Costar 96 well assay plate black plate with clear bottom

For measurements of GFP fluorescence, cultures were inoculated from washed overnight cultures in CDM medium at a 1:50 dilution. Inoculated medium (175 μL) was added to each well along with a 50 μL mineral oil overlay in a Costar^™ 96 well assay plate (black plate with clear bottom; Corning Incorporated) and incubated at 37°C. At intervals of 30 minutes for a total of 18 hours, OD₆₀₀ along with GFP fluorescence (excitation 485/20 nm, emission 528/20 nm) was measured with a Synergy 2 multimode microplate reader (BioTek). Relative expression was calculated by subtracting the background fluorescence of UA159 (mean from six replicates) from raw fluorescence units of the reporter strains and then dividing by OD_600.

Overnight cultures of selected S. mutans strains, grown in CDM medium with addition of a final concentration of 10 μM of synthetic XrpA peptides when noted, where measured for a final OD₆₀₀ and then harvested by centrifugation for their supernatant fraction. 5 mL of the resulting supernatant was then run through QIAGEN (Chatsworth, Calif.) PCR purification columns to capture eDNA present. The eDNA was then eluted off the column with 600 μL water, and 594 μL of this elution was mixed with 5 μL of 50 μM Sytox Green (Invitrogen) to a final concentration of 0.5 μM. After vortexing the solution and incubation for 15 minutes in the dark at room temperature, 200 μL of the stained samples were transferred into a Costar^™ 96 well assay plate (black plate with clear bottom; Corning Incorporated). Fluorescence (excitation 485/20 nm, emission 528/20 nm) was measured with a Synergy 2 multimode microplate reader (BioTek) and the resulting data was then normalized for the measured final OD ₆₀₀ nm resulting in a final arbitrary eDNA release measurement. The data represents 3 independent biological replicates with 3 technical replicates each. Statistical significance was determined by the Student’s T-Test.

For measurements of GFP fluorescence, cells were grown overnight, washed and diluted 1:50 into fresh FMC medium, with or without 2 μM sXIP. Two hundred microliters of the dilution was then loaded on a Costar™ 96-well assay plate (black plate with clear bottom; Corning Incorporated) and incubated at 37°C in a 5% CO₂ aerobic atmosphere. When cells reached an OD₆₀₀ = 0.5, the OD₆₀₀ and fluorescence (excitation 485/20 nm, emission 528/20 nm) were measured with a Synergy 2 multimode microplate reader (BioTek). Relative expression was calculated by subtracting the background fluorescence of UA159 (mean from six replicates) from raw fluorescence units of the reporter strains and then dividing by OD_600.