Burden-driven feedback control of gene expression
Burden-driven feedback control of gene expression
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DOI：
10.1101/177030
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发表时间：
2017-08
期刊：
影响因子：
48
通讯作者：
Francesca Ceroni;Alice Boo;Simone Furini;T. Gorochowski;Olivier Borkowski;Y. Ladak;A. Awan;Charlie Gilbert;G. Stan;T. Ellis
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文献类型：
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作者：
Francesca Ceroni;Alice Boo;Simone Furini;T. Gorochowski;Olivier Borkowski;Y. Ladak;A. Awan;Charlie Gilbert;G. Stan;T. Ellis
细胞使用反馈调节来确保稳健的生长，尽管对资源和不同环境条件的需求波动。然而，来自工程化构建体的外源蛋白的表达是细胞不适应的非天然负担。在这里，我们将RNA-seq与体内试验相结合，以确定当诱导型合成构建体表达时大肠杆菌中发生的主要转录变化。我们观察到，无论构建体如何，与热休克反应相关的天然启动子在响应合成表达时迅速激活表达。使用这些启动子，我们构建了一个基于dCas 9的反馈调节系统，该系统可以自动调节合成构建体的表达以响应负荷。配备有这种通用控制器的细胞保持其天然基因表达的能力，以确保稳健的生长，从而在批量生产中的蛋白质产量方面优于未调节的细胞。据我们所知，这种工程反馈是通用的、基于负担的生物分子控制系统的第一个例子，并且是模块化的、可调的和便携式的。
Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a dCas9-based feedback-regulation system that automatically adjusts the expression of a synthetic construct in response to burden. Cells equipped with this general-use controller maintained their capacity for native gene expression to ensure robust growth and thus outperformed unregulated cells in terms of protein yield in batch production. This engineered feedback is to our knowledge the first example of a universal, burden-based biomolecular control system and is modular, tunable and portable.