Cell-cycle-synchronized, oscillatory expression of a negatively autoregulated gene in E. coli

Engineering genetic networks to be both predictable and robust is a key challenge in synthetic biology. Synthetic circuits must reliably function in dynamic, stochastic and heterogeneous environments 1 , and simple circuits can be studied to refine complex gene-­‐regulation models 2-­‐5. Although robust behaviours such as genetic oscillators have been designed and implemented in prokaryotic 6 and eukaryotic 7 organisms, a priori genetic engineering of even simple networks remains difficult, and many aspects of cell BLOCKIN BLOCKIN and BLOCKIN BLOCKIN molecular BLOCKIN BLOCKIN biology BLOCKIN BLOCKIN critical BLOCKIN BLOCKIN to BLOCKIN BLOCKIN engineering BLOCKIN BLOCKIN robust BLOCKIN BLOCKIN networks BLOCKIN BLOCKIN are BLOCKIN BLOCKIN still inadequately characterized. Particularly, periodic processes such as gene doubling and cell division are rarely considered in gene regulatory models, which may become more important as synthetic biologists utilize new tools for chromosome integration 8,9. We studied a chromosome-­‐integrated, negative-­‐feedback BLOCKIN BLOCKIN circuit BLOCKIN BLOCKIN based BLOCKIN BLOCKIN upon BLOCKIN BLOCKIN the BLOCKIN BLOCKIN bacteriophage BLOCKIN BLOCKIN λ BLOCKIN BLOCKIN transcriptional repressor Cro and observed strong, feedback-­‐dependent oscillations in single-­‐cell time traces. This finding was surprising due to a lack of cooperativity, long delays or fast protein degradation 10. We further show that oscillations are synchronized to the cell cycle by gene duplication, with phase shifts predictably correlating with estimated gene doubling times. Furthermore, we characterized the influence of negative feedback on the magnitude and dynamics of noise in gene expression. Our results show that cell-­‐cycle effects must be accounted for in accurate, predictive models for even simple gene circuits. Cell-­‐cycle-­‐periodic expression of λ Cro also suggests BLOCKIN