Simultaneous Visualization of Multiple Gene Expression in Single Cells Using an Engineered Multicolor Reporter Toolbox and Approach of Spectral Crosstalk Correction

Synthetic biology aims to make biology easier to engineer and focuses on the design and construction of core components that can be modeled, understood, and tuned to meet specific performance criteria, and the assembly of these smaller parts and devices into larger integrated systems to solve specific problems. Here, we designed and engineered a multicolor fluorescent reporter toolbox to simultaneously monitor the activities of multiple genes in single cells. The toolbox contained standardized and well-characterized genetic building blocks for the convenient and reproducible assembly of multiple promoter–reporter fusions (ranging from 1 to 4) into a single plasmid. Given the common problem of spectral crosstalk among multiple fluorescent proteins, we deciphered multiple spectral signatures within cells through a deduced linear unmixing algorithm. Our approach enabled the quantification of gene expression with direct FP concentrations, instead of mix-contributed fluorescence intensities, thus enabling true signal separation with high confidence. This approach performed well in the imaging of mixing cells with single FP labels. Additionally, combining with the multicolor toolbox, we succeeded in simultaneously monitoring the genetic dynamics of four selected quorum-sensing genes in response to the induction of two exogenously added autoinducers and were able to examine gene regulatory connections within the QS signaling network in Pseudomonas aeruginosa. Overall, this synthetic framework (i.e., the genetic toolbox and the well-evaluated approach of spectral correction) will be useful for applied synthetic biology projects, multicolor imaging, and analyzing interactions of multiple genes of natural genetic networks or assembling synthetic ones.