ena-STUDY-IMPERIAL COLLEGE LONDON-25-08-2017-15:01:28:513-615
Synthetic negative feedback circuits using engineered small RNAs
Small RNA (sRNA) are non-coding RNA molecules which can post-transcriptionally regulate gene expression through interaction with messenger RNA (mRNA). Many natural bacterial sRNAs have been identified to date, and a small number of synthetic sRNAs have been designed that can regulate gene expression predictably. The use of sRNAs in synthetic biology has so far focussed on characterising their properties or to silence specific mRNAs for metabolic engineering purposes; they have not been used extensively in circuits. A particular use of sRNA could be in the implementation of feedback control architectures, which are known to endow natural and man-made systems with robust performance in the face of uncertainties. In this paper, we design, model and build two new negative feedback architectures that use translation-inhibiting sRNA modules. The first circuit builds upon the well characterised tet-based autorepressor, allowing fine tuning of the circuit output through the use of an external input molecule that modulates sRNA expression. The second circuit involves an sRNA module that responds to the level of the circuit output - a protein - by regulating the mRNA that encodes for this protein. We use mathematical modelling to predict the output response of both circuits to the external inputs and to predict the noise characteristics of each. Synthetic sRNAs were rationally designed and strong translation-inhibition against each mRNA target validated. The best performing sRNAs were integrated into each negative feedback circuit and qualitatively tested in vivo in Escherichia coli. The properties of both feedback circuits were compared to the autorepressor and found to be in good agreement with those predicted by modelling and found to have many beneficial effects on overall circuit performance. Finally mathematical modelling predicted important increases in noise would occur if these sRNA circuits were split across more than one plasmid, which correlated with our experimental results; this has wider implications for the construction of robust synthetic genetic circuits.
Small RNA (sRNA) are non-coding RNA molecules which can post-transcriptionally regulate gene expression through interaction with messenger RNA (mRNA). Many natural bacterial sRNAs have been identified to date, and a small number of synthetic sRNAs have been designed that can regulate gene expression predictably. The use of sRNAs in synthetic biology has so far focussed on characterising their properties or to silence specific mRNAs for metabolic engineering purposes; they have not been used extensively in circuits. A particular use of sRNA could be in the implementation of feedback control architectures, which are known to endow natural and man-made systems with robust performance in the face of uncertainties. In this paper, we design, model and build two new negative feedback architectures that use translation-inhibiting sRNA modules. The first circuit builds upon the well characterised tet-based autorepressor, allowing fine tuning of the circuit output through the use of an external input molecule that modulates sRNA expression. The second circuit involves an sRNA module that responds to the level of the circuit output - a protein - by regulating the mRNA that encodes for this protein. We use mathematical modelling to predict the output response of both circuits to the external inputs and to predict the noise characteristics of each. Synthetic sRNAs were rationally designed and strong translation-inhibition against each mRNA target validated. The best performing sRNAs were integrated into each negative feedback circuit and qualitatively tested in vivo in Escherichia coli. The properties of both feedback circuits were compared to the autorepressor and found to be in good agreement with those predicted by modelling and found to have many beneficial effects on overall circuit performance. Finally mathematical modelling predicted important increases in noise would occur if these sRNA circuits were split across more than one plasmid, which correlated with our experimental results; this has wider implications for the construction of robust synthetic genetic circuits.