Abstract
This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications.
Highlights
Synthetic biology strives to provide means for designing and building genetic systems that are easier and faster to engineer and, at the same time, allow a predictable and precise control of biological systems
For the experiments presented in section “Core promoter module analysis and engineering of an orthogonal system”, the expression cassette for the sTF42 controlled by the MET17 promoter was integrated into the his3Δ1 locus in three copies and the pBID1 reporter cassettes were integrated into the ura3-52 locus in two copies
In the experiments presented in sections “The development of an external-signal-independent system” and “Regulated sTF42 increases dynamic range of the output signal”, the expression cassettes for the synthetic TF (sTF) with either a weak constitutive TDH3 core promoter or an inducible MET17 promoter were integrated into the his3Δ1 locus in single copy and the pBID2 reporter expression cassettes were integrated in the ura3-52 locus in two copies
Summary
Synthetic biology strives to provide means for designing and building genetic systems that are easier and faster to engineer and, at the same time, allow a predictable and precise control of biological systems. In order to prevent unpredictable interference in the functionality of such systems, the concept of orthogonality is of a great importance. An orthogonal system should provide the designed functions with minimal (or in the best case, absent) cross-talk with the PLOS ONE | DOI:10.1371/journal.pone.0148320. To make the synthetic biology systems designable, it is necessary that they are composed of modular parts. The modularity and orthogonality aspects are essential for our ability to design, apply or transfer the system components for a wide spectrum of applications and host organisms
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