Coy anaerobic chamber

In a typical anaerobic
purification of FtmOx1, ∼30 g wet cell paste was resuspended
in 50 mL of anaerobic buffer (100 mM Tris–HCl, 50 mM NaCl,
pH 7.5) in an anaerobic Coy chamber (Coy Laboratory Products, Inc.,
Grass Lake, USA). Lysozyme (5 mg/mL final concentration), EDTA (5
mM final concentration), and ascorbate (5 mM final concentration)
were then added into the cell suspension, and the mixture was incubated
on ice with gentle stirring for 30 min. The cells were disrupted by
sonication (40 cycles of 3 s bursts) using a VCX130 ultrasonic processor
(SONICS & MATERIALS, Inc., Newtown, USA). The supernatant and
the cell debris were anaerobically separated by centrifugation at
4 °C for 60 min at 12,000 rpm. The resulting supernatant was
mixed with 30 mL of Strep-Tactin resin (IBA Lifesciences GmbH, Göttingen,
Germany) and incubated on ice for 30 min. Then, the column was washed
with washing buffer (100 mM Tris–HCl and 50 mM NaCl, pH 7.5)
until the OD₂₆₀ readout of the eluate was less than 0.05.
The FtmOx1 protein was eluted with elution buffer (2.5 mM desthiobiotin
in 100 mM Tris–HCl and 50 mM NaCl, pH 7.5). The eluted protein
was concentrated to be ∼12 mg/mL, flash-frozen with liquid
nitrogen, and stored at −80 °C. From 31 g of wet cell
paste, ∼300 mg of protein was obtained.

MamC was produced as a recombinant protein following the protocol in Valverde-Tercedor et al.9 (link) The protein was expressed in cultures of Escherichia coli TOP10 growing at 37 °C (Life Technologies, Invitrogen, Grand Island, NY, USA) and induced with isopropyl-β-d-thiogalactopyranoside (IPTG, Fisher BioReagents, Pittsburgh, PA, USA). Guanidinium lysis buffer was used to lysate the cell pellet and, after centrifugation, the supernatant was loaded onto a HiTrap chelating HP column (GE Healthcare, Chicago, IL, USA) and purified under denaturant conditions. MamC was allowed to refold by successive dialyzes. All buffers were procured from Sigma Aldrich, St. Louis, MO, USA.
BMNPs were synthetized inside an anaerobic COY chamber (Coy Laboratory Products, Grass Lake, MI) from the following master solution 2.78 mM Fe(ClO₄)₂, 3.5 mM NaHCO₃, 3.5 mM Na₂CO₃, and 5.56 mM FeCl₃, at pH 9, 10 μg/mL MamC were added. The formation of the nanoparticles occurred in free-drift experiments for 30 days at 25°C and 1 atm total pressure. The precipitates were magnetically concentrated and rinsed with oxygen-free MilliQ water four times. BMNPs were concentrated to 25 mg/mL in HEPES for synthesis 1 and in PBS for synthesis 2 (detailed below).