From Bacillus Subtilis Gene Expression Decomposition to Synthetic Biology

We aimed at decomposing gene expression into distinct and well characterized genetic elements (gene location, TATA box, transcription start, translation initiation region, etc.) that could be rationally assembled to control gene expression across growth conditions. To address this issue, we revisited the contribution of replication, transcription and translation to global mechanisms allowing bacteria to modulate abundance of single proteins with respect to the growth rate. Growth rate has long been regarded as a global variable modulating macromolecular cellular contents but the growth-rate-dependent variation of the transcription machinery abundance was recently shown to clearly influence gene expression.

Regarding translation, we demonstrated that translation efficiency strongly depends on growth rate and that cells can differentially translate individual transcripts as function of the growth rate without dedicated regulators. The mechanism underpinning this translational regulation relies on the selective titration of the free (untranslating) ribosome fraction by the transcript-specific translation initiation regions. We identified this mechanism by developing a knowledge-based, nonlinear, mathematical model of translation, followed by successive cycles of predictions and experimental validations using a combination of specific and global quantitative approaches. Similar modi operandi were adopted to characterize the intrinsic properties of replication and transcription. Integrating knowledge from these three fundamental cellular processes allowed us to acquire genome-wide the quantitative parameters that characterize the native B. subtilis genetic sequences. We then rationally designed a library of synthetic constructs combining a selection of different genetic elements giving rise to any desired gene expression profile across growth conditions.

The intrinsic properties of these hard-wired regulatory layers of gene expression open novel avenues for synthetic biology, particularly in the potential it offers to bypass the need for multiple specific regulators to modulate complex synthetic circuits.