Customized transcription factors that control gene expression in response to small molecules can act as endogenous molecular biosensors and are valuable tools for synthetic biology. We previously engineered the Escherichia coli regulatory protein AraC to respond to non-native inducers such as D-arabinose and triacetic acid lactone. Those prior studies involved the construction and screening of individual 4- or 5-site saturation mutagenesis libraries, followed by iterative rounds of positive- and negative fluorescence-activated cell sorting (FACS). Here we describe an improved screening platform and the isolation of several new and potentially useful AraC variants that respond to vanillin and salicylic acid. To increase throughput and reduce total screening time, selection steps were added to the sorting workflow. Two different site-saturation libraries and a random mutagenesis library were pooled together and >108 variants were subjected to iterative FACS and selection in search of variants responding to a panel of compounds. The new phenolic-sensing variants show responses >100-fold over background and are highly specific towards their target compound. The isolation of these variants further demonstrates the potential for engineering the AraC transcriptional regulatory protein for molecular sensing and reporting, and our improved screening system should prove effective in designing similar biosensors.

中文翻译：

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基于AraC调节蛋白的新型工程酚类生物传感器。

控制响应于小分子的基因表达的定制转录因子可以充当内源性分子生物传感器，并且是合成生物学的宝贵工具。我们之前设计了大肠杆菌调节蛋白AraC，以响应非天然诱导物，例如D-阿拉伯糖和三乙酸内酯。那些先前的研究涉及构建和筛选单个4位或5位饱和诱变文库，然后进行正负荧光激活细胞分选（FACS）的迭代轮次。在这里，我们描述了一种改进的筛选平台，以及对香草醛和水杨酸有反应的几种新的且可能有用的AraC变体的分离。为了增加吞吐量并减少总筛选时间，选择步骤已添加到排序工作流程中。将两个不同的位点饱和文库和一个随机诱变文库合并在一起，并对> 108个变体进行迭代FACS并进行选择，以寻找对一组化合物有反应的变体。新的酚类感应变体显示响应是背景的100倍以上，并且对目标化合物具有高度特异性。这些变体的分离进一步证明了工程化AraC转录调节蛋白用于分子传感和报告的潜力，并且我们改进的筛选系统在设计类似的生物传感器方面应被证明是有效的。比背景高100倍，对目标化合物具有高度特异性。这些变体的分离进一步证明了工程化AraC转录调节蛋白用于分子传感和报告的潜力，我们经过改进的筛选系统在设计类似的生物传感器方面应被证明是有效的。比背景高100倍，对目标化合物具有高度特异性。这些变体的分离进一步证明了工程化AraC转录调节蛋白用于分子传感和报告的潜力，我们经过改进的筛选系统在设计类似的生物传感器方面应被证明是有效的。