Aoki, S. K. | bioRxiv Science

bioRxiv2017

A synthetic integral feedback controller for robust tunable regulation in bacteria

We report on the first engineered integral feedback control system in a living cell. The controller is based on the recently published antithetic integral feedback motif [1] which has been analytically shown to have favorable regulation properties. It is implemented along with test circuitry in Escherichia coli using seven genes and three small-molecule inducers. The closed-loop system is highly tunable, allowing a regulated protein of interest to be driven to a desired level and maintained there with precision. Realized using a sigma/anti-sigma protein pair, the integral controller ensures that regulation is maintained in the face of perturbations that lead to the regulated proteins degradation, thus serving as a proof-of-concept prototype of integral feedback implementation in living cells. When suitably optimized, this integral controller may be utilized as a general-purpose robust regulator for genetic circuits with unknown or partially-known topologies and parameters.

synthetic biology↗

bioRxiv2017

Dynamic blue light-inducible T7 RNA polymerases (Opto-T7RNAPs) for precise spatiotemporal gene expression control

Light has emerged as control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark-state, high expression strength when induced with blue light, or an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we have created a variant, which returns to the inactive dark-state within minutes, once blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. However, minor alterations should be sufficient to allow their use in other species in which the T7 RNAP polymerase and the light-inducible Vivid regulator were shown to be functional, which comprises other bacterial species and eukaryotes such as mammalian cells or yeast. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation, and allow for a more reliable dynamic control of synthetic and natural gene networks.

synthetic biology↗