The supporting online material provides detailed methods and experimental procedures for constructing and analyzing microcolony strains to study the regulation of gene expression by the λ-cascade system. The Cl-YFP fusion protein was constructed using PCR and expressed from a tightly regulated promoter, allowing for the exploration of the full dynamic range of CFP regulation. The O62*-λ-cascade strain was created through site-directed mutagenesis of the P_R promoter, and the symmetric branch strain (MRR) contains CFP and YFP at separate but equivalent loci, each under wild-type P_R promoters. Cultures were grown and induced with anhydrotetracycline (aTc) to control expression, and microcolonies were observed using fluorescence microscopy. Custom software was developed to analyze time-lapse movie data, including segmentation, tracking, and background subtraction. The analysis of cellular fluorescence values and cell division events confirmed negligible protein degradation and photo-bleaching. The distribution of protein production rates was found to be log-normally distributed, and the autocorrelation function of production rates was analyzed, revealing intrinsic noise with an autocorrelation time of less than 10 minutes. The local microenvironment had little effect on the gene expression dynamics, and the production rates were normalized by the cell cycle phase to reduce variability. The regulator dilution method was used to measure repression cooperativity without fluorescent protein fusions, and the apparent CI-YFP concentration was calibrated to determine the concentration of repressor in cells and production rates.The supporting online material provides detailed methods and experimental procedures for constructing and analyzing microcolony strains to study the regulation of gene expression by the λ-cascade system. The Cl-YFP fusion protein was constructed using PCR and expressed from a tightly regulated promoter, allowing for the exploration of the full dynamic range of CFP regulation. The O62*-λ-cascade strain was created through site-directed mutagenesis of the P_R promoter, and the symmetric branch strain (MRR) contains CFP and YFP at separate but equivalent loci, each under wild-type P_R promoters. Cultures were grown and induced with anhydrotetracycline (aTc) to control expression, and microcolonies were observed using fluorescence microscopy. Custom software was developed to analyze time-lapse movie data, including segmentation, tracking, and background subtraction. The analysis of cellular fluorescence values and cell division events confirmed negligible protein degradation and photo-bleaching. The distribution of protein production rates was found to be log-normally distributed, and the autocorrelation function of production rates was analyzed, revealing intrinsic noise with an autocorrelation time of less than 10 minutes. The local microenvironment had little effect on the gene expression dynamics, and the production rates were normalized by the cell cycle phase to reduce variability. The regulator dilution method was used to measure repression cooperativity without fluorescent protein fusions, and the apparent CI-YFP concentration was calibrated to determine the concentration of repressor in cells and production rates.