100 mM AgNO3 85uL buffer
E	Cell free extract 20ul 130ul of buffer	Cell free extract 20ul 130ul of buffer	Cell free extract 20ul 130ul of buffer	Cell free extract 50ul 100ul of buffer	Cell free extract 50ul 100ul of buffer	Cell free extract 50ul 100ul of buffer
F	Cell-free extract 20uL 1.5uL 100 mM AgNO3 128.5uL buffer	Cell-free extract 20uL 1.5uL 100 mM AgNO3 128.5uL buffer	Cell-free extract 20uL 1.5uL 100 mM AgNO3 128.5uL buffer	Cell-free extract 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Cell-free extract 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Cell-free extract 50uL 1.5uL 100 mM AgNO3 98.5uL buffer
G	Cell-free extract 20uL 75uL 10mM AgNO3 55uL buffer	Cell-free extract 20uL 75uL 10mM AgNO3 55uL buffer	Cell-free extract 20uL 75uL 10mM AgNO3 55uL buffer	Cell-free extract 50uL 75uL 10mM AgNO3 25uL buffer	Cell-free extract 50uL 75uL 10mM AgNO3 25uL buffer	Cell-free extract 50uL 75uL 10mM AgNO3 25uL buffer
H	Cell-free extract 20uL 15uL 100 mM AgNO3 115uL buffer	Cell-free extract 20uL 15uL 100 mM AgNO3 115uL buffer	Cell-free extract 20uL 15uL 100 mM AgNO3 115uL buffer	Cell-free extract 50uL 15uL 100 mM AgNO3 85uL buffer	Cell-free extract 50uL 15uL 100 mM AgNO3 85uL buffer	Cell-free extract 50uL 15uL 100 mM AgNO3 85uL buffer

Plate B

	1	2	3	4	5	6
A	Cell free extract 5ul 145ul of buffer	Cell free extract 5ul 145ul of buffer	Cell free extract 5ul 145ul of buffer	Cell free extract 10ul 140ul of buffer	Cell free extract 10ul 140ul of buffer	Cell free extract 10ul 140ul of buffer
B	Cell-free extract 5uL 1.5uL 100 mM AgNO3 143.5uL buffer	Cell-free extract 5uL 1.5uL 100 mM AgNO3 143.5uL buffer	Cell-free extract 5uL 1.5uL 100 mM AgNO3 143.5uL buffer	Cell-free extract 10uL 1.5uL 100 mM AgNO3 138.5uL buffer	Cell-free extract 10uL 1.5uL 100 mM AgNO3 138.5uL buffer	Cell-free extract 10uL 1.5uL 100 mM AgNO3 138.5uL buffer
C	Cell-free extract 5uL 75uL 10mM AgNO3 70uL buffer	Cell-free extract 5uL 75uL 10mM AgNO3 70uL buffer	Cell-free extract 5uL 75uL 10mM AgNO3 70uL buffer	Cell-free extract 10uL 75uL 10mM AgNO3 65uL buffer	Cell-free extract 10uL 75uL 10mM AgNO3 65uL buffer	Cell-free extract 10uL 75uL 10mM AgNO3 65uL buffer
D	Cell-free extract 5uL 15uL 100 mM AgNO3 130uL buffer	Cell-free extract 5uL 15uL 100 mM AgNO3 130uL buffer	Cell-free extract 5uL 15uL 100 mM AgNO3 130uL buffer	Cell-free extract 10uL 15uL 100 mM AgNO3 125uL buffer	Cell-free extract 10uL 15uL 100 mM AgNO3 125uL buffer	Cell-free extract 10uL 15uL 100 mM AgNO3 125uL buffer
E	Supernatant 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Supernatant 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Supernatant 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Supernatant 50uL 75uL 10mM AgNO3 25uL buffer	Supernatant 50uL 75uL 10mM AgNO3 25uL buffer	Supernatant 50uL 75uL 10mM AgNO3 25uL buffer
F	Debris 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Debris 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Debris 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Debris 50uL 75uL 10mM AgNO3 25uL buffer	Debris 50uL 75uL 10mM AgNO3 25uL buffer	Debris 50uL 75uL 10mM AgNO3 25uL buffer
G	1.5uL 10mM AgNO3 148,5uL LB	1.5uL 10mM AgNO3 148,5uL LB	1.5uL 10mM AgNO3 148,5uL LB	75uL 10mM AgNO3 75uL LB	75uL 10mM AgNO3 75uL LB	75uL 10mM AgNO3 75uL LB
H	1.5uL 100 mM AgNO3 148,5uL Cell system	1.5uL 100 mM AgNO3 148,5uL Cell system	1.5uL 100 mM AgNO3 148,5uL Cell system	75uL 10mM AgNO3 75uL Cell system	75uL 10mM AgNO3 75uL Cell system	75uL 10mM AgNO3 75uL Cell system

Plate B continued

	7	8	9	10	11	12
A	Cell free extract 20ul 130ul of buffer	Cell free extract 20ul 130ul of buffer	Cell free extract 20ul 130ul of buffer	Cell free extract 50ul 100ul of buffer	Cell free extract 50ul 100ul of buffer	Cell free extract 50ul 100ul of buffer
B	Cell-free extract 20uL 1.5uL 100 mM AgNO3 128.5uL buffer	Cell-free extract 20uL 1.5uL 100 mM AgNO3 128.5uL buffer	Cell-free extract 20uL 1.5uL 100 mM AgNO3 128.5uL buffer	Cell-free extract 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Cell-free extract 50uL 1.5uL 100 mM AgNO3 98.5uL buffer	Cell-free extract 50uL 1.5uL 100 mM AgNO3 98.5uL buffer
C	Cell-free extract 20uL 75uL 10mM AgNO3 55uL buffer	Cell-free extract 20uL 75uL 10mM AgNO3 55uL buffer	Cell-free extract 20uL 75uL 10mM AgNO3 55uL buffer	Cell-free extract 50uL 75uL 10mM AgNO3 25uL buffer	Cell-free extract 50uL 75uL 10mM AgNO3 25uL buffer	Cell-free extract 50uL 75uL 10mM AgNO3 25uL buffer
D	Cell-free extract 20uL 15uL 100 mM AgNO3 115uL buffer	Cell-free extract 20uL 15uL 100 mM AgNO3 115uL buffer	Cell-free extract 20uL 15uL 100 mM AgNO3 115uL buffer	Cell-free extract 50uL 15uL 100 mM AgNO3 85uL buffer	Cell-free extract 50uL 15uL 100 mM AgNO3 85uL buffer	Cell-free extract 50uL 15uL 100 mM AgNO3 85uL buffer
E	Supernatant 50uL 15uL 100 mM AgNO3 85uL buffer	Supernatant 50uL 15uL 100 mM AgNO3 85uL buffer	Supernatant 50uL 15uL 100 mM AgNO3 85uL buffer	Supernatant 50uL 100uL buffer	Supernatant 50uL 100uL buffer	Supernatant 50uL 100uL buffer
F	Debris 50uL 15uL 100 mM AgNO3 85uL buffer	Debris 50uL 15uL 100 mM AgNO3 85uL buffer	Debris 50uL 15uL 100 mM AgNO3 85uL buffer	Debris 50uL 100uL buffer	Debris 50uL 100uL buffer	Debris 50uL 100uL buffer
G	15uL 100 mM AgNO3 125uL LB	15uL 100 mM AgNO3 125uL LB	15uL 100 mM AgNO3 125uL LB	150ul LB	150ul LB	150ul LB
H	15uL 100 mM AgNO3 135uL Cell system	15uL 100 mM AgNO3 135uL Cell system	15uL 100 mM AgNO3 135uL Cell system	150ul Cell system	150ul Cell system	150ul Cell system

Plate C

	1	2	3	4	5	6
A	15uL 1mM AgNO3 135uL LB	30uL 1mM AgNO3 120uL LB	45uL 1mM AgNO3 105uL LB	6uL 10mM AgNO3 * 144uL LB	7.5uL 10mM AgNO3 * 142.5uL LB	9uL 10mM AgNO3 * 141uL LB
B	15uL 1mM AgNO3 135uL LB	30uL 1mM AgNO3 120uL LB	45uL 1mM AgNO3 105uL LB	6uL 10mM AgNO3 * 144uL LB	7.5uL 10mM AgNO3 * 142.5uL LB	9uL 10mM AgNO3 * 141uL LB
C	15uL 1mM AgNO3 135uL Cell System	30uL 1mM AgNO3 120uL Cell System	45uL 1mM AgNO3 105uL Cell System	6uL 10mM AgNO3 * 144uL Cell System	7.5uL 10mM AgNO3 * 142.5uL Cell System	9uL 10mM AgNO3 * 141uL Cell System
D	15uL 1mM AgNO3 135uL Cell System	30uL 1mM AgNO3 120uL Cell System	45uL 1mM AgNO3 105uL Cell System	6uL 10mM AgNO3 * 144uL Cell System	7.5uL 10mM AgNO3 * 142.5uL Cell System	9uL 10mM AgNO3 * 141uL Cell System
E	6uL 10mM AgNO3 144uL LB	7.5uL 10mM AgNO3 142.5uL LB	9uL 10mM AgNO3 141uL LB	10.5uL 10mM AgNO3 139.5uL LB
F	6uL 10mM AgNO3 144uL LB	7.5uL 10mM AgNO3 142.5uL LB	9uL 10mM AgNO3 141uL LB	10.5uL 10mM AgNO3 139.5uL LB
G	6uL 10mM AgNO3 144uL Cell System	7.5uL 10mM AgNO3 142.5uL Cell System	9uL 10mM AgNO3 141uL Cell System	10.5uL 10mM AgNO3 139.5uL Cell System
H	6uL 10mM AgNO3 144uL Cell System	7.5uL 10mM AgNO3 142.5uL Cell System	9uL 10mM AgNO3 141uL Cell System	10.5uL 10mM AgNO3 139.5uL Cell System

Plate C continued

	7	8	9	10	12
A	10.5uL 10mM AgNO3 * 139.5uL LB	12uL 10mM AgNO3 138uL LB	13.5uL 10mM AgNO3 136.5uL LB	150uL LB	150uL LB
B	10.5uL 10mM AgNO3 * 139.5uL LB	12uL 10mM AgNO3 138uL LB	13.5uL 10mM AgNO3 136.5uL LB	150uL LB	150uL LB
C	10.5uL 10mM AgNO3 * 139.5uL Cell System	12uL 10mM AgNO3 138uL Cell System	13.5uL 10mM AgNO3 136.5uL Cell System	150uL Cell System	150uL Cell System
D	10.5uL 10mM AgNO3 * 139.5uL Cell System	12uL 10mM AgNO3 138uL Cell System	13.5uL 10mM AgNO3 136.5uL Cell System	150uL Cell System	150uL Cell System
E
F
G
H

Plate D

	1	2	3	4	5	6
A	50uL of Cell Extract 100uL of buffer	50uL of Cell Extract 100uL of buffer	50uL of Cell Extract 100uL of buffer	15uL of 10mM AgNO3 50uL of Cell Extract 85uL of buffer	15uL of 10mM AgNO3 50uL of Cell Extract 85uL of buffer	15uL of 10mM AgNO3 50uL of Cell Extract 85uL of buffer
B	50uL of Supernatant 100uL of buffer	50uL of Supernatant 100uL of buffer	50uL of Supernatant 100uL of buffer	15uL of 10mM AgNO3 50uL of Supernatant 85uL of buffer	15uL of 10mM AgNO3 50uL of Supernatant 85uL of buffer	15uL of 10mM AgNO3 50uL of Supernatant 85uL of buffer
C	15uL 1mM AgNO3 135uL LB	15uL 1mM AgNO3 135uL LB	15uL 1mM AgNO3 135uL LB	30uL 1mM AgNO3 120uL LB	30uL 1mM AgNO3 120uL LB	30uL 1mM AgNO3 120uL LB
D	7.5uL 10mM AgNO3 142.5uL LB	7.5uL 10mM AgNO3 142.5uL LB	7.5uL 10mM AgNO3 142.5uL LB	9uL 10mM AgNO3 141uL LB	9uL 10mM AgNO3 141uL LB	9uL 10mM AgNO3 141uL LB
E	13.5uL 10mM AgNO3 136.5uL LB	13.5uL 10mM AgNO3 136.5uL LB	13.5uL 10mM AgNO3 136.5uL LB	150uL LB	150uL LB	150uL LB
F	15uL 1mM AgNO3 135uL Cell System	15uL 1mM AgNO3 135uL Cell System	15uL 1mM AgNO3 135uL Cell System	30uL 1mM AgNO3 120uL Cell System	30uL 1mM AgNO3 120uL Cell System	30uL 1mM AgNO3 120uL Cell System
G	7.5uL 10mM AgNO3 142.5uL Cell System	7.5uL 10mM AgNO3 142.5uL Cell System	7.5uL 10mM AgNO3 142.5uL Cell System	9uL 10mM AgNO3 141uL Cell System	9uL 10mM AgNO3 141uL Cell System	9uL 10mM AgNO3 141uL Cell System
H	13.5uL 10mM AgNO3 136.5uL Cell System	13.5uL 10mM AgNO3 136.5uL Cell System	13.5uL 10mM AgNO3 136.5uL Cell System	150uL Cell System	150uL Cell System	150uL Cell System

