Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Nat Chem Biol. 2019 Feb;15(2):196-204. doi: 10.1038/s41589-018-0168-3. Epub 2018 Nov 26.
Cellular processes are carried out by many genes, and their study and optimization requires multiple levers by which they can be independently controlled. The most common method is via a genetically encoded sensor that responds to a small molecule. However, these sensors are often suboptimal, exhibiting high background expression and low dynamic range. Further, using multiple sensors in one cell is limited by cross-talk and the taxing of cellular resources. Here, we have developed a directed evolution strategy to simultaneously select for lower background, high dynamic range, increased sensitivity, and low cross-talk. This is applied to generate a set of 12 high-performance sensors that exhibit >100-fold induction with low background and cross-reactivity. These are combined to build a single "sensor array" in the genomes of E. coli MG1655 (wild-type), DH10B (cloning), and BL21 (protein expression). These "Marionette" strains allow for the independent control of gene expression using 12 small-molecule inducers.
细胞过程由许多基因执行，对其进行研究和优化需要多个控制杆，以便能够独立控制它们。最常见的方法是通过对小分子作出反应的遗传编码传感器。然而，这些传感器往往不够理想，表现出高背景表达和低动态范围。此外，在一个细胞中使用多个传感器受到交叉对话和细胞资源消耗的限制。在这里，我们开发了一种定向进化策略，以同时选择低背景、高动态范围、更高灵敏度和低交叉对话。这适用于生成一组 12 个高性能传感器，它们具有低背景和交叉反应性的>100 倍诱导。这些传感器被组合在一起，在大肠杆菌 MG1655（野生型）、DH10B（克隆）和 BL21（蛋白质表达）的基因组中构建单个“传感器阵列”。这些“傀儡”菌株允许使用 12 种小分子诱导剂来独立控制基因表达。
