Fine-tuning gene networks using simple sequence repeats
Fine-tuning gene networks using simple sequence repeats
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DOI：
10.1073/pnas.1205693109
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发表时间：
2012-10-16
影响因子：
11.1
通讯作者：
Klavins, Eric
中科院分区：
文献类型：
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作者：
Egbert, Robert G.;Klavins, Eric
复杂的合成基因网络中的参数必须在网络按设计发挥作用之前进行广泛的调整。在这里，我们介绍了一种简单而通用的方法来快速调整大肠杆菌中的基因网络，使用嵌入在核糖体结合位点的间隔区的超变简单序列重复。通过改变重复序列长度，我们生成了表达文库，这些文库可以在1,000倍范围内递增和可预测地对基因表达水平进行采样。我们演示了实用程序的方法，通过创建一个可编程的采样表达式空间的开关库，以平衡开关的两个状态，我们说明了调整的需要，开关的行为是敏感的主机上下文。此外，我们表明，重复的突变率是可控的，在体内的稳定性或有针对性的诱变，这表明一种新的方法来优化基因网络通过定向进化。这种调整方法应该加速工程功能复杂的基因网络的过程。
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.