Department of Electrical Engineering, University of Washington, Seattle, WA 98195, USA.
Proc Natl Acad Sci U S A. 2012 Oct 16;109(42):16817-22. doi: 10.1073/pnas.1205693109. Epub 2012 Aug 27.
The parameters in a complex synthetic gene network must be extensively tuned before the network functions as designed. Here, we introduce a simple and general approach to rapidly tune gene networks in Escherichia coli using hypermutable simple sequence repeats embedded in the spacer region of the ribosome binding site. By varying repeat length, we generated expression libraries that incrementally and predictably sample gene expression levels over a 1,000-fold range. We demonstrate the utility of the approach by creating a bistable switch library that programmatically samples the expression space to balance the two states of the switch, and we illustrate the need for tuning by showing that the switch's behavior is sensitive to host context. Further, we show that mutation rates of the repeats are controllable in vivo for stability or for targeted mutagenesis--suggesting a new approach to optimizing gene networks via directed evolution. This tuning methodology should accelerate the process of engineering functionally complex gene networks.
在复杂的合成基因网络的参数必须经过广泛调整，然后网络才能按设计运行。在这里，我们引入了一种简单而通用的方法，使用核糖体结合位点间隔区中的高突变简单重复序列，在大肠杆菌中快速调整基因网络。通过改变重复长度，我们生成了表达文库，这些文库在 1000 倍的范围内递增且可预测地采样基因表达水平。我们通过创建一个双稳态开关文库来证明该方法的实用性，该文库可以程序化地采样表达空间，以平衡开关的两个状态，我们通过表明开关的行为对宿主环境敏感来说明调整的必要性。此外，我们表明重复序列的突变率可以在体内控制，以实现稳定性或靶向诱变——这表明了一种通过定向进化优化基因网络的新方法。这种调整方法应该会加速工程功能复杂的基因网络的进程。
