Figure 1. Genetic implementation of the Universal Bacterial Expression Resource for cross-species engineering.

(a) The Universal Bacterial Expression Resource (UBER) consists of a mixed feedback loop (MFL) composed of two interlinked transcriptional feedback loops. The T7 RNA polymerase (T7 RNAP) drives an auto-activating positive feedback loop (PFL), while the TetR repressor controls the negative feedback loop (NFL). A core component of UBER is the T7 RNAP cassette that consists of a priming promoter (pr) for basal leaky transcription, a T7-TetO pr (T7 pr with flanking TetO operator sites) for controlling the PFL, a tuneable cross-species RBS, the T7 RNAP-coding sequence, and a set of intrinsic and T7 terminator sequences. The other core component is the TetR cassette that consists of a T7 pr, a tuneable cross-species RBS, the TetR-coding sequence and a set of intrinsic and T7 terminator sequences. The cross-species RBS is designed computationally to have similar translation rates in both Gram-negative and Gram-positive bacteria. The T7 RNAP produced in the system is used to drive the transcription of an output gene or operon of interest. RBS translation initiation rates were calculated using the RBS Calculator v2.0. (b) UBER achieves cross-species expression of a GFP reporter protein or a multi-enzyme pathway in (Bs) Bacillus subtilis, (Pp) Pseudomonas putida and (Ec) Escherichia coli. Here the enzymes CrtEBI synthesize the terpenoid neurosporene. The translation rates of the T7 RNAP and TetR ribosome-binding sites are shown alongside the copy numbers of the UBER and output modules. GFP fluorescence points and bars are the mean and s.d. of three measurements. Neurosporene content points and bars are the mean and s.d. of two measurements.