Plate D continued

	7	8	9	10	11	12
A	75uL of 10mM AgNO3 50uL of Cell Extract 25uL of buffer	75uL of 10mM AgNO3 50uL of Cell Extract 25uL of buffer	75uL of 10mM AgNO3 50uL of Cell Extract 25uL of buffer	15uL of 100 mM AgNO3 50uL of Cell Extract 85uL of buffer	15uL of 100 mM AgNO3 50uL of Cell Extract 85uL of buffer	15uL of 100 mM AgNO3 50uL of Cell Extract 85uL of buffer
B	75uL of 10mM AgNO3 50uL of Supernatant 25uL of buffer	75uL of 10mM AgNO3 50uL of Supernatant 25uL of buffer	75uL of 10mM AgNO3 50uL of Supernatant 25uL of buffer	15uL of 100 mM AgNO3 50uL of Supernatant 85uL of buffer	15uL of 100 mM AgNO3 50uL of Supernatant 85uL of buffer	15uL of 100 mM AgNO3 50uL of Supernatant 85uL of buffer
C	45uL 1mM AgNO3 105uL LB	45uL 1mM AgNO3 105uL LB	45uL 1mM AgNO3 105uL LB	6uL 10mM AgNO3 144uL LB	6uL 10mM AgNO3 144uL LB	6uL 10mM AgNO3 144uL LB
D	10.5uL 10mM AgNO3 139.5uL LB	10.5uL 10mM AgNO3 139.5uL LB	10.5uL 10mM AgNO3 139.5uL LB	12uL 10mM AgNO3 138uL LB	12uL 10mM AgNO3 138uL LB	12uL 10mM AgNO3 138uL LB
E
F	45uL 1mM AgNO3 105uL Cell System	45uL 1mM AgNO3 105uL Cell System	45uL 1mM AgNO3 105uL Cell System	6uL 10mM AgNO3 144uL Cell System	6uL 10mM AgNO3 144uL Cell System	6uL 10mM AgNO3 144uL Cell System
G	10.5uL 10mM AgNO3 139.5uL Cell System	10.5uL 10mM AgNO3 139.5uL Cell System	10.5uL 10mM AgNO3 139.5uL Cell System	12uL 10mM AgNO3 138uL Cell System	12uL 10mM AgNO3 138uL Cell System	12uL 10mM AgNO3 138uL Cell System
H

Materials

• 20 mL Escherichia coli (E. coli) BL21 overnight culture in Lysogeny Nitrate Broth.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 10 mM aqueous AgNO₃ solution.
• Lysogeny Nitrate Broth.
• 150 mM Triton X-100.
• 100 mM Tween 20.
• 100 mM SDS.
• 100 mM Nonidet.
• 50 mL Falcon tubes.
• Sterile flat-bottom 24-well plate with lid.
• 0.2 µm Syringe filter.
• 15 mL Syringe.
• Parafilm.
• 1 mL plastic cuvettes.

Equipment

• Shaking Incubator.
• Incubator.
• Tabletop centrifuge.
• Tecan spark microplate reader.
• UV-vis NIR spectrophotometer.

Protocol

1. Dilute the overnight culture down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
3. Once the cultures have reached this phase:
   1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
   2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as Supernatant.
4. Add supernatant, or Lysogeny Nitrate Broth into the intended wells of the 24-well plates according to the layouts below (e.g. 1000 µL).
5. Add 10 mM aqueous AgNO₃ solution (e.g. 5 µL) into the intended wells of the 24-well plates according to the layouts below.
6. Add 150 mM Triton X-100, 100 mM Tween 20, 100 mM SDS, or 100 mM Nonidet (e.g. 5 µL) into the intended wells of the 24-well plates according to the layouts below.
7. Seal the 24-well plates with parafilm and scan absorbance from 350 nm to 1000 nm with a microplate reader.
8. Place 24-well plates in a (non-shaking) incubator set to 37°C for 72 hours and scan absorbance from 350 nm to 1000 nm with a microplate reader.
9. After 72 hours, add 5 µL of 100 mM aqueous HAuCl4 solution (final concentration of 0.5 mM) into the intended wells of the 24-well plates with AgNO₃ according to the layouts below.
10. Scan absorbance from 350 nm to 1000 nm with a microplate reader.
11. Place 24-well plates back into a (non-shaking) incubator set to 37°C for 24 hours.
12. Scan absorbance from 350 nm to 1000 nm with a microplate reader.

24-Well Plates

Plate 1

	1	2	3	4	5	6
A	Trition 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 3.33 ul Trition (150mM)	Trition 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 3.33 ul Trition (150mM)	Trition 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 3.33 ul Trition (150mM)	Trition 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 10 ul Trition (150mM)	Trition 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 10 ul Trition (150mM)	Trition 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 10 ul Trition (150mM)
B	Trition 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 16.66 ul Trition (150mM)	Trition 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 16.66 ul Trition (150mM)	Trition 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 16.66 ul Trition (150mM)	Trition 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 23.33 ul Trition (150mM)	Trition 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 23.33 ul Trition (150mM)	Trition 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 23.33 ul Trition (150mM)
C	Tween 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 5 ul Tween (100mM)	Tween 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 5 ul Tween (100mM)	Tween 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 5 ul Tween (100mM)	Tween 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 15 ul Tween (100mM)	Tween 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 15 ul Tween (100mM)	Tween 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 15 ul Tween (100mM)
D	Tween 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 25 ul Tween (100mM)	Tween 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 25 ul Tween (100mM)	Tween 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 25 ul Tween (100mM)	Tween 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 35 ul Tween (100mM)	Tween 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 35 ul Tween (100mM)	Tween 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 35 ul Tween (100mM)

Plate 2

	1	2	3	4	5	6
A	SDS 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 5 ul SDS (100mM)	SDS 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 5 ul SDS (100mM)	SDS 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 5 ul SDS (100mM)	SDS 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 15 ul SDS (100mM)	SDS 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 15 ul SDS (100mM)	SDS 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 15 ul SDS (100mM)
B	SDS 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 25 ul SDS (100mM)	SDS 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 25 ul SDS (100mM)	SDS 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 25 ul SDS (100mM)	SDS 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 35 ul SDS (100mM)	SDS 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 35 ul SDS (100mM)	SDS 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 35 ul SDS (100mM)
C	Nonidet 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 5 ul Nonidet (100mM)	Nonidet 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 5 ul Nonidet (100mM)	Nonidet 1:1 1 mL Supernatant 50 ul 10 mM AgNO3 5 ul Nonidet (100mM)	Nonidet 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 15 ul Nonidet (100mM)	Nonidet 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 15 ul Nonidet (100mM)	Nonidet 1:3 1 mL Supernatant 50 ul 10 mM AgNO3 15 ul Nonidet (100mM)
D	Nonidet 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 25 ul Nonidet (100mM)	Nonidet 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 25 ul Nonidet (100mM)	Nonidet 1:5 1 mL Supernatant 50 ul 10 mM AgNO3 25 ul Nonidet (100mM)	Nonidet 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 35 ul Nonidet (100mM)	Nonidet 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 35 ul Nonidet (100mM)	Nonidet 1:7 1 mL Supernatant 50 ul 10 mM AgNO3 35 ul Nonidet (100mM)

Plate 3

	1	2	3	4	5	6
A	Trition 1:1 1 mL Medium 50 ul 10 mM AgNO3 3.33 ul Trition (150mM)	Trition 1:1 1 mL Medium 50 ul 10 mM AgNO3 3.33 ul Trition (150mM)	Trition 1:3 1 mL Medium 50 ul 10 mM AgNO3 10 ul Trition (150mM)	Trition 1:3 1 mL Medium 50 ul 10 mM AgNO3 10 ul Trition (150mM)	Trition 1:5 1 mL Medium 50 ul 10 mM AgNO3 16.66 ul Trition (150mM)	Trition 1:5 1 mL Medium 50 ul 10 mM AgNO3 16.66 ul Trition (150mM)
B	Trition 1:7 1 mL Medium 50 ul 10 mM AgNO3 23.33 ul Trition (150mM)	Trition 1:7 1 mL Medium 50 ul 10 mM AgNO3 23.33 ul Trition (150mM)	Tween 1:1 1 mL Medium 50 ul 10 mM AgNO3 5 ul Tween (100mM)	Tween 1:1 1 mL Medium 50 ul 10 mM AgNO3 5 ul Tween (100mM)	Tween 1:3 1 mL Medium 50 ul 10 mM AgNO3 15 ul Tween (100mM)	Tween 1:3 1 mL Medium 50 ul 10 mM AgNO3 15 ul Tween (100mM)
C	Tween 1:5 1 mL Medium 50 ul 10 mM AgNO3 25 ul Tween (100mM)	Tween 1:5 1 mL Medium 50 ul 10 mM AgNO3 25 ul Tween (100mM)	Tween 1:7 1 mL Medium 50 ul 10 mM AgNO3 35 ul Tween (100mM)	Tween 1:7 1 mL Medium 50 ul 10 mM AgNO3 35 ul Tween (100mM)	SDS 1:1 1 mL Medium 50 ul 10 mM AgNO3 5 ul SDS (100mM)	SDS 1:1 1 mL Medium 50 ul 10 mM AgNO3 5 ul SDS (100mM)
D	SDS 1:3 1 mL Medium 50 ul 10 mM AgNO3 15 ul SDS (100mM)	SDS 1:3 1 mL Medium 50 ul 10 mM AgNO3 15 ul SDS (100mM)	SDS 1:5 1 mL Medium 50 ul 10 mM AgNO3 25 ul SDS (100mM)	SDS 1:5 1 mL Medium 50 ul 10 mM AgNO3 25 ul SDS (100mM)	SDS 1:7 1 mL Medium 50 ul 10 mM AgNO3 35 ul SDS (100mM)	SDS 1:7 1 mL Medium 50 ul 10 mM AgNO3 35 ul SDS (100mM)

Plate 4

	1	2	3	4	5	6
A	Nonidet 1:1 1 mL Medium 50 ul 10mM AgNO3 5 ul Nonidet (100mM)	Nonidet 1:1 1 mL Medium 50 ul 10mM AgNO3 5 ul Nonidet (100mM)	Nonidet 1:3 1 mL Medium 50 ul 10mM AgNO3 15 ul Nonidet (100mM)	Nonidet 1:3 1 mL Medium 50 ul 10mM AgNO3 15 ul Nonidet (100mM)	Nonidet 1:5 1 mL Medium 50 ul 10mM AgNO3 25 ul Nonidet (100mM)	Nonidet 1:5 1 mL Medium 50 ul 10mM AgNO3 25 ul Nonidet (100mM)
B	Nonidet 1:7 1 mL Medium 50 ul 10mM AgNO3 35 ul Nonidet (100mM)	Nonidet 1:7 1 mL Medium 50 ul 10mM AgNO3 35 ul Nonidet (100mM)	1 mL Medium	1 mL Medium	1 mL Supernatant	1 mL Supernatant
C	Medium 50 ul 10mM AgNO3	Medium 50 ul 10mM AgNO3	Supernatant 50 ul 10mM AgNO3	Supernatant 50 ul 10mM AgNO3	Trition 1 mL Medium 10 ul Trition (150mM)	Trition 1 mL Medium 10 ul Trition (150mM)
D	Tween 1 mL Medium 15 ul Tween (100mM)	Tween 1 mL Medium 15 ul Tween (100mM)	SDS 1 mL Medium 15 ul SDS (100mM)	SDS 1 mL Medium 15 ul SDS (100mM)	Nonidet 1 mL Medium 15 ul Nonidet (100mM)	Nonidet 1 mL Medium 15 ul Nonidet (100mM)

Materials

• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Lysogeny Broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Mueller Hinton Broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Tryptic Soy Broth (+150 µg/mL Ampicillin).
• Lysogeny Broth (+150 µg/mL Ampicillin).
• Mueller Hinton Broth (+150 µg/mL Ampicillin).
• Tryptic Soy Broth (+150 µg/mL Ampicillin).
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 100 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 100 mM aqueous AgNO₃ solution.
• 50 mL Falcon tubes.
• 15 mL Falcon tubes.
• 1 mL plastic cuvettes.
• 0.2 µm Syringe filter.
• 15 mL Syringe.

Equipment

• Tabletop centrifuge.
• Shaking incubator.
• UV-vis NIR spectrophotometer.

