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From 2013.igem.org

Contents 1 29-04 - 05-05-13 1.1 Reproduction of Johnsson et al. Paper 1.1.1 Aim 1.1.2 Preparations 1.1.3 Gold Precipitation on ACM Plates 1.1.4 Result 1.1.5 Gold Precipitation in D. acidovorans Supernatants 1.1.6 Result 1.1.7 Gold Precipitation in D. acidovorans Supernatants at different Gold Chloride Concentrations 1.1.8 Result

29-04 - 05-05-13

Reproduction of Johnsson et al. Paper

Aim

Reproduction of Johnsson et al. paper: Precipitating gold with D.acidovorans on agar plate, in culture, the medium of the culture and possibly purified delftibactin.

Preparations

• Obtain D. acidovorans from DSMZ
• Prapare ACM medium as described
• Prepare ACM agar plates

Gold Precipitation on ACM Plates

• Culture D. acidovorans for 3 days on agar plates at 30°C
• Overlay agar plates with 0.2% HAuCl₄ in 0.5 % soft agarose

Result

ACM agar plate with D. acidovorans (left) overlaid with 0.2% HAuCl4 in 0.5% agarose

ACM agar plate with D. acidovorans (left) overlaid with 0.2% HAuCl4 in 0.5% agarose

• Gold precipitation on ACM agar plate worked even better than described in paper

Gold Precipitation in D. acidovorans Supernatants

• Culture D. acidovorans for 3 days in ACM plates
• Centrifuge bacteria in liquid culture (10 min 6,000 rpm)
• Add 0.2% HAuCl₄ to supernatant and medium control

Result

D. acidovorans Supernatant (top) and ACM negative control (bottom) with (left to right) 600µl, 400µl, 200µl, 100µl, 50µl, 0µl 0.2% HAuCl4. Far right: top: uncentrifuged solution culture with 600µl 0.2% HAuCl4, bottom: ACM with 600µl HAuCl4

The supernatant/ medium with gold was centrifuged after 2 h reaction time. left to right: supernatant with 600µl 0.2% HAuCl4, ACM with 600µl 0.2% HAuCl4, supernatant with 400µl 0.2% HAuCl4, ACM with 400µl 0.2% HAuCl4, supernatant with 200µl 0.2% HAuCl4, ACM with 200µl 0.2% HAuCl4

• Gold precipitation with D. acidovorans supernatant worked well

Gold Precipitation in D. acidovorans Supernatants at different Gold Chloride Concentrations

• Culture D. acidovorans for 3 days in ACM plates
• Centrifuge bacteria in liquid culture (10 min 6,000 rpm)
• Add HAuCl₄ to supernatant and medium control

Result

Sequences of movie showing gold precipitation in D. acidovorans supernatant using gold concentrations ranging from 0 to 2.55 µg/ml

• Gold precipitation with supernatant worked at very low concentration Gold
• Gold precipitation with unpurified culture worked as well