Integrase-mediated differentiation circuits improve evolutionary stability of burdensome and toxic functions in E. coli

Abstract

Application of synthetic biology is limited by the capacity of cells to faithfully execute burdensome engineered functions in the face of Darwinian evolution. Division of labor, both metabolic and reproductive, are underutilized in confronting this barrier. To address this, we developed a serine-integrase based differentiation circuit that allows control of the population composition through tuning of the differentiation rate and number of cell divisions differentiated cells can undergo. We applied this system to T7 RNAP-driven expression of a fluorescent protein, and demonstrate both increased duration of circuit function and total production for high burden expression. While T7 expression systems are typically used for high-level short-term expression, this system enables longer duration production, and could be readily applied to burdensome or toxic products not readily produced in bacteria.

Dynamic cybergenetic control of bacterial co-culture composition via optogenetic feedback

“…However, our method still suffered from the eventual fixation of escape mutations after around 80 generations, which limits the time window in which we can maintain compositional control over the community. Loss-offunction mutations are inevitable in circuits that reduce the fitness of the host, although some strategies have been recently proposed for improving genetic stability and delaying the onset of mutations 63,64 . However, it is worth noting that the use of a real-time feedback can, in principle, prolong the stability of the system by adapting the lightinput profile to counteract the effect of certain mutations, a behavior we likely observe in our closed-loop experiments after around 35 h (Fig.…”

Dynamic cybergenetic control of bacterial co-culture composition via optogenetic feedback

“…However, our method still suffered from the eventual fixation of escape mutations after around 80 generations, which limits the time window in which we can maintain compositional control over the community. Loss-of-function mutations are inevitable in circuits that reduce the fitness of the host, although some strategies have been recently proposed for improving genetic stability and delaying the onset of mutations (56,57).…”

“…However, our method still suffered from the eventual fixation of escape mutations after around 80 generations, which limits the time window in which we can maintain compositional control over the community. Loss-of-function mutations are inevitable in circuits that reduce the fitness of the host, although some strategies have been recently proposed for improving genetic stability and delaying the onset of mutations(56, 57). However, it is worth noting that the use of a real-time feedback can, in principle, prolong the stability of the system by adapting the light-input profile to counteract the effect of certain mutations, a behavior we likely observe in our closed-loop experiments after around 35h (Figures 6C-D, S14, S15).…”