Protocol

1. Dilute the overnight cultures down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
3. Once the cultures have reached this phase:
   1. For Escherichia coli (E. coli) BL21-pET16b in Lysogeny Broth:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as BL21-supernatant Lysogeny Broth.
   2. For E. coli BL21-pET16b in Mueller Hinton Broth:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as BL21-supernatant Mueller Hinton Broth.
   3. For E. coli BL21-pET16b in Tryptic Soy Broth:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as BL21-supernatant Tryptic Soy Broth.
4. Add the 5 mL of intended supernatant (e.g. BL21-supernatant Mueller Hinton), or intended media (e.g. Tryptic Soy Broth) into the assigned 15 mL falcon tubes according to the layout below.
5. Add 100 mM aqueous HAuCl₄ solution (e.g. 10 µL) into assigned 15 mL falcon tubes according to the layout below.
6. Add 100 mM aqueous AgNO₃ solution (e.g. 5 µL) into assigned 15 mL falcon tubes according to the layout below.
7. Place in a shaking incubator set to 37°C at 200 rpm for 24 hours.
8. Scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.

15 mL Falcon Tubes

MH1: 5mL medium	LB1: 5mL medium	TSBS1: 5mL medium
MH2: 5mL supernatant from BL21 WT	LB2: 5mL supernatant from BL21 WT	TSBS2: 5mL supernatant from BL21 WT
MH3: 5mL medium + 3mM HAuCl4 + 0.3mM AgNO3	LB3: 5mL medium + 3mM HAuCl4 + 0.3mM AgNO3	TSBS3: 5mL medium + 3mM HAuCl4 + 0.3mM AgNO3
MH4: 5mL medium + 3mM HAuCl4	LB4: 5mL medium + 3mM HAuCl4	TSBS4: 5mL medium + 3mM HAuCl4
MH5: 5mL medium + 0.3mM AgNO3	LB5: 5mL medium + 0.3mM AgNO3	TSBS5: 5mL medium + 0.3mM AgNO3
MH6: 5mL supernatant + 3mM HAuCl4 + 0.3mM AgNO3	LB6: 5mL supernatant + 3mM HAuCl4 + 0.3mM AgNO3	TSBS6: 5mL supernatant + 3mM HAuCl4 + 0.3mM AgNO3
MH7: 5mL supernatant + 0.3mM AgNO3	LB7: 5mL supernatant + 0.3mM AgNO3	TSBS7: 5mL supernatant + 0.3mM AgNO3
MH8: 5mL supernatant + 3mM HAuCl4	LB8: 5mL supernatant + 3mM HAuCl4	TSBS8: 5mL supernatant + 3mM HAuCl4

Materials

• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Lysogeny Broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Lysogeny Nitrate Broth (+150 µg/mL Ampicillin).
• Lysogeny Broth (+150 µg/mL Ampicillin).
• Lysogeny Nitrate Broth (+150 µg/mL Ampicillin).
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 10 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 10 mM aqueous AgNO₃ solution.
• 50 mL Falcon tubes.
• 100 mL Flasks.
• 0.2 µm Syringe filter.
• 15 mL Syringe.
• 1 mL plastic cuvettes.

Equipment

• Tabletop centrifuge.
• Shaking incubator.
• UV-vis NIR Spectrophotometer.

Protocol

1. Dilute the overnight cultures down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
3. Once the cultures have reached this phase:
   1. For Escherichia coli (E. coli) BL21-pET16b in Lysogeny Broth:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as BL21-supernatant Lysogeny Broth.
   2. For E. coli BL21-pET16b in Lysogeny Nitrate Broth:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as BL21-supernatant Lysogeny Nitrate Broth.
4. Add Lysogeny Broth, Lysogeny Nitrate Broth, BL21-supernatant Lysogeny Broth, or BL21-supernatant Lysogeny Nitrate Broth, into intended flasks according to the layout below (e.g. 100µL).
5. Add 10 mM aqueous HAuCl₄ solution (e.g. 5 µL) into assigned flasks according to the layout below.
6. Add 10 mM aqueous AgNO₃ solution (e.g. 1 µL) into assigned flasks according to the layout below.
7. Place flasks in a shaking incubator set to 37°C at 200 rpm for 24 hours.
8. After 24 hours, add 10 mM aqueous HAuCl₄ solution (e.g. 5 µL) into assigned flasks according to the layout below.
9. Scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.

Flasks

LB Flasks

Flask	LB	BL21 supernatant LB	10mM AgNO3	10mM HAuCl4
L1	20 mL
L2	18 mL		2 mL	After 1d: 0.2 mL
L3	17.8 mL		0.2 mL	2 mL
L4		20 mL
L5		18 mL	2 mL	After 1d: 0.2 mL
L6		17.8 mL	0.2 mL	2 mL

LB-Nitrate Flasks

Flask	LB-NO3	BL21 supernatant LB-NO3	10mM AgNO3	10mM HAuCl4
N1	20 mL
N2	18 mL		2 mL	After 1d: 0.2 mL
N3	17.8 mL		0.2 mL	2 mL
N4		20 mL
N5		18 mL	2 mL	After 1d: 0.2 mL
N6		17.8 mL	0.2 mL	2 mL

Materials

• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 10 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 10 mM aqueous AgNO₃ solution.
• Ascorbic acid (AA, 99%, Acros).
  • 100 mM aqueous ascorbic acid.
• ddH₂O.
• 1.5 mL Eppendorf Tubes.
• 1 mL plastic cuvettes.

Equipment

• Vortex mixer.
• UV-vis NIR spectrophotometer.

Protocol

First Experiment

1. Add 966 µL of ddH₂O into a 1.5 mL Eppendorf tube.
2. Add 20 µL of 10 mM aqueous HAuCl₄ solution to the sample.
3. Add 10 µL of 10 mM aqueous AgNO₃ solution to the sample.
4. Briefly vortex the Eppendorf tube with the sample.
5. Add 4 µL of 100 mM aqueous ascorbic acid into the sample.
6. Vortex the Eppendorf tube with the sample for 20 seconds.
7. Transfer the sample into a 1 mL cuvette and scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.

Second Experiment (Less AgNO₃ initially, add HAuCl₄ 10 minutes after Ascorbic Acid)

1. Add 984 µL of ddH₂O into a 1.5 mL Eppendorf tube.
2. Add 2 µL of 10 mM aqueous AgNO₃ solution to the sample.
3. Briefly vortex the Eppendorf tube with the sample.
4. Add 4 µL of 100 mM aqueous ascorbic acid into the sample.
5. Vortex the Eppendorf tube with the sample for 20 seconds.
6. After 10 minutes add 10 µL of 10 mM aqueous HAuCl₄ solution into the sample.
7. Transfer the sample into a 1 mL cuvette and scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.

Third Experiment (Add HAuCl₄ after Ascorbic Acid)

1. Add 966 µL of ddH₂O into a 1.5 mL Eppendorf tube.
2. Add 20 µL of 10 mM aqueous AgNO₃ solution to the sample.
3. Briefly vortex the Eppendorf tube with the sample.
4. Add 4 µL of 100 mM aqueous ascorbic acid into the sample.
5. Vortex the Eppendorf tube with the sample for 20 seconds.
6. After 10 minutes add 2 µL of 10 mM aqueous HAuCl₄ solution into the sample.
7. Transfer the sample into a 1 mL cuvette and scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.

Materials

• 20 mL Escherichia coli (E. coli) BL21 overnight culture in Lysogeny Broth.
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 100 mM aqueous HAuCl₄ solution.
  • 10 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 100 mM aqueous AgNO₃ solution.
  • 10 mM aqueous AgNO₃ solution.
• 0.01 M Luminol in NaOH solution (1x dilution).
  • + 10x dilution
  • + 100x dilution
• Lysogeny Broth.
• 50 mL Flasks.
• 1 mL plastic cuvettes.

Equipment

• Shaking incubator.
• UV-vis NIR spectrophotometer.

Protocol

1. Dilute the overnight culture down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
3. Transfer 9 mL of culture, or Lysogeny Broth into flasks according to the layout below.
4. Add 1 mL of 100 mM aqueous AgNO₃ solution, or 1 mL of 10 mM aqueous AgNO₃ solution into flasks according to the layout below.

Flasks

1	9 mL culture
	1 mL AgNO3 (10 mM)
2	9 mL culture
	1 mL AgNO3 (10 mM)
3	9 mL culture
	1 mL AgNO3 (10 mM)
4	9 mL LB
	1 mL AgNO3 (10 mM)
5	9 mL culture
	1 mL AgNO3 (100 mM)
6	9 mL culture
	1 mL AgNO3 (100 mM)
7	9 mL culture
	1 mL AgNO3 (100 mM)
8	9 mL LB
	1 mL AgNO3 (100 mM)

1. Place flasks in a shaking incubator set to 37°C at 200 rpm for 72 hours.
2. Remove 1.5 mL of culture + salt mixture from each flask.
3. Add 1 mL of 100 mM aqueous HAuCl₄ solution, or 1 mL of 10 mM aqueous HAuCl₄ solution into flasks according to the layout below.
4. Add 0.5 mL of (1x dilution) Luminol solution, 0.5 mL of (10x dilution) Luminol solution, or 0.5 mL of (100x dilution) Luminol solution into flasks according to the layout below.
5. Place flasks back in a shaking incubator set to 37°C at 200 rpm for 72 hours.
6. Scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.

Flasks

	To add	Final contents of flask
1	1 mL HAuCl4 (10 mM)	1 mM AgNO3, 1 mM HAuCl4 , 0.5 mM Luminol
	0.5 mL luminol (undiluted)
2	1 mL HAuCl4 (10 mM)	1 mM AgNO3 , 1 mM HAuCl4 , 0.05 mM Luminol
	0.5 mL luminol (10x diluted)
3	1 mL HAuCl4 (10 mM)	1 mM AgNO3, 1 mM HAuCl4 , 0.005 mM Luminol
	0.5 mL luminol (100x diluted)
4	1 mL HAuCl4 (10 mM)	1 mM AgNO3, 1 mM HAuCl4 , 0.05 mM NaOH
	0,5 mL 0,1M NaOH
5	1 mL HAuCl4 (100 mM)	10 mM AgNO3, 10 mM HAuCl4 , 0.5 mM Luminol
	0.5 mL luminol (undiluted)
6	1 mL HAuCl4 (100 mM)	10 mM AgNO3, 10 mM HAuCl4 , 0.05 mM Luminol
	0.5 mL luminol (10x diluted)
7	1 mL HAuCl4 (100 mM)	10 mM AgNO3, 10 mM HAuCl4 , 0.005 mM Luminol
	0.5 mL luminol (100x diluted)
8	1 mL HAuCl4 (100 mM) after 3 days	10 mM AgNO3, 10 mM HAuCl4 , 0.05 mM NaOH
	0,5 mL 0,1M NaOH

Materials

• ddH₂O.
• Baking soda.
• Na₂CO₃ · H₂O.
• Ammoniumcarbonate (NH₄)CO₃.
• CuSO₄· 5H₂O.
• Luminol.
• 3% H₂O₂.
• 100 mL Beaker.
• Dark Bottle.
• 15 mL Falcon tubes.

Protocol

Buffer

1. Make buffer:
   1. Add 10 mL dH₂O to a 100 mL beaker.
   2. Slowly add:
   3. 105.12 mg baking soda.
   4. 20.5 mg Na₂CO₃ · H₂O.
   5. 2.2 mg Ammoniumcarbonate (NH₄)CO₃.
   6. 1.75 mg CuSO₄· 5H₂O.
   7. Add dH₂O to a total volume of 30 mL in the beaker.
   8. Dilute 10x.
2. Make a 0.15% H₂O₂ solution:
   1. Mix 3 mL 3% H₂O₂ with 57 mL ddH₂O and store in a bottle.
3. Make Luminol Solutions
   1. Mix 0.088 mg luminol in 1 mL ddH₂O in a 15 mL falcon tube. Add 9 mL of buffer to this solution. This will be sample 1.
   2. Transfer 1 mL of Luminol Experiment flask 1 into a 15 mL falcon tube. Add 9 mL of buffer to this solution. This will be sample 2.
   3. Transfer 1 mL of Luminol Experiment flask 2 into a 15 mL falcon tube. Add 9 mL of buffer to this solution. This will be sample 3.
   4. Transfer 1 mL of Luminol Experiment flask 3 into a 15 mL falcon tube. Add 9 mL of buffer to this solution. This will be sample 4.
   5. Transfer 1 mL of Luminol Experiment flask 4 into a 15 mL falcon tube. Add 9 mL of buffer to this solution. This will be sample 5.
   6. Transfer 1 mL of Luminol Experiment flask 5 into a 15 mL falcon tube. Add 9 mL of buffer to this solution. This will be sample 6.
   7. Transfer 1 mL of Luminol Experiment flask 6 into a 15 mL falcon tube. Add 9 mL of buffer to this solution. This will be sample 7.
   8. Transfer 1 mL of Luminol Experiment flask 7 into a 15 mL falcon tube. Add 9 mL of buffer to this solution. This will be sample 8.
   9. Transfer 1 mL of Luminol Experiment flask 8 into a 15 mL falcon tube. Add 9 mL of buffer to this solution. This will be sample 9.
4. For each sample, transfer 10 mL of solution into a dark bottle, then add 5 mL of 0.15% H₂O₂ solution to the same bottle.
5. Check for luminescence.

