National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi 214112, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
ACS Synth Biol. 2023 Jul 21;12(7):2157-2167. doi: 10.1021/acssynbio.3c00246. Epub 2023 Jun 23.
In synthetic biology, the precise control of gene expression is challenging due to the limited orthogonality of expression elements. Here, to address this issue and improve the reusability of genetic elements, we developed a bicistronic expression cassette in based on a leaderless promoter lacking a 5'UTR. The created leaderless bicistronic design (BCD) significantly improved the orthogonality of expression elements across different genes of interest. We also explored the importance of the fore-cistron and SD motif in maintaining the strength of leaderless BCDs. Additionally, we established a library containing 55,901 fore-cistrons and demonstrated that the regulatory range of gene expression in leaderless BCDs can be broader by modifying the fore-cistron sequence. This study provides a novel synthetic biology tool based on leaderless BCD for fine-tuning gene expression in using fore-cistrons. Moreover, the strategy developed here can also be applied to improve the performance of other leaderless promoters in other bacteria.
在合成生物学中，由于表达元件的正交性有限，精确控制基因表达具有挑战性。在这里，为了解决这个问题并提高遗传元件的可重用性，我们基于缺乏 5'UTR 的无启动子的 leaderless 开发了一种双顺反子表达盒。所创建的无启动子双顺反子设计（BCD）显著提高了不同感兴趣基因的表达元件的正交性。我们还探讨了前顺反子和 SD 基序在维持无启动子 BCD 强度方面的重要性。此外，我们建立了一个包含 55901 个前顺反子的文库，并通过修饰前顺反子序列证明了无启动子 BCD 中基因表达的调控范围可以更宽。本研究提供了一种基于无启动子 BCD 的新型合成生物学工具，用于使用前顺反子在 中精细调节基因表达。此外，这里开发的策略也可以应用于提高其他细菌中其他无启动子的性能。
