add multifluidics assay and protocol

assays/MillifluidicsExperiment/protocols/MillifluidicsExperimentProtocol.md

+## Millifluidics experiment
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+Bacterial incubation in root exudates was performed in a millifluidics system (MilliDrop Analyzer, MilliDrop, www.millidrop.com). This drop-based system allows incubation of bacteria in very small volumes of root exudates or growth medium. In brief, bacteria and exudates or growth medium are combined in wells of a 96-well plate using a pipetting robot Freedom Evo 100 (Tecan). Droplets of approximately 100–200 nl are then sucked in from the wells of the loading plate by a tip on the robotic arm of the MilliDrop Analyzer, generating hundreds of droplets within an oil-filled tube, separated by air spacers. During incubation, the droplet ‘train’ moves back and forth, so that during each round, each droplet passes a detector that counts the droplets. Culture droplets are collected after the experiment and subjected to community profiling.
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+The mixed community *LjAt*-SC1 was used and was essentially prepared as described above for the in planta experiments, adjusted to OD600 of 0.1 and used as input for preparation of the loading plate. Pure exudates (pH between 7.0 and 8.0) or a defined M9 + carbon growth medium (1× M9 salts including phosphate buffer, 1 mM magnesium sulfate, 0.3 mM calcium chloride, 1× vitamin B solution and artificial root exudates, pH 7.0) was used for incubation. Vitamin B solution contained 0.4 mg l−1 4-aminobenzoic acid, 1 mg l−1 nicotinic acid, 0.5 mg l−1 calcium-d-pantothenate, 1.5 mg l−1 pyridoxine hydrochloride, 1 mg l−1 thiamine hydrochloride, 0.1 mg l−1 biotin and 0.1 mg l−1 folic acid (modified from Pfennig, 1978). Artificial root exudates (modified from Baudoin, E. et al., 2003) were composed of 0.9 mM glucose, 0.9 mM fructose, 0.2 mM sucrose, 0.8 mM succinic acid, 0.6 mM sodium lactate, 0.3 mM citric acid, 0.9 mM serine, 0.9 mM alanine and 0.5 mM glutamic acid. Bacteria were incubated for 3 d, during which the pH of the cultures stayed stable. Droplets were collected in 6-µl amounts, and DNA isolated via quick alkaline lysis (Bai, Y. et al., 2015), which consisted of addition of 10 µl of buffer 1 (25 mM NaOH, 0.2 mM EDTA, pH 12), incubation at 95 °C for 30 min, addition of 10 µl of buffer 2 (40 mM Tris-HCl at pH 7.5) and storage at −20 °C.
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