Materials

• 3x 20 mL Escherichia coli (E. coli) BL21 overnight culture in Lysogeny Broth.
• 3x 20 mL Escherichia coli (E. coli) BL21 overnight culture in Lysogeny Broth Low Salt.
• 3x 20 mL Escherichia coli (E. coli) BL21 overnight culture in Tryptic Soy Broth.
• 3x 20 mL Escherichia coli (E. coli) BL21 overnight culture in Mueller Hinton Broth.
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 100 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 100 mM aqueous AgNO₃ solution.
• Lysogeny Broth.
• Lysogeny Broth Low Salt.
• Tryptic Soy Broth.
• Mueller Hinton Broth.
• 2 mL Eppendorf tubes
• 50 mL Falcon Tubes
• 0.2 µm Syringe filter.
• 15 mL Syringe.
• Sterile flat-bottom 24-well plate with lid.
• Parafilm.
• 1 mL plastic cuvettes.

Equipment

• Shake incubators.
• Tabletop centrifuge.
• Refrigerated micro centrifuge.
• Rod sonicator (MS-72 probe).
• UV-vis NIR spectrophotometer.

Protocol

1. Dilute the overnight cultures down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
3. Once the cultures have reached this phase: (three biological replicates)
   1. For Escherichia coli (E. coli) BL21 in Lysogeny Broth:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube (save the pellet for the next step). This will now be referred to as BL21-supernatant Lysogeny Broth.
      3. Resuspend the pellet in 1 mL of Lysogeny Broth. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)). Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes. This will be referred to as BL21-lysate Lysogeny Broth.
   2. For E. coli BL21 in Lysogeny Broth Low Salt:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube (save the pellet for the next step). This will now be referred to as BL21-supernatant Lysogeny Broth Low Salt.
      3. Resuspend the pellet in 1 mL of Lysogeny Broth Low Salt. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)). Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes. This will be referred to as BL21-lysate Lysogeny Broth Low Salt.
   3. For E. coli BL21 in Mueller Hinton Broth:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube (save the pellet for the next step). This will now be referred to as BL21-supernatant Mueller Hinton Broth.
      3. Resuspend the pellet in 1 mL of Mueller Hinton Broth. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)). Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes. This will be referred to as BL21-lysate Mueller Hinton Broth.
   4. For E. coli BL21 in Tryptic Soy Broth:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube (save the pellet for the next step). This will now be referred to as BL21-supernatant Tryptic Soy Broth.
      3. Resuspend the pellet in 1 mL of Tryptic Soy Broth. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)). Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes. This will be referred to as BL21-lysate Tryptic Soy Broth.
4. Add supernatant, media, or lysate (e.g. 987.5 µL) into the intended wells of the 24-well plates according to the layout below.
5. Add 100 mM aqueous HAuCl₄ solution (e.g. 10 µL) into the intended wells of the 24-well plates according to the layout below.
6. Add 100 mM aqueous AgNO₃ solution (e.g. 5 µL) (Making sure to use the ratio of 1.85:1 for Gold:Silver) into the intended wells of the 24-well plates according to the layout below.
7. Seal the 24-well plates with parafilm and place it into a shaking incubator set to 37°C at 200 rpm for 24 hours.
8. Scan absorbance from 350 nm to 1000 nm with a microplate reader.

24-Well Plates

Plate 1

	1	2	3	4	5	6
A	1 mL LB medium	971.5 µL LB medium + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	1 mL LB medium BL21 supernatant	1 mL LB medium BL21 lysate	971.5 µL LB medium BL21 supernatant 1 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL LB medium BL21 supernatant 2 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4
B	971.5 µL LB medium BL21 supernatant 3 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL LB medium BL21 lysate 1 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL LB medium BL21 lysate 2 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL LB medium BL21 lysate 3+ 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4
C	1 mL TSBS medium	971.5 µL TSBS medium + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	1 mL TSBS Bl21 supernatant	1 mL TSBS medium BL21 lysate	971.5 µL TSBS BL21 supernatant 1+ 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL TSBS BL21 supernatant 2 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4
D	971.5 µL TSBS BL21 supernatant 3+ 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL TSBS medium BL21 lysate 1+ 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL TSBS medium BL21 lysate 2+ 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL TSBS medium BL21 lysate 3 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4

Plate 2

	1	2	3	4	5	6
A	1 mL MH medium	971.5 µL MH medium + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	1 mL MH medium BL21 supernatant	1 mL MH medium BL21 lysate	971.5 µL MH medium BL21 supernatant 1 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL MH medium BL21 supernatant 2 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4
B	971.5 µ MH medium BL21 supernatant 3 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL MH medium BL21 lysate 1 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL MH medium BL21 lysate 2 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL MH medium BL21 lysate 3 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4
C	1 mL LBls medium	971.5 µL LBls medium + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	1 mL LBls Bl21 supernatant	1 mL LBls medium BL21 lysate	971.5 µL LBls BL21 supernatant 1 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL LBls BL21 supernatant 2+ 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4
D	971.5 µL LBls BL21 supernatant 3 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL LBls medium BL21 lysate 1 + 10 µL 100 mM AgNO3 +18.5 µL 100 mM HAuCl4	971.5 µL LBls medium BL21 lysate 2 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4	971.5 µL LBls medium BL21 lysate 3 + 10 µL 100 mM AgNO3 + 18.5 µL 100 mM HAuCl4

Materials

• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 10 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 10 mM aqueous AgNO₃ solution.
• Ascorbic acid (AA, 99%, Acros).
  • 100 mM aqueous ascorbic acid.
• Sodium acetate.
• Acetic acid.
• Sodium phosphate dibasic heptahydrate.
• Sodium phosphate monobasic monohydrate.
• Trizma hydrochloride.
• Trizma base.
• 10 M HCl.
• 10 M NaOH.
• ddH₂O.
  • Adjusted to pH 5 with Acetate buffer pH 5
  • Adjusted to pH 7 with Phosphate buffer pH 7
  • Adjusted to pH 9 with Tris buffer pH 9
• 1.5 mL Eppendorf tube.
• 1 mL plastic cuvettes.

Equipment

• Vortex mixer.
• UV-vis NIR spectrophotometer.

Protocol

1. For 100 mM acetate buffer pH 5:
   1. In a suitable container, add 800 mL ddH₂O.
   2. Add 5.772 grams of sodium acetate to the solution.
   3. Add 1.778 grams of acetic acid to the solution.
   4. Adjust pH with 10M HCl to pH 5.
   5. Add ddH₂O to a final volume of 1 L.
2. For 100 mM phosphate buffer pH 7:
   1. In a suitable container, add 800 mL ddH₂O.
   2. Add 15.487 grams of sodium phosphate dibasic heptahydrate to the solution.
   3. Add 5.827 grams of sodium phosphate monobasic monohydrate to the solution.
   4. Adjust pH with 10 M HCl or 10 M NaOH to pH 7.
   5. Add ddH₂O to a final volume of 1 L.
3. For 100 mM tris buffer pH 9:
   1. In a suitable container, add 800 mL ddH₂O.
   2. Add 1.011 grams of trizma hydrochloride to the solution.
   3. Add 11.337 grams of trizma base to the solution.
   4. Adjust pH with 10 M NaOH to pH 9.
4. Add the appropriate volume (total volume to be 1 mL) of ddH₂O at the intended pH into a 1.5 mL Eppendorf tube (e.g. 966 µL) according to the layout below.
5. Add 10 mM aqueous HAuCl₄ solution (e.g. 20 µL) according to the layout below.
6. Add 10 mM aqueous AgNO₃ solution (e.g. 10 µL) according to the layout below.
7. Briefly vortex the Eppendorf tube with the sample.
8. Add 4 µL of 100 mM aqueous ascorbic acid into the sample.
9. Vortex the Eppendorf tube with the sample for 20 seconds.
10. Transfer the sample into a 1 mL cuvette and quickly scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.

    Eppendorf Tubes

    	pH	10 mM HAuCl4 (µL)	10 mM AgNO3 (µL)
    1	5	15	10
    2	9	15	10
    3	5	25	10
    4	9	25	10
    5	5	20	5
    6	9	20	5
    7	5	20	15
    8	9	20	15
    9	7	15	5
    10	7	25	5
    11	7	15	15
    12	7	25	15
    13	7	20	10
    14	7	20	10
    15	7	20	10

Materials

• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Meuller Hinton Broth (+150 µg/mL Ampicillin).
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 10 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 10 mM aqueous AgNO₃ solution.
• Mueller Hinton Broth (+150 µg/mL Ampicillin).
• Mueller Hinton Broth.
• Sterile flat-bottom 24-well plate with lid.
• 50 mL Falcon tubes.
• 0.2 µm Syringe filter.
• 15 mL Syringe.
• Parafilm.
• 1 mL plastic cuvettes.

Equipment

• Tabletop centrifuge.
• Shake incubator.
• Tecan spark microplate reader.
• UV-vis NIR spectrophotometer.

Protocol

1. Dilute the overnight culture down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
3. Once the culture has reached this phase transfer the contents to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
4. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as supernatant.
5. Add supernatant or Mueller Hinton Broth into the intended wells (e.g. 485 µL) of the 24-well plates according to the layout below.
6. Add 100 mM aqueous HAuCl₄ solution (e.g. 10 µL) into the intended wells of the 24-well plates according to the layout below.
7. Add 100 mM aqueous AgNO₃ solution (e.g. 5 µL) (Making sure to use the ratio of 1.85:1 for Gold:Silver) into the intended wells of the 24-well plates according to the layout below.
8. Seal the 24-well plates with parafilm. Place 24-well plates in a shaking incubator set to 37°C at 200 rpm for 24 hours.
9. Scan absorbance from 350 nm to 1000 nm with a microplate reader.

24-Well Plates

Plate 1

	1	2	3	4	5	6
A	500μL MH	500μL MH	500μL MH	500μL Supernatant of WT BL21 in MH	500μL Supernatant of WT BL21 in MH	500μL Supernatant of WT BL21 in MH
B	492μL MH 5μL 100 mM HAuCl4 2.7μL 100 mM AgNO3	492μL MH 5μL 100 mM HAuCl4 2.7μL 100 mM AgNO3	492μL MH 5μL 100 mM HAuCl4 2.7μL 100 mM AgNO3	492μL Supernatant of WT BL21 in MH 5μL 100 mM HAuCl4 2.7μL 100 mM AgNO3	492μL Supernatant of WT BL21 in MH 5μL 100 mM HAuCl4 2.7μL 100 mM AgNO3	492μL Supernatant of WT BL21 in MH 5μL 100 mM HAuCl4 2.7μL 100 mM AgNO3
C	488μL MH 7.5μL 100 mM HAuCl4 4.05μL 100 mM AgNO3	488μL MH 7.5μL 100 mM HAuCl4 4.05μL 100 mM AgNO3	488μL MH 7.5μL 100 mM HAuCl4 4.05μL 100 mM AgNO3	488μL Supernatant of WT BL21 in MH 7.5μL 100 mM HAuCl4 4.05μL 100 mM AgNO3	488μL Supernatant of WT BL21 in MH 7.5μL 100 mM HAuCl4 4.05μL 100 mM AgNO3	488μL Supernatant of WT BL21 in MH 7.5μL 100 mM HAuCl4 4.05μL 100 mM AgNO3
D	485μL MH 10μL 100 mM HAuCl4 5.41μL 100 mM AgNO3	485μL MH 10μL 100 mM HAuCl4 5.41μL 100 mM AgNO3	485μL MH 10μL 100 mM HAuCl4 5.41μL 100 mM AgNO3	485μL Supernatant of WT BL21 in MH 10μL 100 mM HAuCl4 5.41μL 100 mM AgNO3	485μL Supernatant of WT BL21 in MH 10μL 100 mM HAuCl4 5.41μL 100 mM AgNO3	485μL Supernatant of WT BL21 in MH 10μL 100 mM HAuCl4 5.41μL 100 mM AgNO3

