Formvar carbon 200 mesh grids

Cells expressing ARGs, or purified GVs, were exchanged into water or 10 mM HEPES pH8.0 with 150 mM NaCl, respectively, via 3 rounds of buoyancy purification and buffer exchange as described above. Samples were deposited on Formvar/carbon 200 mesh grids (Ted Pella) that were rendered hydrophilic by glow discharging (Emitek K100X). For purified GVs, 2% uranyl acetate was added for staining. The samples were then imaged on a FEI Tecnai T12 transmission electron microscope equipped with a Gatan Ultrascan CCD. Images were processed with FIJI ³⁵ .

Cells expressing ARGs were placed into 10 mM HEPES pH 8.0 following exposure to ultrasound with the same protocol described above for the bacteria cell viability experiments, except that the agarose was cast with 10 mM HEPES pH 8.0 instead of PBS. Samples were deposited on Formvar-carbon 200 mesh grids (Ted Pella) that were rendered hydrophilic by glow discharging (Emitek K100X). 2% uranyl acetate was added for staining. The samples were then imaged on a FEI Tecnai T12 transmission electron microscope equipped with a Gatan Ultrascan CCD.

GV samples were diluted to OD₅₀₀ ∼ 0.2 in 10 mM HEPES buffer and spotted on Formvar/Carbon 200 mesh grids (Ted Pella, Redding, CA), which were rendered hydrophilic by glow discharging (Emitek K100X). Unclustered Mega GVs were negatively stained using 2% uranyl acetate. Images were acquired using the Tecnai T12 LaB6 120 kV transmission electron microscope (TEM) equipped with a Gatan Ultrascan 2k × 2k CCD camera.

GVs at OD_500,PS ~ 0.2 were prepared in a buffer of 10 mM HEPES, 150 mM NaCl (pH 8) and spotted on Formvar/Carbon 200 mesh grids (Ted Pella) that were rendered hydrophilic by glow discharging (Emitek K100X). GV samples were negatively stained using 2% uranyl acetate. Images were acquired using a Tecnai T12 LaB6 120kV TEM.

The protocols to express and purify GVs were described previously.^{72 (link)} Briefly, among the three genotypes of GVs used in this study, Ana GVs and Halo GVs were obtained from their native hosts, Anabaena flos-aquae (CCAP strain 1403/13 F) and Halobacteria NRC-1 (Carolina Biological Supply), respectively, and Mega GVs were obtained by heterologously expressing the GV genes from Bacillus megaterium in E. coli. Following the harvest and lysis of the cells, GVs were purified through multiple rounds of centrifugally assisted floatation. Mega GVs, which are natively clustered after purification from bacteria, underwent additional steps of unclustering by 6M urea treatment, centrifugally assisted floatation and dialysis. The concentration of GVs was quantified by the pressure-sensitive optical density at 500-nm wavelength (OD_500,PS). OD_500,PS was then converted to the molar concentration using the relation of 114, 47.3 and 2030 pM/OD_500,PS for Ana, Halo and Mega GVs, respectively (Supplementary Table 1). For TEM images, GVs were diluted to OD_{500, PS} ~ 0.2 and then spotted on Formvar/Carbon 200 mesh grids (Ted Pella) that were rendered hydrophilic by glow discharging (Emitek K100X). After negative staining using 2% uranyl acetate, the samples were imaged on a Tecnai T12 LaB6 120 kV TEM.