Beadbeater

Yeast cultures were grown in YE medium at 30°C to OD₆₀₀ = 0.8, collected by centrifugation at 3000r ≤ m at 4°C for 5 min and washed with 1x CXS buffer (50 mM HEPES, pH 7.0, 0,20 mM KCl, 1 mM MgCl2, 2 mM EDTA pH7.5 containing an anti-proteolitic tablet (Roche, Ref 05892791001). Lysates were obtained via mechanical breakage with acid-treated glass beads (Sigma), using a BeadBeater homogenizer for 10 repetitions at 4.5V of 30 s on, 30 s off on ice cycles. The samples were centrifuged at 10,000 g for 20 min at 4°C and extracts were recovered by pipetting into a new tube. Protein concentration was determined via spectroscopy using Bradford reagent. 270 μg of proteins were loaded per sample on a 10% SDS-PAGE gel and transferred via wet Western blot transfer. 1° antibodies used: α-GFP 1:1000 dilution (Mouse, Roche, Cat.No. 11814460001), α-Tubulin (TAT1, Mouse, 1:5000 dilution), 2° antibodies α-mouse (HRP detection, Promega, W4021). The mean intensity quantification of 4 independent experiments is shown in Figure 4—figure supplement 1B. Before averaging, values were corrected for the corresponding background and presented as a ratio of α-GFP to α-tubulin signal.

Stool specimens were collected in sterile containers and examined within 24 h. Stools were stored at 2 to 8°C while in transit to the laboratory. Each fresh stool specimen was aliquoted into multiple tubes. All samples were analyzed by traditional microbiological tests for known bacterial, viral, and eukaryotic pathogens. Details of these methods can be found in Panchalingam et al. [43 (link)] DNA was isolated using a bead beater with 3 mm diameter solid glass beads (sigma Life Science), and subsequently 0.1 mm zirconium beads (BIO-SPEC Inc.) to disrupt cells. The cell slurry was then centrifuged at 16,000 g for 1 min, the supernatant removed and processed using the Qiagen QIAamp® DNA stool extraction kit. Extracted DNA was precipitated with 3 M sodium acetate and ethanol and the DNA shipped to the USA.