Plate 2

	1	2	3	4	5	6
A	481μL MH 12.5μL 100 mM HAuCl4 6.76μL 100 mM AgNO3	481μL MH 12.5μL 100 mM HAuCl4 6.76μL 100 mM AgNO3	481μL MH 12.5μL 100 mM HAuCl4 6.76μL 100 mM AgNO3	481μL Supernatant of WT BL21 in MH 12.5μL 100 mM HAuCl4 6.76μL 100 mM AgNO3	481μL Supernatant of WT BL21 in MH 12.5μL 100 mM HAuCl4 6.76μL 100 mM AgNO3	481μL Supernatant of WT BL21 in MH 12.5μL 100 mM HAuCl4 6.76μL 100 mM AgNO3
B	477μL MH 15μL 100 mM HAuCl4 8.11μL 100 mM AgNO3	477μL MH 15μL 100 mM HAuCl4 8.11μL 100 mM AgNO3	477μL MH 15μL 100 mM HAuCl4 8.11μL 100 mM AgNO3	477μL Supernatant of WT BL21 in MH 15μL 100 mM HAuCl4 8.11μL 100 mM AgNO3	477μL Supernatant of WT BL21 in MH 15μL 100 mM HAuCl4 8.11μL 100 mM AgNO3	477μL Supernatant of WT BL21 in MH 15μL 100 mM HAuCl4 8.11μL 100 mM AgNO3
C	473μL MH 17.5μL 100 mM HAuCl4 9.46μL 100 mM AgNO3	473μL MH 17.5μL 100 mM HAuCl4 9.46μL 100 mM AgNO3	473μL MH 17.5μL 100 mM HAuCl4 9.46μL 100 mM AgNO3	473μL Supernatant of WT BL21 in MH 17.5μL 100 mM HAuCl4 9.46μL 100 mM AgNO3	473μL Supernatant of WT BL21 in MH 17.5μL 100 mM HAuCl4 9.46μL 100 mM AgNO3	473μL Supernatant of WT BL21 in MH 17.5μL 100 mM HAuCl4 9.46μL 100 mM AgNO3
D	469μL MH 20μL 100 mM HAuCl4 10.81μL 100 mM AgNO3	469μL MH 20μL 100 mM HAuCl4 10.81μL 100 mM AgNO3	469μL MH 20μL 100 mM HAuCl4 10.81μL 100 mM AgNO3	469μL Supernatant of WT BL21 in MH 20μL 100 mM HAuCl4 10.81μL 100 mM AgNO3	469μL Supernatant of WT BL21 in MH 20μL 100 mM HAuCl4 10.81μL 100 mM AgNO3	469μL Supernatant of WT BL21 in MH 20μL 100 mM HAuCl4 10.81μL 100 mM AgNO3

Materials

• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Meuller Hinton Broth (+150 µg/mL Ampicillin).
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 10 mM aqueous HAuCl₄solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 10 mM aqueous AgNO₃ solution.
• Sodium acetate.
• Acetic acid.
• Sodium phosphate dibasic heptahydrate.
• Sodium phosphate monobasic monohydrate.
• Trizma hydrochloride.
• Trizma base.
• 10 M HCl.
• 10 M NaOH.
• Mueller Hinton Broth (+150 µg/mL Ampicillin).
• Mueller Hinton Broth.
• Sterile flat-bottom 24-well plate with lid.
• ddH₂O.
• 50 mL Falcon tubes.
• 0.2 µm Syringe filter.
• 15 mL Syringe.
• Parafilm.
• 1 mL plastic cuvettes.

Equipment

• Tabletop centrifuge.
• Shaking incubators.
• Tecan spark microplate reader.
• UV-vis NIR spectrophotometer.

Protocol

1. For 100 mM acetate buffer pH 5:
   1. In a suitable container, add 800 mL ddH₂O.
   2. Add 5.772 grams of sodium acetate to the solution.
   3. Add 1.778 grams of acetic acid to the solution.
   4. Adjust pH with 10M HCl to pH 5.
   5. Add ddH₂O to a final volume of 1 L.
2. For 100 mM phosphate buffer pH 7:
   1. In a suitable container, add 800 mL ddH₂O.
   2. Add 15.487 grams of sodium phosphate dibasic heptahydrate to the solution.
   3. Add 5.827 grams of sodium phosphate monobasic monohydrate to the solution.
   4. Adjust pH with 10 M HCl or 10 M NaOH to pH 7.
   5. Add ddH₂O to a final volume of 1 L.
3. For 100 mM tris buffer pH 9:
   1. In a suitable container, add 800 mL ddH₂O.
   2. Add 1.011 grams of trizma hydrochloride to the solution.
   3. Add 11.337 grams of trizma base to the solution.
   4. Adjust pH with 10 M NaOH to pH 9.
4. Dilute the overnight culture down to an OD600 of 0.2.
5. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
6. Once the culture has reached this phase transfer the contents to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
7. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as supernatant.
8. Adjust the pH of the supernatant to pH 5 by adding 500 µL of Acetate buffer (pH 5) to 3 mL of the supernatant. This will be pH 5 supernatant.
9. Adjust the pH of Mueller Hinton Broth to pH 5 by adding 285 µL of Acetate buffer (pH 5) and 30 µL NaOH to 3 mL of Mueller Hinton Broth. This will be pH 5 Mueller Hinton Broth.
10. Adjust the pH of the supernatant to pH 7 by adding 330 µL of Phosphate buffer (pH 7) and 2 µL of NaOH to 3 mL of the supernatant. This will be pH 7 supernatant.
11. Adjust the pH of Mueller Hinton Broth to pH 7 by adding 20µL Phosphate buffer (pH 7) and 10 µL NaOH to 3 mL Mueller Hinton Broth. This will be pH 7 Mueller Hinton Broth.
12. Adjust the pH of the supernatant to pH 9 by adding 20 µL of Tris buffer (pH 9) and 20 µL NaOH to 3 mL of the supernatant. This will be pH 9 supernatant.
13. Adjust the pH of Mueller Hinton Broth to pH 9 by adding 45 µL of NaOH to 3 mL of Mueller Hinton Broth. This will be pH 9 Mueller Hinton Broth.
14. Add the correct pH supernatant or Mueller Hinton Broth into the intended wells of the 24-well plates (e.g. 485 µL pH 5) according to the layout below.
15. Add 100 mM aqueous HAuCl₄ solution (e.g. 10 µL) into the intended wells of the 24-well plates according to the layout below.
16. Add 100 mM aqueous AgNO₃ solution (e.g. 5 µL) (Making sure to use the ratio of 1.85:1 for Gold:Silver) into the intended wells of the 24-well plates according to the layout below.
17. Seal the 24-well plates with parafilm.
18. Place the 24-well plates in 3 different shake incubators for 24 hours.
    1. 25°C at 200 rpm
    2. 37°C at 200 rpm
    3. 49°C at 200 rpm
19. Scan absorbance from 350 nm to 1000 nm with a microplate reader.

24-Well Plates

Plate 1 25°C

	1	2	3	4	5	6
A	485 uL Supernatant 1 pH=7 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL Supernatant 2 pH=7 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL MH pH=7 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	500uL pH 5 supernatant 1	500uL pH 7 supernatant 1	500uL pH 9 supernatant 1
B	454 uL Supernatant 1 pH=7 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL Supernatant 2 pH=7 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL MH pH=7 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	500uL pH 5 MH	500uL pH 7 MH	500uL pH 9 MH
C	469 uL Supernatant 1 pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 2 pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL MH pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	485 uL ddH2O pH=5 10.0 uL100 mM HAuCl4 5.41 uL AgNO3	485 uL ddH2O pH=7 10.0 uL100 mM HAuCl4 5.41 uL AgNO3	485 uL ddH2O pH=9 10.0 uL100 mM HAuCl4 5.41 uL AgNO3
D	469 uL Supernatant 1 pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 2 pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL MH pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL ddH2O pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL ddH2O pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL ddH2O pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3

Plate 2 25°C

	1	2	3	4	5	6
A	485 uL Supernatant 3 pH=7 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL MH pH=7 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	500uL pH 5 supernatant 3	500uL pH 7 supernatant 3	500uL pH 9 supernatant 3
B	454 uL Supernatant 3 pH=7 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL MH pH=7 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	500uL pH 5 MH	500uL pH 7 MH	500uL pH 9 MH
C	469 uL Supernatant 3 pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL MH pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3
D	469 uL Supernatant 3 pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL MH pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	454 uL ddH2O pH=5 30.0 uL100 mM HAuCl4 16.22 uL AgNO3	454 uL ddH2O pH=7 30.0 uL100 mM HAuCl4 16.22 uL AgNO3	454 uL ddH2O pH=9 30.0 uL100 mM HAuCl4 16.22 uL AgNO3

Plate 1 37°C

	1	2	3	4	5	6
A	485 uL Supernatant 1 pH=5 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL Supernatant 2 pH=5 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL Supernatant 3 pH=5 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL MH pH=5 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	500uL pH 5 supernatant 1	500uL pH 9 supernatant 1
B	485 uL Supernatant 1 pH=9 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL Supernatant 2 pH=9 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL Supernatant 3 pH=9 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL MH pH=9 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	500uL pH 5 supernatant 2	500uL pH 9 supernatant 2
C	454 uL Supernatant 1 pH=5 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL Supernatant 2 pH=5 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL Supernatant 3 pH=5 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL MH pH=5 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	500uL pH 5 supernatant 3	500uL pH 9 supernatant 3
D	454 uL Supernatant 1 pH=9 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL Supernatant 2 pH=9 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL Supernatant 3 pH=9 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL MH pH=9 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	500uL pH 5 MH	500uL pH 9 MH

Plate 2 37°C

	1	2	3	4	5	6
A	469 uL Supernatant 1 pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 2 pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 3 pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL MH pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3
B	469 uL Supernatant 1 pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 2 pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 3 pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	500uL pH 7 supernatant 1	500uL pH 7 supernatant 2	500uL pH 7 supernatant 3
C	469 uL Supernatant 1 pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 2 pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 3 pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL ddH2O pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	485 uL ddH2O pH=5 10.0 uL100 mM HAuCl4 5.41 uL AgNO3	485 uL ddH2O pH=9 10.0 uL100 mM HAuCl4 5.41 uL AgNO3
D	500uL pH 5 MH	500uL pH 7 MH	500uL pH 9 MH		454 uL ddH2O pH=5 30.0 uL100 mM HAuCl4 16.22 uL AgNO3	454 uL ddH2O pH=9 30.0 uL100 mM HAuCl4 16.22 uL AgNO3

Plate 1 49°C

	1	2	3	4	5	6
A	469 uL Supernatant 1 pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 2 pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 3 pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	500uL pH 5 supernatant 1	500uL pH 7 supernatant 1	500uL pH 9 supernatant 1
B	469 uL Supernatant 1 pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 2 pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL Supernatant 3 pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	500uL pH 5 supernatant 2	500uL pH 7 supernatant 2	500uL pH 9 supernatant 2
C	485 uL Supernatant 1 pH=7 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL Supernatant 2 pH=7 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL Supernatant 3 pH=7 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	500uL pH 5 Supernatant 3	500uL pH 7 Supernatant 3	500uL pH 9 Supernatant 3
D	454 uL Supernatant 1 pH=7 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL Supernatant 2 pH=7 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL Supernatant 3 pH=7 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	500uL pH 5 MH	500uL pH 7 MH	500uL pH 9 MH

Plate 2 49°C

	1	2	3	4	5	6
A	469 uL MH pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL MH pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	500uL pH 5 MH	500uL pH 7 MH	500uL pH 9 MH
B	469 uL MH pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL MH pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	485 uL ddH2O pH=5 10.0 uL100 mM HAuCl4 5.41 uL AgNO3	485 uL ddH2O pH=7 10.0 uL100 mM HAuCl4 5.41 uL AgNO3	485 uL ddH2O pH=9 10.0 uL100 mM HAuCl4 5.41 uL AgNO3
C	485 uL MH pH=7 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	485 uL MH pH=7 10.0 uL 100 mM HAuCl4 5.41 uL 100 mM AgNO3	469 uL ddH2O pH=5 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL ddH2O pH=7 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3	469 uL ddH2O pH=9 20.0 uL 100 mM HAuCl4 10.81 uL 100 mM AgNO3
D	454 uL MH pH=7 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL MH pH=7 30.0 uL 100 mM HAuCl4 16.22 uL 100 mM AgNO3	454 uL ddH2O pH=5 30.0 uL100 mM HAuCl4 16.22 uL AgNO3	454 uL ddH2O pH=7 30.0 uL100 mM HAuCl4 16.22 uL AgNO3	454 uL ddH2O pH=9 30.0 uL100 mM HAuCl4 16.22 uL AgNO3

Materials

• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Mueller Hinton broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b-copA overnight culture in Mueller Hinton broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b-napA overnight culture in Mueller Hinton broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli BL21-pET16b-metallothionein overnight culture in Mueller Hinton broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-copA overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-napA overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-cueO overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-melA overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 100 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 100 mM aqueous AgNO₃ solution.
• Mueller Hinton Broth (+150 µg/mL Ampicillin).
• Mueller Hinton Broth (+25 µg/mL Chloramphenicol).
• Sodium phosphate dibasic heptahydrate.
• Sodium phosphate monobasic monohydrate.
• 10 M HCl.
• 10 M NaOH.
• Sterile flat-bottom 24-well plate with lid.
• 50 mL Falcon tubes.
• 15 mL Falcon tubes.
• 0.2 µm Syringe filter.
• 15 mL Syringe.
• 1 mL plastic cuvettes.

Equipment

• Shaking incubator.
• Tabletop centrifuge.
• Refrigerated micro centrifuge.
• Rod sonicator (MS-72 probe).
• UV-vis NIR spectrophotometer.

