Supragingival plaque from the maxillary molars of four healthy volunteers was collected about 24 hours after tooth brushing. The plaque was washed with prereduced sterile PBS and combined for overnight growth in Shi medium (56) at 37°C anaerobically (10% H2, 10% CO2, and 80% N2). The complex microbial community was frozen in 10% glycerol and served as the stock for our biofilm integration experiments. For biofilm integration, the ex vivo plaque community was grown overnight in Shi medium and diluted to an absorbance of 0.1 (λ = 600 nm) in fresh Shi medium. S. gordonii strains containing the JHMD1 cassette were also grown to an absorbance of 0.1 and 2 μL were inoculated into 2 mL of the plaque community. The inoculated culture was grown overnight using saliva-coated 12-well polystyrene culture plates (Corning). The planktonic cells were aspirated, and the surface-attached biofilm was washed once with 500 μl of pre-reduced sterile PBS. Biofilms that remained attached to the wells were collected for DNA isolation with DNeasy PowerLyzer PowerSoil Kit (Qiagen). S. gordonii presence in the ex vivo community was determined by qPCR amplification of the JHMD1 cassette with primer pairs pheSermAM Fwd and Rev (table S4) and normalized to total 16S ribosomal RNA gene DNA, which was amplified with primer pair (BAC1 and BAC2) (56). The ΔΔCt method was used to calculate relative abundance.

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