Cells can be programmed to monitor and react to their environment using genetic circuits. Design automation software maps a desired circuit function to a DNA sequence, a process that requires units of gene regulation (gates) that are simple to connect and behave predictably. This poses a challenge for eukaryotes due to their complex mechanisms of transcription and translation. To this end, we have developed gates for yeast (Saccharomyces cerevisiae) that are connected using RNA polymerase flux as the signal carrier and are insulated from each other and host regulation. They are based on minimal constitutive promoters (~120 base pairs), for which rules are developed to insert operators for DNA-binding proteins. Using this approach, we constructed nine NOT/NOR gates with nearly identical response functions and 400-fold dynamic range. In circuits, they are transcriptionally insulated from each other by placing ribozymes downstream of terminators to block nuclear export of messenger RNAs resulting from RNA polymerase readthrough. Based on these gates, Cello 2.0 was used to build circuits with up to 11 regulatory proteins. A simple dynamic model predicts the circuit response over days. Genetic circuit design automation for eukaryotes simplifies the construction of regulatory networks as part of cellular engineering projects, whether it be to stage processes during bioproduction, serve as environmental sentinels or guide living therapeutics.

可以对细胞进行编程，以使用遗传电路监控其环境并对其做出反应。设计自动化软件将所需的电路功能映射到 DNA 序列，这一过程需要易于连接且行为可预测的基因调控单元（门）。由于其复杂的转录和翻译机制，这对真核生物提出了挑战。为此，我们开发了酵母（Saccharomyces cerevisiae) 使用 RNA 聚合酶通量作为信号载体连接，并且彼此绝缘且不受宿主调节。它们基于最小的组成型启动子（约 120 个碱基对），为此制定了规则以插入 DNA 结合蛋白的操作符。使用这种方法，我们构建了九个具有几乎相同的响应函数和 400 倍动态范围的 NOT/NOR 门。在电路中，它们通过将核酶置于终止子下游以阻止由 RNA 聚合酶通读产生的信使 RNA 的核输出，从而在转录上相互隔离。基于这些门，Cello 2.0 被用于构建具有多达 11 种调节蛋白的电路。一个简单的动态模型可以预测几天内的电路响应。