Protocol

1. For 100 mM phosphate buffer pH 7:
   1. In a suitable container, add 800 mL ddH₂O.
   2. Add 15.487 grams of sodium phosphate dibasic heptahydrate to the solution.
   3. Add 5.827 grams of sodium phosphate monobasic monohydrate to the solution.
   4. Adjust pH with 10 M HCl or 10 M NaOH to pH 7.
   5. Add ddH₂O to a final volume of 1 L.
2. Dilute the overnight cultures down to an OD600 of 0.2.
3. Grow diluted cultures in a shaking incubator set to 37°C until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
4. Once the cultures have reached this phase:
   1. For Escherichia coli (E. coli) BL21-pET16b:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as BL21-supernatant.
   2. For E. coli BL21-pET16b-copA:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as copA-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as copA-induced lysate.
   3. For E. coli BL21-pET16b-napA:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as napA-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as napA-induced lysate.
   4. For E. coli BL21-pET16b-metallothionein:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as metallothionein(MT)-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as metallothionein(MT)-induced lysate.
   5. For E. coli BL21-ASKA-copA:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as ASKA-copA-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as ASKA-copA-induced lysate.
   6. For E. coli BL21-ASKA-napA:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as ASKA-napA-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as ASKA-napA-induced lysate.
   7. For E. coli BL21-ASKA-cueO:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as ASKA-cueO-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as ASKA-cueO-induced lysate.
   8. For E. coli BL21-ASKA-melA:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as ASKA-melA-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as ASKA-melA-induced lysate.
5. Add intended supernatant (e.g. BL21-supernatant) or Mueller Hinton broth into the intended wells of a 24-well plate (e.g. 485 µL) according to the layout below.
6. Add intended lysate (e.g. ASKA-melA-induced lysate) into the intended wells of a 24-well plate (e.g. 25 µL) according to the layout below.
7. Add 100 mM aqueous HAuCl₄ solution (e.g. 10 µL) into the intended wells of a 24-well plate according to the layout below.
8. Add 100 mM aqueous AgNO₃ solution (e.g. 10 µL) into the intended wells of a 24-well plate according to the layout below.
9. Place 24-well plate into a shaking incubator set to 37°C at 200 rpm for 24 hours.
10. Scan absorbance from 350 nm to 1000 nm with a microplate reader.
    

24-Well Plate

Plate 1

	1	2	3	4	5	6
A	500μL MH	500μL BL21 Sup. MH	475μL BL21 Sup. MH 25ul lysate BL21 in MH	475μL MH 25ul lysate BL21 in MH
B	476.9μL MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	476.9ul BL21 Sup. MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL MH 25ul lysate ASKA-copA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH 25ul lysate ASKA-copA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL MH 25ul lysate ASKA-napA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH 25ul lysate ASKA-napA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3
C	451.9μL MH 25ul lysate ASKA-cueO in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH 25ul lysate ASKA-cueO in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	446.9μL MH 25ul lysate BL21 in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3 5μL L-DOPA	446.9μL BL21 Sup. MH 25ul lysate ASKA-cueO in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3 5μL L-DOPA	446.9μL MH 25ul lysate ASKA-melA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3 5μL L-DOPA	451.9μL BL21 Sup. MH 25ul lysate ASKA-melA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3
D	451.9μL MH 25ul lysate TR-copA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH 25ul lysate TR-copA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL MH 25ul lysate TR-napA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH 25ul lysate TR-napA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL MH 25ul lysate TR-MT in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH 25ul lysate TR-MT in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3

Materials

• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Mueller Hinton broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b-copA overnight culture in Mueller Hinton broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b-napA overnight culture in Mueller Hinton broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b-metallothionein overnight culture in Mueller Hinton broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-copA overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-napA overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-cueO overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-melA overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 100 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 100 mM aqueous AgNO₃ solution.
• Mueller Hinton Broth (+150 µg/mL Ampicillin).
• Mueller Hinton Broth (+25 µg/mL Chloramphenicol).
• Sodium phosphate dibasic heptahydrate.
• Sodium phosphate monobasic monohydrate.
• 10 M HCl.
• 10 M NaOH.
• Sterile flat-bottom 24-well plate with lid.
• 50 mL Falcon tubes.
• 15 mL Falcon tubes.
• 0.2 µm Syringe filter.
• 15 mL Syringe.
• 1 mL plastic cuvettes.

Equipment

• Shaking incubator.
• Tabletop centrifuge.
• Refrigerated micro centrifuge.
• Rod sonicator (MS-72 probe).
• UV-vis NIR spectrophotometer.

Protocol

1. For 100 mM phosphate buffer pH 7:
   1. In a suitable container, add 800 mL ddH₂O.
   2. Add 15.487 grams of sodium phosphate dibasic heptahydrate to the solution.
   3. Add 5.827 grams of sodium phosphate monobasic monohydrate to the solution.
   4. Adjust pH with 10 M HCl or 10 M NaOH to pH 7.
   5. Add ddH₂O to a final volume of 1 L.
2. Dilute the overnight cultures down to an OD600 of 0.2.
3. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
4. Once the cultures have reached this phase:
   1. For Escherichia coli (E. coli) BL21-pET16b:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as BL21-supernatant MH-Nitrate.
   2. For E. coli BL21-pET16b-copA:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as copA-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as copA-induced lysate.
   3. For E. coli BL21-pET16b-napA:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as napA-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as napA-induced lysate.
   4. For E. coli BL21-pET16b-metallothionein:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as MT-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as MT-induced lysate.
   5. For E. coli BL21-ASKA-copA:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as ASKA-copA-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as ASKA-copA-induced lysate.
   6. For E. coli BL21-ASKA-napA:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as ASKA-napA-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as ASKA-napA-induced lysate.
   7. For E. coli BL21-ASKA-cueO:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as ASKA-cueO-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 30 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as ASKA-cueO-induced lysate.
   8. For E. coli BL21-ASKA-melA:
      1. Transfer 5 mL from flasks into a 15 mL falcon tube and centrifuge at 4000 rpm for 10 minutes. This will be the uninduced sample. Take the supernatant of centrifuged uninduced sample and filter sterilize it with a 0.2 µm filter into a new falcon tube. This will now be referred to as ASKA-melA-uninduced-supernatant. Use immediately or store at 4°C.
      2. For the rest of the culture, add IPTG to an end concentration of 1 mM. Place culture back in a shaking incubator for 4 hours.
      3. After 4 hours, split the contents of the culture into two appropriately sized falcon tubes and centrifuge at 4000 rpm for 10 minutes.
      4. Discard the supernatant. Add 10 mL of phosphate buffer to the pellet.
      5. Sonicate with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
      6. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes.
      7. This will not be referred to as ASKA-melA-induced lysate.
5. Add intended supernatant (e.g. BL21-supernatant MH-Nitrate) or Mueller Hinton Nitrate broth into the intended wells of a 24-well plate (e.g. 485 µL) according to the layout below.
6. Add the intended lysate (e.g. ASKA-melA-induced lysate) into the intended wells of a 24-well plate (e.g. 25 µL) according to the layout below.
7. Add 100 mM aqueous HAuCl₄ solution (e.g. 10 µL) into intended wells of a 24-well plate according to the layout below.
8. Add 100 mM aqueous AgNO₃ solution (e.g. 5 µL) into intended wells of a 24-well plate according to the layout below.
9. Place the 24-well plate into a shaking incubator set to 37°C at 200 rpm for 24 hours.
10. Scan absorbance from 350 nm to 1000 nm with a microplate reader.

24-Well Plate

Plate 1

	1	2	3	4	5	6
A	500μL MH	500μL MH-Nitrate	476.9uL MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	476.9uL MH-Nitrate 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	475μL BL21 Sup. MH 25ul lysate BL21 in MH	475μL BL21 Sup. MH-Nitrate 25ul lysate BL21 in MH
B	476.9ul BL21 Sup. MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	476.9ul BL21 Sup. MH-Nitrate 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH 25ul lysate ASKA-copA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH-Nitrate 25ul lysate ASKA-copA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH 25ul lysate ASKA-napA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH-Nitrate 25ul lysate ASKA-napA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3
C	451.9μL BL21 Sup. MH 25ul lysate ASKA-cueO in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH-Nitrate 25ul lysate ASKA-cueO in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	446.9μL BL21 Sup. MH 25ul lysate BL21 in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3 5μL L-DOPA	446.9μL BL21 Sup. MH-Nitrate 25ul lysate BL21 in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3 5μL L-DOPA	446.9μL BL21 Sup. MH 25ul lysate ASKA-melA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3 5μL L-DOPA	446.9μL BL21 Sup. MH-Nitrate 25ul lysate ASKA-melA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3 5μL L-DOPA
D	451.9μL BL21 Sup. MH 25ul lysate TR-copA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH-Nitrate 25ul lysate TR-copA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH 25ul lysate TR-napA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH-Nitrate 25ul lysate TR-napA in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH 25ul lysate TR-MT in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3	451.9μL BL21 Sup. MH-Nitrate 25ul lysate TR-MT in MH 15μL 100 mM HAuCl4 8.1μL 100 mM AgNO3

Materials

• 20 mL Escherichia coli (E. coli) BL21-pET16b-copA overnight culture in Mueller Hinton broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b-napA overnight culture in Mueller Hinton broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b-metallothionein overnight culture in Mueller Hinton broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-copA overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-napA overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-cueO overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• 20 mL Escherichia coli (E. coli) BL21-ASKA-melA overnight culture in Mueller Hinton broth (+25 µg/mL Chloramphenicol).
• Mueller Hinton Broth (+150 µg/mL Ampicillin).
• Mueller Hinton Broth (+25 µg/mL Chloramphenicol).
• Coomasie brilliant blue solution.
• 1 mL plastic cuvettes.
• Destaining solution (30% ethanol, 10% glacial acetic acid).
• Laemmli Buffer (loading buffer) (1x)
  • 50 mM Tris HCl, pH 6.8
  • 1% (w/v) SDS
  • 25% (w/v) glycerol
  • 1% (w/v) ß-Mercaptoethanol
  • 0.01% Bromophenol blue (add this on the day, right before use)
• 12.5% Separation Gels
  • 30% acryl/Bisacryl 4100 µL
  • 1.5 M Tris-HCl pH 8.8 2500 µL
  • ddH₂O 3195 µL
  • 10% SDS 100 µL
  • 10% APS 100 µL
  • TEMED (add last, starts polymerisation) 5 µL

    Total volume 10 mL

• 5% Stacking Gels
  • 30% acryl/Bisacryl 666 µL
  • 1.5 M Tris-HCl pH 8.8 1000 µL
  • ddH₂O 2250 µL
  • 10% SDS 40 µL
  • 10% APS 40 µL
  • TEMED (add last, starts polymerisation) 4 µL

    Total volume 4 mL

Equipment

• Tabletop centrifuge.
• Shaking incubator.
• Electrophoresis cells.
• Vortex mixer.
• UV-vis NIR spectrophotometer.

Protocol

Making the gel

1. Add the spacer/comb and mark the plate +/- 1cm beneath it. Now remove the spacer.
2. Clean the glass plates and spacers of the gel casting unit with ddH₂O and ethanol. Now dry the plates and spacers.
3. Assemble the plates with the spacers on a stable surface.
4. Test for leakage with ddH₂O and drain.
5. Prepare the resolving gel solutions:
   • Laemmli buffer 1x
   • 12.5% separation gels
6. Pour the gel solution into the plates assembled with spacers. To maintain an even horizontal surface, overlay the gel with a bit of isopropanol.
7. Allow the gel to set for 30 minutes at room temperature.
8. Prepare stacking gel solution
   • 5% stacking gels
9. Remove layer of isopropanol with filter paper.
10. Add stacking gel solution until it overflows.
11. Insert comb immediately ensuring no air bubbles are trapped in the gel or near the wells.
12. Allow the gel to set for 30 minutes at room temperature.

Making the samples

1. Dilute the overnight cultures down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
   1. For induced cultures add 1 mM IPTG at this stage and wait another 2 hours.
3. Take a 1 mL sample of cultures and centrifuge for 2 minutes at max speed. Samples need to be used for SDS analysis immediately or need to be stored at -20°C for later use.
4. Add 200 µL of 1x Laemmli buffer to samples and vortex to resuspend the pellet.
5. Heat samples in a heating block for 10 minutes at 95°C. Samples need to be used for SDS analysis immediately or need to be stored at -20°C for later use.
   1. If this yields a sticky/viscous sample, centrifuge for 5 minutes at max speed and use the supernatant.
6. Load 10 µL of sample and 5 µL of protein ladder to the gel and run at 150V until the bands hit the bottom.
7. Stain the gels by shaking them overnight in coomasie brilliant blue solution.
8. Destain the gels by shaking for two hours in a destaining solution containing 30% ethanol and 10% glacial acetic acid.
9. Image the gels with geldoc using visible light.

Materials

• pJET 1.2/blunt plasmid.
• DNA/gBlocks (ours were ordered from IDT).
• Rapid ligation buffer.
• Ligase.
• ddH₂O.
• 1.5 mL Eppendorf tube.

