Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Department of Electrical and Computer Engineering, Boston University, Boston, MA, USA.
Nat Microbiol. 2020 Nov;5(11):1349-1360. doi: 10.1038/s41564-020-0757-2. Epub 2020 Aug 3.
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.
细胞可以通过遗传电路被编程来监测和响应其环境。设计自动化软件将所需的电路功能映射到 DNA 序列上，这个过程需要单元的基因调控（门），这些门易于连接且行为可预测。由于真核生物转录和翻译的复杂机制，这对真核生物来说是一个挑战。为此，我们开发了用于酵母（酿酒酵母）的门，它们使用 RNA 聚合酶流作为信号载体进行连接，并且彼此隔离，不受宿主调控的影响。这些门基于最小的组成型启动子（~120 个碱基对），为其开发了插入 DNA 结合蛋白的操纵子的规则。使用这种方法，我们构建了九个 NOT/NOR 门，它们具有几乎相同的响应功能和 400 倍的动态范围。在电路中，通过在终止子下游放置核酶来阻断 RNA 聚合酶通读产生的信使 RNA 的核输出，从而使它们在转录上彼此隔离。基于这些门，使用 Cello 2.0 构建了具有多达 11 种调控蛋白的电路。一个简单的动态模型可以预测电路在数天内的响应。真核生物的遗传电路设计自动化简化了调控网络的构建，作为细胞工程项目的一部分，无论是在生物生产过程中进行阶段处理、作为环境监测器还是指导活体治疗。
