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无细胞体系中的表达优化和合成基因网络。

Expression optimization and synthetic gene networks in cell-free systems.

机构信息

Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Bethel Valley Road, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.

出版信息

Nucleic Acids Res. 2012 Apr;40(8):3763-74. doi: 10.1093/nar/gkr1191. Epub 2011 Dec 17.

Abstract

Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion.

摘要

合成生物学通过对生物功能的正向工程设计,为各种应用带来了巨大的前景。该领域的大多数研究都集中在使用活细胞上,但无细胞方法提供了更简单、更灵活的环境。在这里,我们通过在无细胞环境中对 TetR 和 LacI 可抑制 T7 启动子的变体进行特征描述,并研究决定表达效率的序列元件,评估基于 T7 启动子驱动表达的无细胞调控系统。利用得到的构建体,我们探索了不同的方法来构建调控系统,从而在大肠杆菌提取物和最小 PURE 系统中实现了诱导型负反馈,该系统由转录和翻译所需的纯化蛋白组成。尽管负反馈元件在许多自然和工程系统中很常见且必不可少,但这种简单的构建块以前尚未在无细胞环境中实现。作为最后一步,我们证明了使用我们的无细胞方法开发的反馈系统也可以在活大肠杆菌中实现,这说明了使用无细胞表达快速开发合成生物学中活细胞系统的潜力。我们对无细胞组件的定量特征描述和负反馈的演示体现了以自下而上的方式利用生物功能的重要步骤。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1174/3333853/66a490f1cbe0/gkr1191f1.jpg

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