Abstract
The rational design and realisation of simple-to-use genetic control elements that are modular, orthogonal and robust is essential to the construction of predictable and reliable biological systems of increasing complexity. To this effect, we introduce modular Artificial RNA interference (mARi), a rational, modular and extensible design framework that enables robust, portable and multiplexed post-transcriptional regulation of gene expression in Escherichia coli. The regulatory function of mARi was characterised in a range of relevant genetic contexts, demonstrating its independence from other genetic control elements and the gene of interest, and providing new insight into the design rules of RNA based regulation in E. coli, while a range of cellular contexts also demonstrated it to be independent of growth-phase and strain type. Importantly, the extensibility and orthogonality of mARi enables the simultaneous post-transcriptional regulation of multi-gene systems as both single-gene cassettes and poly-cistronic operons. To facilitate adoption, mARi was designed to be directly integrated into the modular BASIC DNA assembly framework. We anticipate that mARi-based genetic control within an extensible DNA assembly framework will facilitate metabolic engineering, layered genetic control, and advanced genetic circuit applications.
Highlights
Synthetic biology aims to make the engineering of biological systems more predictable, efficient and reliable [1,2,3]
We focus on expanding the toolbox of modular biomolecular control elements that function at the post-transcriptional level, small non-coding RNAs
We report the development, characterisation, and implementation of a modular post-transcriptional regulation system based on trans-encoded small non-coding RNAs (sRNAs)
Summary
Synthetic biology aims to make the engineering of biological systems more predictable, efficient and reliable [1,2,3]. To leverage the use of sRNAs in genetic circuits constructed via a modular DNA assembly method, we set out to design a universal, modular framework for the facile implementation of trans-acting sRNA-based control using verified, re-usable target sequences, namely modular Artificial RNA interference (mARi). BASIC DNA assembly provides a modular, standardised and automatable [33] framework for genetic structure based on orthogonal, computationally designed linkers [34] (Figure 1) We exploited this by targeting the DNA linkers used in the construction of gene expression cassettes and operons. In our mARi design, a modified seed sequence specific to the linker upstream of the target Gene of Interest (GOI) was fused to a native sRNA scaffold containing a host factor-1 (Hfq) binding site [17,27] (Figures 1A and 2A). MARi sequence variants were further expanded and their implementation was demonstrated for simultaneous regulation in a multi-gene system with different genetic architectures
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