Fig. 1: Shuffling upstream promoter elements (UPEs) and terminators by Cre-LoxPsym recombination for in vivo expression diversification.
a The gene expression modulator (GEM) design. 5’ and 3’ GEM consist of an array of UPEs and terminators, respectively, separated by LoxPsym sites. Different promoter and terminator parts have different strengths (color scale). Upon induction of Cre recombination, blocks in each GEM are shuffled, resulting in a pool of GEM variants that differently influence expression of the gene of interest (GOI). Different, orthogonal LoxPsym sites restrict recombination to one GEM module. b Inducing recombination of GEMs generates a cell population with diversified GOI expression levels (different colors). c Effect of introducing LoxPsym (5’-ATAACTTCGTATATTATATAATATACGAAGTTAT-3’) at the TDH3 promoter on yECitrine fluorescence. LoxPsym was placed either directly upstream of the start codon (ATG), transcription start site (TSS) or TATA box, each time in combination with a LoxPsym site upstream of the promoter region. The strength of the native promoter (yellow) and LoxPsym-following core promoter (pink) was also measured. Constructs were tested in combination with terminator CYC1 and integrated at the CAN1 locus of the laboratory strain BY4741-mCherry (the control strain, gray). Histograms represent concatenated populations of three biological repeats. Statistics by analysis of variance and two-sided Tukey multiple comparisons of means (exact p-values in Supplementary Data 1, p values LoxPsym position). Colors of p values indicate the population used for comparison (‘***p < 0.001, ‘ns’ p > 0.1). d yECitrine fluorescence versus mRNA abundance, measured via qPCR (ΔCT value). Dots represent average of three biological replicates, colors correspond to (c). Horizontal and vertical error bars represent standard error and standard deviation, respectively. e Effect of flanking a yeast terminator (HIS5 (purple) and CYC1 (blue)) with LoxPsym on yECitrine fluorescence. Constructs were tested in combination with promoter TPI1 and integrated at the CAN1 locus of the laboratory strain BY4741-mCherry. Histograms show concatenated data of three biological repeats. Statistics similar to (c). f Normalized fluorescence versus mRNA abundance of yECitrine, colors correspond to (e), dots and error bars similar as (c). Source data for this figure are provided as a Source Data file.
