Vecchione Stefano, Fritz Georg
LOEWE Center for Synthetic Microbiology, Philipps-University Marburg, Hans-Meerwein Str. 6, 35032 Marburg, Germany.
J Biol Eng. 2019 Nov 28;13:92. doi: 10.1186/s13036-019-0218-8. eCollection 2019.
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 and related bacteria by site-specific recombination at different phage attachment () 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 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 sites into the genome of . 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 .
合成生物学严重依赖于快速且简单的DNA工程技术，如连接酶循环反应（LCR）、吉布森组装法和金门组装法，所有这些方法都能实现快速的多片段DNA组装。金门组装法的一项重大改进是模块化克隆（MoClo）系统，该系统允许从可重复使用的部件中简单地进行文库扩增和遗传回路的组合构建。然而，MoClo系统的一个局限性在于所有回路都是在低拷贝和中拷贝质粒中组装的，而缺乏一条通向染色体整合的快速途径。为了克服这一瓶颈，我们在此利用了条件复制、整合和模块化（CRIM）质粒，这些质粒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形式整合到大肠杆菌及其相关细菌的染色体中。
通过将MoClo系统的模块化与CRIM质粒的特性相结合，我们创建了一组32种新型CRIMoClo质粒，并对它们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，我们将一个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。通过分析这些回路的行为，我们发现表达水平基本相同，这表明这些位点具有正交性。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位点进行快速且可靠的顺序整合。利用四个抗性盒，该程序在每轮整合之间不需要重组事件。最后，我们高效地组装并在基因组中整合了合成的ECF σ因子/抗σ开关，表明在中拷贝质粒上编码的回路所观察到的生长缺陷得到了缓解。
CRIMoClo系统能够从可重复使用的、与MoClo兼容的部件中生成遗传回路，并将它们整合到大肠杆菌基因组的4个正交att位点中。利用四个不同的抗性模块，CRIMoClo系统允许进行简单、快速且可靠的多次整合。此外，利用CRIMoClo质粒和MoClo可重复使用的部件，我们有效地整合并减轻了质粒携带回路的毒性。最后，由于CRIMoClo框架具有高度灵活性，可以同时利用质粒携带的回路和染色体整合的回路。这提高了我们排列多个遗传模块的能力，并使得在大肠杆菌中更容易设计复杂的合成代谢途径。