Protocol

1. Add 1 µL of pJET 1.2/blunt plasmid to an eppendorf tube.
2. Add 4 µL of gBlock DNA.
   1. Metallothionein
   2. CopA
   3. NapA
3. Add 4 µL of rapid ligation buffer.
4. Add 1 µL of ligase.
5. Add 10 µL of ddH₂O.
6. Incubate at room temperature for 1 hour.

Material

• Competent Escherichia coli (E. coli) BL21 or DH5𝛼 cells.
• Ligation mix.
• Lysogeny broth.
• Petri dishes
• Lysogeny broth agar.
• Ampicillin.
• 70% Ethanol.
• Spreader.

Equipment

• Incubator.
• Heating block or water bath at 42°C.
• Bunsen burner.

Protocol

1. Take 125 μL of competent cells out of -80°C freezer and put on ice.
2. Add 10 μL of ligation mix to the competent cells (about 1 μg of DNA). Leave on ice for 15-30min. Also have 125 μL of competent cells without ligation mix as a control.
3. Heat shock at 42 °C for 90 seconds and put back on ice for 2 min.
4. Add 500 µl LB next to the Bunsen burner to keep sterile and place in incubate set to 37 °C for 60 min without shaking.
5. Plate transformation mix on LB-plates + ampicillin (150 μg/ml end concentration) and incubate o/n at 37 °C.
   1. Plate 1: Pipette 100 µL transformation mix onto the plate and disperse with spreader.
   2. Plate 2: Spin down remaining transformation mix (6000 rpm, 2 min), discard supernatant until 100 µL, resuspend with pipet and plate with spreader.
   3. Plate 3: Pipette 100 µL control onto the plate and disperse with spreader.

Material

• Appropriate restriction enzymes.
• Appropriate buffer (if using NEB look at the manufacturers scheme).
• DNA (that needs to be cut).
• ddH₂O.

Equipment

• Vortex mixer
• Incubator at 37°C

Protocol

1. Add 400 ng of DNA to an Eppendorf tube. Take 200 ng if you are doing a diagnostic restriction digest.
2. Add 4µL buffer ( 2 µL for diagnostic)
   1. Find the appropriate buffer for the restriction enzyme.
   2. 10x concentrated .
3. Add 1 µL restriction enzyme (0.5 for diagnostic). Add enzymes last and keep on ice always.
4. Bring to a volume of 40mL with ddH₂O. Bring to 20mL for diagnostic.
5. Mix by vortexing or pipetting up and down. If mixing by vortexing, centrifuge quickly before incubation.
6. Incubate for at least 1 hour at 37 °C.
7. Use for downstream application or check on gel for diagnostic.

Materials

• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Lysogeny Nitrate Broth (+150 µg/mL Ampicillin).
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 100 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 100 mM aqueous AgNO₃ solution.
• Lysogeny Nitrate Broth (+150 µg/mL Ampicillin).
• ddH₂O.
• 1.5 mL Eppendorf tubes.
• 50 mL Falcon tubes.
• 50 mL Flasks.
• Sterile flat-bottom 24-well plate with lid.
• 1 mL plastic cuvettes.
• 0.2 µm Syringe filter.
• 15 mL Syringe.

Equipment

• Shaking incubator.
• Tabletop centrifuge.
• Tecan spark microplate reader.
• UV-vis NIR spectrophotometer.

Protocol

1. Dilute the overnight culture down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
3. Once the culture has reached this phase transfer the contents to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
4. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as supernatant.
5. Add supernatant, Lysogeny Nitrate Broth, or ddH₂O into the intended wells in a 24-well plate (e.g. 485 µL) according to the layout below.
6. Add supernatant, Lysogeny Nitrate Broth, or ddH₂O into the assigned Eppendorf tubes (e.g. 971 µL) according to the layout below.
7. Add supernatant, Lysogeny Nitrate Broth, or ddH₂O into the assigned flasks (e.g. 9.7 mL) according to the layout below.
8. Add 100 mM aqueous HAuCl₄ solution (e.g. 10 µL) into intended wells in a 24-well plate according to the layout below.
9. Add 100 mM aqueous HAuCl₄ solution (e.g. 10 µL) into assigned Eppendorf tubes according to the layout below.
10. Add 100 mM aqueous HAuCl₄ solution (e.g. 10 µL) into assigned flasks according to the layout below.
11. Add 100 mM aqueous AgNO₃ solution (e.g. 5 µL) (Making sure to use the ratio of 1.85:1 for Gold:Silver) into intended wells in a 24-well plate according to the layout below.
12. Add 100 mM aqueous AgNO₃ solution (e.g. 5 µL) (Making sure to use the ratio of 1.85:1 for Gold:Silver) into assigned Eppendorf tubes according to the layout below.
13. Add 100 mM aqueous AgNO₃ solution (e.g. 5 µL) (Making sure to use the ratio of 1.85:1 for Gold:Silver) into assigned flasks according to the layout below.
14. Scan absorbance for all containers:
    1. Using a microplate reader to scan absorbance from 350 nm to 1000 nm for the 24-well plate.
    2. Use a UV-vis spectrophotometer for the Eppendorf tubes by transferring 1 mL of the sample into a 1 mL cuvette and scanning absorbance from 350 nm to 1000 nm.
    3. Use a UV-vis spectrophotometer for the flasks by transferring 1 mL of the sample into a 1 mL cuvette and scanning absorbance from 350 nm to 1000 nm.

24-Well Plate

	1	2	3	4	5	6
A	1.584 mL Supernatant + 24.75 µL 100 mM AgNO3 + 41.25 µL 100 mM HAuCl4	1.584 mL Supernatant + 24.75 µL 100 mM AgNO3 + 41.25 µL 100 mM HAuCl4	1.584 mL Supernatant + 24.75 µL 100 mM AgNO3 + 41.25 µL 100 mM HAuCl4	*1:10 dilution of previous wells* 1.35 mL LB-Nitrate + 150 µL sample from well A1	*1:10 dilution of previous wells* 1.35 mL LB-Nitrate + 150 µL sample from well A2	*1:10 dilution of previous wells* 1.35 mL LB-Nitrate + 150 µL sample from well A3
B	1.5 mL LB-Nitrate	1.5 mL LB-Nitrate	1.5 mL LB-Nitrate	1.5 mL Supernatant	1.5 mL Supernatant	1.5 mL Supernatant
C	1.44 mL LB-Nitrate + 22.5 µL 100 mM AgNO3 + 37.5 µL 100 mM HAuCl4	1.44 mL LB-Nitrate + 22.5 µL 100 mM AgNO3 + 37.5 µL 100 mM HAuCl4	1.44 mL LB-Nitrate + 22.5 µL 100 mM AgNO3 + 37.5 µL 100 mM HAuCl4	1.44 mL ddH20 + 22.5 µL 100 mM AgNO3 + 37.5 µL 100 mM HAuCl4	1.44 mL ddH20 + 22.5 µL 100 mM AgNO3 + 37.5 µL 100 mM HAuCl4	1.44 mL ddH20 + 22.5 µL 100 mM AgNO3 + 37.5 µL 100 mM HAuCl4
D	1.494 mL LB-Nitrate + 2.25 µL 100 mM AgNO3 + 3.75 µL 100 mM HAuCl4	1.494 mL LB-Nitrate + 2.25 µL 100 mM AgNO3 + 3.75 µL 100 mM HAuCl4	1.494 mL LB-Nitrate + 2.25 µL 100 mM AgNO3 + 3.75 µL 100 mM HAuCl4	1.494 mL ddH20 + 2.25 µL 100 mM AgNO3 + 3.75 µL 100 mM HAuCl4	1.494 mL ddH20 + 2.25 µL 100 mM AgNO3 + 3.75 µL 100 mM HAuCl4	1.494 mL ddH20 + 2.25 µL 100 mM AgNO3 + 3.75 µL 100 mM HAuCl4

50 mL Flasks

1	9.6 mL Supernatant + 250 µL 100 mM HAuCl4 + 150 µL 100 mM AgNO3
2	9.6 mL LB-Nitrate + 250 µL 100 mM HAuCl4 + 150 µL 100 mM AgNO3
3	9.6 mL ddH2O + 250 µL 100 mM HAuCl4 + 150 µL 100 mM AgNO3
4	10 mL Supernatant

1.5 mL Eppendorf tubes

1	1.4 mL Supernatant + 37.5 µL 100 mM HAuCl4 + 22.5 µL 100 mM AgNO3
2	9.6 mL LB-Nitrate + 37.5 µL 100 mM HAuCl4 + 22.5 µL 100 mM AgNO3
3	9.6 mL ddH2O + 37.5 µL 100 mM HAuCl4 + 22.5 µL 100 mM AgNO3
4	1.5 mL Supernatant
5	1.5 mL LB-Nitrate

Materials

• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 10 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 10 mM aqueous AgNO₃ solution.
• Ascorbic acid (AA, 99%, Acros).
  • 100 mM aqueous ascorbic acid.
• ddH₂O.
• 1.5 mL Eppendorf Tubes.
• 1 mL plastic cuvettes.

Equipment

• Vortex mixer.
• UV-vis NIR spectrophotometer.

Protocol

1. Add 970 µL of ddH₂O into a 1.5 mL Eppendorf tube.
2. Add 20 µL of 10 mM aqueous HAuCl₄ solution.
3. Add 10 µL of 10 mM aqueous AgNO₃ solution.
4. Briefly vortex the Eppendorf tube with the sample.
5. Add 4 µL of 100 mM aqueous ascorbic acid into the sample.
6. Vortex the Eppendorf tube with the sample for 20 seconds. This will be sample 1 (dilution 0).
7. Transfer sample 1 into a 1 mL cuvette and scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.
8. Set up a dilution series:
   1. Transfer 500 µL of sample 1 into a new Eppendorf tube and add 500 µL of ddH₂O and vortex briefly. This will be sample 2 (dilution 1/2).
   2. Transfer sample 2 into a 1 mL cuvette and scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.
   3. Transfer 500 µL of sample 2 into a new Eppendorf tube and add 500 µL of ddH₂O and vortex briefly. This will be sample 3 (dilution 1/4).
   4. Transfer sample 3 into a 1 mL cuvette and scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.
   5. Transfer 500 µL of sample 3 into a new Eppendorf tube and add 500 µL of ddH₂O and vortex briefly. This will be sample 4 (dilution 1/8).
   6. Transfer sample 4 into a 1 mL cuvette and scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.
   7. Transfer 500 µL of sample 4 into a new Eppendorf tube and add 500 µL of ddH₂O and vortex briefly. This will be sample 5 (dilution 1/16).
   8. Transfer sample 5 into a 1 mL cuvette and scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.
   9. Transfer 500 µL of sample 5 into a new Eppendorf tube and add 500 µL of ddH₂O and vortex briefly. This will be sample 6 (dilution 1/32).
   10. Transfer sample 6 into a 1 mL cuvette and scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.

Materials

• ddH₂O.
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 10 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 10 mM aqueous AgNO₃ solution.
• Sterile flat-bottom 24-well plate with lid.

Equipment

• UV-vis NIR spectrophotometer.
• Tecan spark microplate reader.
• Shaking incubator.

Protocol

1. Add the appropriate volume (total volume to be 500 µL) of ddH₂O into the intended wells in a 24-well plate (e.g. 486 µL) according to the layout below.
2. Add 10 mM aqueous HAuCl₄ solution (e.g. 9 µL) into the intended wells in a 24-well plate according to the layout below.
3. Add 10 mM aqueous AgNO₃ solution (e.g. 5 µL) into the intended wells in a 24-well plate according to the layout below.
4. Place the 24-well plate into a shaking incubator at 37°C for 24 hours.
5. Scan absorbance from ₂350 nm to 1000 nm with a microplate reader.

24-Well Plate

Plate 1

	1	2	3	4	5	6
A	500 µL ddH2O	500 µL ddH2O	500 µL ddH2O
B	495 µL ddH2O + 5 µL 10 mM AgNO3	495 µL ddH2O + 5 µL 10 mM AgNO3	495 µL ddH2O + 5 µL 10 mM AgNO3
C	491 µL ddH2O + 9 µL 10 mM HauCl4	491 µL ddH2O + 9 µL 10 mM HauCl4	491 µL ddH2O + 9 µL 10 mM HauCl4
D	486 µL ddH2O + 5 µL 10 mM AgNO3 + 9 µL 10 mM HauCl4	486 µL ddH2O + 5 µL 10 mM AgNO3 + 9 µL 10 mM HauCl4	486 µL ddH2O + 5 µL 10 mM AgNO3 + 9 µL 10 mM HauCl4

Materials

• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros).
  • 10 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros).
  • 10 mM aqueous AgNO₃ solution.
• Ascorbic acid (AA, 99%, Acros).
  • 100 mM aqueous ascorbic acid.
• ddH₂O.
• Sterile flat-bottom 24-well plate with lid.
• Parafilm.

Equipment

• Tecan spark microplate reader.

