Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

中文翻译：

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用于大肠杆菌合成电路的模块化组装和正交染色体整合的 CRIMoClo 质粒

合成生物学在很大程度上依赖于快速简单的 DNA 工程技术，例如连接酶循环反应 (LCR)、吉布森组装和金门组装，所有这些都允许快速、多片段的 DNA 组装。模块化克隆 (MoClo) 系统代表了 Golden Gate 组装的一项重大改进，该系统允许简单的文库传播和从可重复使用的部件组合构建遗传电路。然而，MoClo 系统的一个限制是所有电路都组装在低拷贝和中等拷贝质粒中，而缺乏染色体整合的快速途径。为了克服这个瓶颈，我们在这里利用了条件复制、整合和模块化 (CRIM) 质粒，它可以通过在不同噬菌体附着（att）位点的位点特异性重组以单拷贝整合到大肠杆菌和相关细菌的染色体中。通过将 MoClo 系统的模块化与 CRIM 质粒特性相结合，我们创建了一组 32 种新型 CRIMoClo 质粒，并对其在合成生物学应用中的适用性进行了基准测试。使用 CRIMoClo 质粒，我们将给定的遗传回路组装并整合到四个选定的噬菌体附着位点。分析这些电路的行为，我们发现基本相同的表达水平，表明基因座的正交性。使用 CRIMoClo 质粒和四种不同的报告系统，我们展示了一个框架，该框架允许在四个选定的 att 位点进行快速可靠的顺序整合。利用四个电阻盒，该程序不需要在每轮整合之间进行重组事件。最后，我们高效地组装和基因组整合合成 ECF σ 因子/抗 σ 开关，表明在中等拷贝质粒上编码的电路中观察到的生长缺陷得到缓解。CRIMoClo 系统能够从可重复使用的、与 MoClo 兼容的部分生成遗传回路，并将它们整合到大肠杆菌基因组的 4 个正交 att 位点中。CRIMoClo 系统使用四种不同的电阻模块，可实现简单、快速和可靠的多重集成。此外，利用 CRIMoClo 质粒和 MoClo 可重复使用的部分，我们有效地整合并减轻了质粒携带电路的毒性。最后，由于 CRIMoClo 框架允许高度灵活性，可以同时利用质粒携带和染色体集成电路。这增加了我们置换多个遗传模块的能力，并允许更容易地设计大肠杆菌中复杂的合成代谢途径。