A
volume of 3 mL of BL21 cells containing either a metalloid reductase
gene or GFP reporter gene was grown separately in 10 mL culture tubes
overnight containing LB medium (Teknova) supplemented with Kanamycin
at 25 μg/mL. The following morning, the culture was added to
a 125 mL Erlenmeyer flask containing LB medium supplemented with Kanamycin
(25 μg/mL). The cells were grown for 2.5 h, and 100 mM Na2SeO3 (Alfa Aesar, 98+%) was added to reach a final
concentration of 5 mM. The cells were collected by centrifuging for
20 min at 4000 rpm and 4 °C after 3 h of growth with selenite.
The cells were washed with 20 mM Tris (pH 7.4) (Fischer) three times
followed by resuspension in 1 mL of fixing solution (2% glutaraldehyde
(25% Sigma-Aldrich) and 2.5% formaldehyde); the fixing solution was
allowed to react for 12 h at 4 °C. The fixing solution was centrifuged
and the pellet was washed five times in 20 mM Tris (pH 7.4). The cells
were resuspended in 1 mL of 20 mM Tris (pH 7.4). The aliquots (4 μL)
were mounted on 400 mesh Cu grids with 50 nm C coating and washed
two times with H2O. The dry-mounted cells on transmission
electron microscopy grids were loaded onto a STEM holder. The STEM
images were taken with a JEOL JSM-6500-F scanning electron microscope
at an accelerating voltage of 15 kV.
volume of 3 mL of BL21 cells containing either a metalloid reductase
gene or GFP reporter gene was grown separately in 10 mL culture tubes
overnight containing LB medium (Teknova) supplemented with Kanamycin
at 25 μg/mL. The following morning, the culture was added to
a 125 mL Erlenmeyer flask containing LB medium supplemented with Kanamycin
(25 μg/mL). The cells were grown for 2.5 h, and 100 mM Na2SeO3 (Alfa Aesar, 98+%) was added to reach a final
concentration of 5 mM. The cells were collected by centrifuging for
20 min at 4000 rpm and 4 °C after 3 h of growth with selenite.
The cells were washed with 20 mM Tris (pH 7.4) (Fischer) three times
followed by resuspension in 1 mL of fixing solution (2% glutaraldehyde
(25% Sigma-Aldrich) and 2.5% formaldehyde); the fixing solution was
allowed to react for 12 h at 4 °C. The fixing solution was centrifuged
and the pellet was washed five times in 20 mM Tris (pH 7.4). The cells
were resuspended in 1 mL of 20 mM Tris (pH 7.4). The aliquots (4 μL)
were mounted on 400 mesh Cu grids with 50 nm C coating and washed
two times with H2O. The dry-mounted cells on transmission
electron microscopy grids were loaded onto a STEM holder. The STEM
images were taken with a JEOL JSM-6500-F scanning electron microscope
at an accelerating voltage of 15 kV.