REINFORCED CLOSTRIDIAL MEDIUM (RCM)

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	-The bacterial growth media were supplemented with erythromycin (100 µg ml−1), chloramphenicol (20 µg ml−1) or colisitin sulfate (20 µg ml−1) as appropriate. -pH 6.8 ± 0.2.

Protocol tips
-The bacterial growth media were supplemented with erythromycin (100 µg ml−1), chloramphenicol (20 µg ml−1) or colisitin sulfate (20 µg ml−1) as appropriate. -pH 6.8 ± 0.2.

Publication protocol

The recipient cultures for MPM were prepared by inoculating Brain Heart Infusion (BHI, Difco™) supplemented with colistin sulfate with a raw stool sample collected from a healthy 2-year-old female child. The donor had not taken antibiotics during the 3-month period prior to collection. The child was recruited as part of a study into the link between the gut microbiota and type 1 diabetes susceptibility. All study samples were collected in accordance with the recommendations of the Mater Health Services Human Research Ethics Committee (HREC/13/MHS/21/AM02). All subjects gave written informed consent in accordance with the Declaration of Helsinki, with written consent provided from parents or legal guardians for all subjects <13 years. The protocol was approved by the Mater Health Services HREC. E. faecalis was cultured in BHI and the Escherichia coli ST18 donor strain for MPM was cultured in BHI supplemented with δ-aminolevulinic acid (100 µg ml−1). The E. coli cloning strains were grown in LB and F. prausnitzii A2-165 was cultured in anaerobic Reinforced Clostridial Medium (RCM, Oxoid™) buffered with salt solutions 2 and 3 (18). F. prausnitzii cultures were routinely manipulated in a Coy vinyl anaerobic chamber with an oxygen free atmosphere (85% N2:10% CO2:5% H2). Both E. coli ST18 and JM109 competent cells were prepared by the rubidium chloride method (19) while Invitrogen™ E. coli DH5 α competent cells were purchased from ThermoFisher Scientific. The bacterial growth media were supplemented with erythromycin (100 µg ml−1), chloramphenicol (20 µg ml−1) or colisitin sulfate (20 µg ml−1) as appropriate.

Bacterial growth was measured by the increase in optical density at 600 nm (OD600 nm). Specific growth rates [μ (h−1)] were calculated by log10 transformation of the OD600 nm measurements and plotting a trendline (R2 > 0.97) for the linear phase of growth corresponding to exponential (EX) growth phase. Then μ was calculated using the equation: μ = (slope of the line × 2.3).

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