Protocol

1. Add 1 mL of ddH₂O into the intended wells of a 24-well plate according to the layout below.
2. Add 10 mM of aqueous HAuCl₄ solution (e.g. 20 µL) into the intended wells of a 24-well plate according to the layout below.
3. Add 10 mM aqueous AgNO₃ solution (e.g. 11 µL) into the intended wells of a 24-well plate according to the layout below.
4. Add 100 mM aqueous ascorbic acid (e.g. 2 µL) into the intended wells of a 24-well plate according to the layout below.
5. Seal the 24-well plate with parafilm.
6. Place 24-well plate in microplate reader for dynamic measurements for 24 hours.
   • Microplate reader settings:
     • Shake for 15 seconds at 810 rpm at a 2-centimeter amplitude between measurements.
     • Absorbance scan from 350 nm to 1000 nm.

24-Well Plate

Plate 1

	1	2	3	4	5	6
A	1mL ddH2O	1mL ddH2O	1mL ddH2O	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 1μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 1μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 1μL Ascorbic Acid
B	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 5μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 5μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 5μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 2μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 2μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 2μL Ascorbic Acid
C	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 6μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 6μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 6μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 3μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 3μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 3μL Ascorbic Acid
D	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 7μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 7μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 7μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 4μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 4μL Ascorbic Acid	1000μL ddH2O 20μL 10 mM HAuCl4 11μL 10 mM AgNO3 4μL Ascorbic Acid

Materials

• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Lysogeny Broth (+150 µg/mL Ampicillin).
• Kanamycin (50 mg/mL).
• Chloramphenicol (50 mg/mL).
• Lysogeny Broth Agar Plates with 150 µg/ml Ampicillin.
• 15 mL or 50 mL Falcon tubes.
• 0.2 µm Syringe filter.
• 15 mL Syringe.

Equipment

• Tabletop centrifuge.
• Incubator.

Protocol

1. Transfer contents of overnight culture into a falcon tube and centrifuge for 10 minutes at full speed.
2. Transfer 2 mL of the supernatant into three separate 2 mL Eppendorf tubes. Label the Eppendorf tubes appropriately.
3. In Eppendorf tube 1 add 20 µL of Kanamycin (50 mg/mL).
4. In Eppendorf tube 2 add 20 µL of Chloramphenicol (50 mg/mL).
5. Filter sterilize the contents of Eppendorf tube 3 through a 0.2 µm filter using a 15 mL syringe into a new labeled Eppendorf tube.
6. Pour contents of each Eppendorf tube onto individually labeled Lysogeny Broth agar plates containing 150 µg/ml of Ampicillin.
7. Place Lysogeny Broth agar plates into a 37°C incubator overnight.

Materials

• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Lysogeny Broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Lysogeny Broth Low Salt (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Lysogeny Broth Nitrate (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Mueller Hinton Broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Mueller Hinton Nitrate Broth (+150 µg/mL Ampicillin).
• Lysogeny Broth (+150 µg/mL Ampicillin).
• Lysogeny Broth Low Salt (+150 µg/mL Ampicillin).
• Lysogeny Broth Nitrate (+150 µg/mL Ampicillin).
• Mueller Hinton Broth (+150 µg/mL Ampicillin).
• Mueller Hinton Nitrate Broth (+150 µg/mL Ampicillin).
• 1 mL plastic cuvettes.
• Laemmli Buffer (loading buffer) (1x)
  • 50 mM Tris HCl, pH 6.8
  • 1% (w/v) SDS
  • 25% (w/v) glycerol
  • 1% (w/v) ß-Mercaptoethanol
  • 0.01% Bromophenol blue (add this on the day, right before use)
• 12.5% Separation Gels
  • 30% acryl/Bisacryl 4100 µL
  • 1.5 M Tris-HCl pH 8.8 2500 µL
  • ddH₂O 3195 µL
  • 10% SDS 100 µL
  • 10% APS 100 µL
  • TEMED (add last, starts polymerisation) 5 µL

    Total volume 10 mL

• 5% Stacking Gels
  • 30% acryl/Bisacryl 666 µL
  • 1.5 M Tris-HCl pH 8.8 1000 µL
  • ddH₂O 2250 µL
  • 10% SDS 40 µL
  • 10% APS 40 µL
  • TEMED (add last, starts polymerisation) 4 µL

    Total volume 4 mL

Equipment

• Tabletop centrifuge.
• Shake incubators.
• Electrophoresis cells.
• Vortex mixer.

Protocol

Making the gel

1. Add the spacer/comb and mark the plate +/- 1cm beneath it. Now remove the spacer.
2. Clean the glass plates and spacers of the gel casting unit with ddH₂O and ethanol. Now dry the plates and spacers.
3. Assemble the plates with the spacers on a stable surface.
4. Test for leakage with ddH₂O and drain.
5. Prepare the resolving gel solutions:
   1. Laemmli buffer 1x
   2. 12.5% separation gels
6. Pour the gel solution into the plates assembled with spacers. To maintain an even horizontal surface, overlay the gel with a bit of isopropanol.
7. Allow the gel to set for 30 minutes at room temperature.
8. Prepare stacking gel solution
   1. 5% stacking gels
9. Remove layer of isopropanol with filter paper.
10. Add stacking gel solution until it overflows.
11. Insert comb immediately ensuring no air bubbles are trapped in the gel or near the wells.
12. Allow the gel to set for 30 minutes at room temperature.

Making the samples

1. Dilute the overnight cultures down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
3. Take a 1 mL sample of cultures and centrifuge for 2 minutes at max speed. Samples need to be used for SDS analysis immediately or need to be stored at -20°C for later use.
4. Add 200 µL of 1x Laemmli buffer to samples and vortex to resuspend the pellet.
5. Heat samples in a heating block for 10 minutes at 95°C. Samples need to be used for SDS analysis immediately or need to be stored at -20°C for later use.
   1. If this yields a sticky/viscous sample, centrifuge for 5 minutes at max speed and use the supernatant.
6. Load 10 µL of sample and 5 µL of protein ladder to the gel and run at 150V until the bands hit the bottom.
7. Stain the gels by shaking them overnight in coomasie brilliant blue solution.
8. Destain the gels by shaking for two hours in a destaining solution containing 30% ethanol and 10% glacial acetic acid.
9. Image the gels with geldoc using visible light.

Materials

• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Lysogeny Broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Mueller Hinton Broth (+150 µg/mL Ampicillin).
• 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Tryptic Soy Broth (+150 µg/mL Ampicillin).
• Lysogeny Broth media components in solution:
  • Tryptone - 10g/L solution
  • Yeast Extract - 5g/L solution
  • NaCl - 10g/L solution
  • Nitrate - 1g/L solution *for Lysogeny broth nitrate*
• Mueller Hinton Broth media components in solution:
  • Dehydrated infusion from beef (substituted with brain heart infusion) - 300g/L solution
  • Casein hydrolysate (substituted with Tryptone) - 17.5g/L solution
  • Starch - 1.5g/L solution
• Tryptic Soy Broth media components in solution:
  • Tryptone - 17g/L solution
  • Soy (substituted with yeast extract) - 3g/L solution
  • NaCl - 5g/L solution
  • Dipotassium Phosphate (KHPO) - 2.5g/L solution
  • Glucose - 2.5g/L solution
• 2xYT media components in solution:
  • Tryptone - 16g/L solution
  • Yeast Extract - 10g/L solution
  • NaCl - 5g/L solution
• Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂0, 99.9%, Acros)
  • 100 mM aqueous HAuCl₄ solution.
• Silver nitrate (AgNO₃, 99%, Acros)
  • 100 mM aqueous AgNO₃ solution.
• Lysogeny Broth.
• Lysogeny Broth unautoclaved.
• Mueller Hinton Broth.
• Mueller Hinton Broth unautoclaved.
• Tryptic Soy Broth.
• Tryptic Soy Broth unautoclaved.
• ddH₂O.
• 50 mL Falcon tubes.
• Sterile flat-bottom 24-well plate with lid.
• 0.2 µm Syringe filter.
• 15 mL Syringe.
• Parafilm.
• 1 mL plastic cuvettes.

Equipment

• Shaking Incubator.
• Tabletop centrifuge.
• UV-vis NIR spectrophotometer.

Protocol

1. Dilute the overnight cultures down to an OD600 of 0.2.
2. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
3. Once the cultures have reached this phase:
   1. For Escherichia coli (E. coli) BL21-pET16b in Lysogeny Broth:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as BL21-supernatant Luria Broth.
   2. For E. coli BL21-pET16b in Mueller Hinton Broth:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as BL21-supernatant Mueller Hinton Broth.
   3. For E. coli BL21-pET16b in Tryptic Soy Broth:
      1. Transfer the contents of the culture to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
      2. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as BL21-supernatant Tryptic Soy Broth.
4. Add either ddH₂O, media component solution (e.g. Glucose), autoclaved or unautoclaved media (e.g. Mueller Hinton Broth), or supernatant (e.g. BL21-supernatant Tryptic Soy Broth) into the intended wells of the 24-well plates (e.g. 485 µL) according to the layout below.
5. Add 100 mM aqueous HAuCl₄ solution (e.g. 10 µL) into the intended wells of the 24-well plates according to the layout below.
6. Add 100 mM aqueous AgNO₃ solution (e.g. 5 µL) into the intended wells of the 24-well plates according to the layout below.
7. Seal the 24-well plates with parafilm and place them into a shaking incubator set to 37°C at 200 rpm for 24 hours.
8. Scan absorbance from 350 nm to 1000 nm with a microplate reader.

24-Well Plate

Plate 1

	1	2	3	4	5	6
A	D1-1: 500μL ddH2O	D1-2: 500μL ddH2O	T1-1: 500μL Tryptone	T1-2: 500μL Tryptone	Y1-1: 500μL Yeast Extract	Y1-2: 500μL Yeast Extract
B	D2-1: 483.5μL ddH2O 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	D2-2: 483.5 ddH2O 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	T2-1: 483.5μL DI Tryptone 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	T2-2: 483.5μL DI Tryptone 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	Y2-2: 483.5μL Yeast Extract 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	Y2-2: 483.5μL Yeast Extract 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3
C	K1-1: 500μL KHPO	K1-2: 500μL KHPO	G1-1: 500μL Glucose	G1-2: 500μL Glucose	S1-1: 500μL Starch	S1-2: 500μL Starch
D	K2-1: 483.5μL KHPO 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	K2-2: 483.5 KHPO 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	G2-1: 483.5μL Glucose 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	G2-2: 483.5 Glucose 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	S2-1: 483.5μL Starch 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	S2-2: 483.5 Starch 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3

Plate 2

	1	2	3	4	5	6
A	N1-1: 500μL NaCl	N1-2: 500μL NaCl	BHI1-1: 500μL BHI	BHI1-2: 500μL BHI	NI1-1: 500μL Nitrate	NI1-2: 500μL Nitrate
B	N2-1: 483.5μL NaCl 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	N2-2: 483.5 NaCl 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	BHI2-1: 483.5μL BHI 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	BHI2-2: 483.5 BHI 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	NI2-1: 483.5μL Nitrate 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	NI2-2: 483.5 Nitrate 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3
C	LB-A1-1: 500μL LB-A	LB-A1-2: 500μL LB-A	LB-S1-1: 500μL LB-S	LB-S1-2: 500μL LB-S	LB-NA1-1: 500μL LB-NA	LB-NA1-2: 500μL LB-NA
D	LB-A2-1: 483.5μL LB-A 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	LB-A2-2: 483.5 LB-A 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	LB-S2-1: 483.5μL LB-S 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	LB-S2-2: 483.5 LB-S 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	LB-NA2-1: 483.5μL LB-NA 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	LB-NA2-2: 483.5 LB-NA 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3

Plate 3

	1	2	3	4	5	6
A	MH-NA1-1: 500μL MH-NA	MH-NA1-2: 500μL MH-NA	MH-A1-1: 500μL MH-A	MH-A1-2: 500μL MH-A	MH-S1-1: 500μL MH-S	MH-S1-2: 500μL MH-S
B	MH-NA2-1: 483.5μL MH-NA 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	MH-NA2-2: 483.5 MH-NA 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	MH-A2-1: 483.5μL MH-A 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	MH-A2-2: 483.5 MH-A 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	MH-S2-1: 483.5μL MH-S 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	MH-S2-2: 483.5 MH-S 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3
C	TSBS-NA1-1: 500μL TSBS-NA	TSBS-NA1-2: 500μL TSBS-NA	TSBS-A1-1: 500μL TSBS-A	TSBS-A1-2: 500μL TSBS-A	TSBS-S1-1: 500μL TSBS-S	TSBS-S1-2: 500μL TSBS-S
D	TSBS-NA2-1: 483.5μL TSBS-NA 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	TSBS-NA2-2: 483.5 TSBS-NA 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	TSBS-A2-1: 483.5μL TSBS-A 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	TSBS-A2-2: 483.5 TSBS-A 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	TSBS-S2-1: 483.5μL TSBS-S 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3	TSBS-S2-2: 483.5 TSBS-S 15μL 100 mM HAuCl4 1.5μL 100 mM AgNO3