Genetic circuits have many applications, from guiding living therapeutics to ordering process in a bioreactor, but to be useful they have to be genetically stable and not hinder the host. Encoding circuits in the genome reduces burden, but this decreases performance and can interfere with native transcription. We have designed genomic landing pads in Escherichia coli at high‐expression sites, flanked by ultrastrong double terminators. DNA payloads >8 kb are targeted to the landing pads using phage integrases. One landing pad is dedicated to carrying a sensor array, and two are used to carry genetic circuits. NOT/NOR gates based on repressors are optimized for the genome and characterized in the landing pads. These data are used, in conjunction with design automation software (Cello 2.0), to design circuits that perform quantitatively as predicted. These circuits require fourfold less RNA polymerase than when carried on a plasmid and are stable for weeks in a recA⁺ strain without selection. This approach enables the design of synthetic regulatory networks to guide cells in environments or for applications where plasmid use is infeasible.

中文翻译：

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高度绝缘的大肠杆菌基因组着陆垫中携带的稳定遗传电路的精确设计。


遗传电路有很多应用，从指导活体治疗到生物反应器中的排序过程，但要有用，它们必须在遗传上稳定且不妨碍宿主。基因组中的编码电路减轻了负担，但这会降低性能并可能干扰天然转录。我们在大肠杆菌的高表达位点设计了基因组着陆垫，两侧是超强双终止子。使用噬菌体整合酶将 >8 kb 的 DNA 有效负载靶向着陆垫。一个着陆垫专门用于承载传感器阵列，两个着陆垫用于承载遗传电路。基于阻遏物的 NOT/NOR 门针对基因组进行了优化，并在着陆垫中进行了表征。这些数据与设计自动化软件 (Cello 2.0) 结合使用，设计出按预期定量运行的电路。这些电路所需的 RNA 聚合酶比质粒携带的 RNA 聚合酶少四倍，并且无需选择即可在recA ⁺菌株中稳定数周。这种方法可以设计合成调控网络，以在无法使用质粒的环境或应用中引导细胞。