Synthetic negative feedback circuits using engineered small RNAs
Synthetic negative feedback circuits using engineered small RNAs
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DOI：
10.1101/184473
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发表时间：
2017-09
影响因子：
14.9
通讯作者：
Ciaran L. Kelly;Andreas W. K. Harris;Harrison Steel;E. J. Hancock;J. Heap;A. Papachristodoulou
中科院分区：
文献类型：
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作者：
Ciaran L. Kelly;Andreas W. K. Harris;Harrison Steel;E. J. Hancock;J. Heap;A. Papachristodoulou
负反馈控制可以使自然、生物和人造技术系统在面对不确定性时具有稳健的性能。到目前为止，合成生物反馈电路主要依赖于转录因子，并受到诸如负担过大和缺乏灵活可调整性等限制。小RNA(SRNAs)是一类非编码的RNA分子，可通过与信使RNA(信使RNA)的相互作用在转录后调节基因的表达。在本文中，我们首次提出了两种新的负反馈结构的设计、建模和构造，这两种结构使用了合理设计的、翻译抑制的SRNA模块。第一个电路建立在特征良好的基于tet的自动抑制器的基础上，允许通过使用调节sRNA表达的外部输入分子来微调电路输出。第二个回路涉及一个与回路的输出蛋白直接负反馈的sRNA，调节编码该蛋白质的mRNA以响应输出蛋白的浓度。随机和确定性建模指导了这两种电路的设计和优化实现，实验数据与模型预测结果吻合较好。这项工作中提出的详细和特征良好的电路可以集成到更大、更复杂的合成生物电路、路径和系统中。
Negative feedback control is known to endow natural biological and man-made technological systems with robust performance in the face of uncertainties. To date synthetic biological feedback circuits have predominantly relied upon transcription factors and suffer from limitations such as excessive burden and lack of flexible tunability. Small RNAs (sRNAs) are non-coding RNA molecules which can post-transcriptionally regulate gene expression through interaction with messenger RNA (mRNA). In this paper, we present the design, modelling and construction of two new negative feedback architectures that use rationally-designed, translation-inhibiting sRNA modules for the first time. 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 in direct negative feedback with the output protein of the circuit, regulating the mRNA encoding this protein in response to output protein concentration. Stochastic and deterministic modelling guided the design and optimal implementations of both circuits, and the experimental data obtained compared well with model predictions. The detailed and well-characterised circuits presented in this work can be integrated into larger, more complex, synthetic biological circuits, pathways and systems.