The DYNOME project tackles the challenging task of monitoring gene-expression dynamics in individual bacterial cells at single-molecule resolution. Its goal is to detect the expression of single fluorescent molecules in bacteria and to use the resulting expression trajectories to extract kinetic rates for the underlying gene-expression steps—such as transcription and translation—across large cell populations.To achieve this goal, my group and I designed and built a dedicated super-resolution microscope. Standard instruments, including TIRF (total internal reflection fluorescence) microscopes, lack the long-term stability required to maintain nanometer-level precision over many hours. In addition, our system needed to image cells and track individual fluorescent molecules simultaneously, enabling us to follow gene expression in moving and potentially dividing cells.
Over the course of the project, we overcame numerous technical challenges and completed the DYNOME microscope in several phases. The first version was finished early and initial experiments yielded promising gene-expression data in bacterial cells. However, technical challenges in later funding periods necessitated major upgrades to ensure robust a performance of the system under our experimental conditions. The system is now in its final form, and new experiments with genetically engineered E. coli are currently underway.
Beyond the construction of the microscope, the DYNOME project also required the development of suitable bacterial model systems. To this end, we created a library of E. coli strains that chromosomally express two spectrally distinct fluorescent proteins. These proteins are produced either independently or in a partially coupled manner (by fusions at the mRNA or protein level), a design that will allow us to disentangle the respective contributions of transcriptional and translational noise.
Within the scope of DYNOME, we also developed a new analytical framework for photon-counting experiments (Terterov et al., 2025, Nature Communications, 16, 5537). The DYNOME project is built on the analogy between photon counting in in-vitro single-molecule fluorescence experiments and molecule counting inside living cells, and we anticipate that this new method will facilitate the computation of bias-free correlation functions in our in-cell measurements.
