Stringency (low leak) is one of the most important specifications required for genetic circuits and induction systems, but it is challenging to evolve without sacrificing the maximum output level. This problem also comes from the absence of truly tunable negative selection methods. This paper reports that stringently switching variants can sometimes emerge with surprising frequency upon mutations. We randomly mutated the previously generated leaky variants of LuxR, the quorum-sensing transcription activator from Vibrio fischeri, to restore the stringency. We found as much as 10-20% of the entire population exhibited significantly improved signal-to-noise ratios compared with their parents. This indicated that these mutants arose by the loss of folding capability by accumulating destabilizing mutations, not by introducing rare adaptive mutations, thereby becoming AHL-dependent folders. Only four rounds of mutagenesis and ON-state selection resulted in the domination of the entire population by the improved variants with low leak, without direct selection pressure for stringency. With this surprising frequency, conversion into the "ligand-addicted folders" should be one of the prevailing modes of evolving stringency both in the laboratory and in nature, and the workflow described here provides a rapid and versatile method of improving the signal-to-noise ratio of various genetic switches.

中文翻译：

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没有关闭状态选择的LuxR vibrio fischeri Quorum法定度传感器的严格度的定向进化。

严格性（低泄漏）是遗传回路和诱导系统所需的最重要的规范之一，但是要在不牺牲最大输出水平的前提下发展其挑战性。这个问题也来自缺乏真正可调的负选择方法。本文报道，有时突变后，严格转换的变异有时会以惊人的频率出现。我们随机突变了先前产生的LuxR的泄漏变异体，即Fischio fischeri的群体感应转录激活因子，以恢复严格性。我们发现与他们的父母相比，多达10-20％的人口表现出明显改善的信噪比。这表明这些突变体是通过积累破坏稳定的突变而不是通过引入稀有的适应性突变而丧失折叠能力而产生的，从而成为依赖AHL的文件夹。仅经过四轮诱变和ON状态选择，就导致了整个种群被泄漏低的改良变种所主导，而没有严格的直接选择压力。以这种令人惊讶的频率，转换为“配体上瘾的文件夹”应该是实验室和自然界中严格性发展的主要模式之一，此处描述的工作流程提供了一种快速，通用的方法来改善信号强度。各种遗传开关的噪声比。