Tween 80

The construction of the droplet flow reactor has been previously reported in detail^{34 (link),35 (link)}. Briefly, this system is composed of two tanks (505 μL), the inside solutions of which were mixed by magnetic stirrers (Fig. 1). The tanks were attached to electromagnetic coils to control the stirring intensity. The oil phase including mineral oil (Sigma) and surfactants (2% Span 80 (v/v, Wako) and 3% Tween 80 (v/v, Wako)) and the aqueous phase including the E.coli translation system were supplied into the first tank with two syringe pumps. The oil phase was used after saturation with some solutes as described previously¹⁷ . In the first tank, droplets were prepared at 2 °C and supplied to the second tank. In the second tank, droplets contain the genomic and parasitic RNAs and fusions and divisions among the droplets were induced by mixing. Droplets in the second tank were removed at the same rate as the supply rate (dilution rate). The mixing intensities were controlled by changing the spinning period of the magnetic stirrers in the tanks. The details of the droplet flow reactor are shown in Fig. S6.

As host cells, we used two strains of E. coli, namely JM109 (Yanisch-Perron et al., 1985 (link)) for the construction of plasmids and Rosetta^TM 2 (Merck Millipore, Darmstadt, Germany) for the expression of the target genes. The cells were grown in 2 mL of Luria–Bertani (LB) medium (Bertani, 1951 (link)) at 37°C with shaking at 180 rpm. All transformants were maintained in LB medium solidified with 1.5% (w/v) Bacto^® agar (BD Biosciences Japan, Tokyo, Japan) in the presence of 100 μg/mL sodium ampicillin, 50 μg/mL chloramphenicol, or 50 μg/mL spectinomycin dihydrochloride pentahydrate, depending on the selection markers on the plasmids. To exogenously supply fatty acids to the culture of E. coli cells, 1 mM of each sodium salt of palmitoleic acid (16:1Δ9, Wako Pure Chemicals, Osaka, Japan), 18:1Δ9 (Tokyo Chemical Industry, Tokyo, Japan), linoleic acid (18:2Δ9,12, Funakoshi, Tokyo, Japan), γ-linolenic acid (18:3Δ6,9.12, Sigma-Aldrich Japan, Tokyo, Japan), α-linolenic acid (18:3Δ9,12,15, Funakoshi), or vaccenic acid (18:1Δ11, Sigma-Aldrich Japan) was added to the liquid LB medium. Corynebacterium urealyticum ATCC 43042 was grown on R agar¹ with 0.5% (v/v) TWEEN 80 (Wako Pure Chemicals) and incubated at 37°C for 18 h.

The numbers of viable C. difficile, C. butyricum, and Bacteroides in 1 g of feces were quantified by culture method. The feces were suspended and diluted in phosphate buffer containing 0.05% L-cystain hydrochloride hydrate (Sigma–Aldrich, St. Louis, MO, United States) and 0.05% Tween80 (Wako Pure Chemicals Industries, Ltd.), and 50 μL of appropriate diluent was spread onto agar medium and cultured. CCFA agar (OXOID, Hampshire, United Kingdom) supplemented with 0.1% sodium taurocholate (Wako Pure Chemicals Industries, Ltd.), C. butyricum-selective medium (Sato and Tanaka, 1997 (link)), and NBGT agar (Mitsuoka, 1978 ) were used for selective counting for C. difficile, C. butyricum, and Bacteroides, respectively. An anaerobic chamber (10% CO₂, 10% H₂, and N₂ to balance) was used for anaerobic manipulation and cultivation.

After the olfactory bulb and cerebellum were cut from the extracted brain, the brain was separated into the left brain and the right brain. Next, 5 mL/g tissue of lysis buffer was added to the left brain. Lysis buffer was composed by Ethylenediamine-N,N,N’,N’-tetra acetic acid (EDTA), disodium salt di-hydrated (DOJINDO laboratories, Kumamoto, Japan), Triton×100 (Sigma-Aldrich Co., Milwaukee, WI, USA), Tween 80 (FUJIFILM Wako Chemicals, Osaka, Japan), NaCl (FUJIFILM Wako Chemicals, Osaka, Japan), and Tris-HCl (pH 7.5) (Nippon gene, Tokyo, Japan) in distilled water. The left brain was cut into fine pieces with scissors and homogenized at 10,000 rpm for 3 min in a homogenizer (polytron^® PT3100, Kinematica, A.A.; Luzern, Switzerland). The homogenized liquid was fractioned into a 1-mL tube, which was then centrifuged for 30 min at 12,000 rpm. Then, 400 μL of the supernatant was used as a sample solution to determine the concentration of TNF-α. The sample was stored at −20 °C until measurement. TNF-α concentration was measured in accordance with the protocol of the ELISA kit. The limit of detection range for rat TNF-α ELISA kit was 12.5–800 pg/mL.