*2.12. Real-Time PCR*

To further analyze the potential antimicrobial effect of *B. subtilis* grown in MMRS medium, we tested the expression of genes that could be affected during mitigating biofilm formation by *S. aureus*. A starter culture of *B. subtilis* was prepared during overnight growth at 30 ◦C, 150 rpm, in LB medium. For generating the antimicrobial substance producing a suspension of *B. subtilis*, a portion of starter culture was introduced (by 1:100 ratio) into either MMRS or LB medium (as a control medium for a low antimicrobial substance production). The samples were incubated for 6 h at 37 ◦C, 50 rpm. Next, 2 mL from each sample was collected and centrifuged at 5000× *g* for 10 min. The RNA was harvested using the RNAeasy kit (QIAGEN, Hilden, Germany) following the manufacturer's protocol. The RNA concentration was measured by means of a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). A complementary DNA (cDNA) was synthesized from 1 μg of RNA in a reverse transcription reaction using a qScript cDNA Synthesis Kit (Quantabio, Beverly, MA, USA) according to the manufacturer's instructions. All cDNA samples were stored at −20 ◦C. The RT-PCR reactions (final volume = 20.0 μL) consisted of 2 μL of cDNA template, 10 μL of fast SYBR green master mix, 1 μL of suspension of each primer, and 7 μL of RNase free water. Forward and reverse PCR primers (Table S2, Supplementary Materials) were designed using the Primer express software and were synthesized by Hylabs (Rehovot, Israel). DNA was amplified with the Applied Biosystems StepOne™ Real-Time PCR System (Life technologies, Foster, CA, USA) under the following PCR conditions: initial denaturation for 2 min at 95 ◦C and subsequent 40 PCR cycles (95 ◦C for 3 s, 60 ◦C for 30 s, and 95 ◦C for 15 s). The RNA samples without reverse transcriptase were used as negative control, to confirm that there was no DNA contamination in the RNA samples. The expression levels of the tested genes (*fenA, srfA*) were normalized using the 16S ribosomal RNA (rRNA) and *rpoB* genes as the endogenous controls (Table S2, Supplementary Materials).
