Carbon sources screening assay

<p><span>The FACS optimized GFPmut3 </span>^{<span>69</span>}<span> was fused to promoter of interest and cloned into a plasmid pSC101. <a name="_Hlk156916219"></a><span>For the first screening, </span>overnight cultures of the strain carrying the screening system were diluted 200X in MOPS Rich <span>(Teknova EZ rich defined medium) supplemented with carbenicillin for plasmid maintenance. </span><span>The phenotype Microarray (Biolog) plates PM1 and PM2B (carbon sources), and PM3B, for a total of 198 molecules including 7 nucleosides and 6 nucleotides were used for molecules screening. Each well was filled with 100 &micro;l of inoculated media and mixed by pipetting. Media were transferred to 96 well dark-bottom plates (Thermo Scientific). GFP fluorescence was followed on the Tecan Infinite 200 PRO (Life Science) at 37&deg;C for 8 hours. Fluorescence induction by the substrate was calculated using the ratio </span>fluorescence (t8h-t0h) over growth (t8h-t0h OD_600nm).</span></p> <p><span>For flow cytometry quantification, </span><span>overnight cultures in MH of strain carrying the screening system were diluted 200X in rich MOPS <span>(Teknova EZ rich defined medium), or MH supplemented with carbenicillin for plasmid maintenance<span> and grown overnight, and the molecule tested at 0.5% (except uracil: 0.1%, limit of solubility). Fluorescence was read on 5 &micro;l of overnight cultures diluted in 200 &micro;l of PBS with the B1 laser. </span></span></span></p>