Toolbox of Characterized Genetic Parts for Staphylococcus aureus
Toolbox of Characterized Genetic Parts for Staphylococcus aureus
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DOI：
10.1021/acssynbio.3c00325
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发表时间：
2023-12-08
影响因子：
4.7
通讯作者：
Andrews，Lauren B.
中科院分区：
文献类型：
--
作者：
Rondthaler，Stephen N.;Sarker，Biprodev;Andrews，Lauren B.
金黄色葡萄球菌是一种重要的临床细菌，广泛存在于人体相关微生物中，是多种疾病的致病菌。然而，S.金黄色葡萄球菌由于这种非典型革兰氏阳性细菌的有限的特征遗传部分而难以用合成生物学方法处理。此外，S. aureus依赖于繁琐而低效的克隆策略。在这里，我们报告的第一个标准化的遗传零件工具箱。金黄色葡萄球菌，其包括表征的启动子、核糖体结合位点、终止子和来自多种细菌的质粒复制子，用于精确控制基因表达。建立了S的标准相对表达单位（REU）。使用质粒参照对金黄色葡萄球菌进行鉴定，并使用S对标准化雷乌斯中的遗传部分进行表征。金黄色ATCC 12600。我们构建了启动子和终止子部分质粒，它们与有效的IIS型DNA组装策略相容，以有效地构建多部分DNA构建体。构建了24个组成型启动子的文库，并在金黄色葡萄球菌中表征，其显示出380倍的活性范围。该启动子文库还在枯草芽孢杆菌中进行了测定（122倍活性范围），以证明组成型启动子在这些革兰氏阳性细菌之间的可转移性。通过应用迭代设计-构建-测试-学习循环，我们展示了使用我们的工具箱在金黄色葡萄球菌中合理设计和工程化四环素传感器，使用PXyl-TetOaTc诱导型启动子，实现了25.8倍的诱导。这个工具箱极大地扩展了革兰氏阳性细菌越来越多的遗传部分，并将使研究人员能够利用合成生物学方法来研究和设计金黄色葡萄球菌的细胞过程。
Staphylococcus aureusis an important clinical bacterium prevalent in human-associated microbiomes and the cause of many diseases. However,S. aureushas been intractable to synthetic biology approaches due to limited characterized genetic parts for this nonmodel Gram-positive bacterium. Moreover, genetic manipulation ofS. aureushas relied on cumbersome and inefficient cloning strategies. Here, we report the first standardized genetic parts toolbox forS. aureus, which includes characterized promoters, ribosome binding sites, terminators, and plasmid replicons from a variety of bacteria for precise control of gene expression. We established a standard relative expression unit (REU) forS. aureususing a plasmid reference and characterized genetic parts in standardized REUs usingS. aureusATCC 12600. We constructed promoter and terminator part plasmids that are compatible with an efficient Type IIS DNA assembly strategy to effectively build multipart DNA constructs. A library of 24 constitutive promoters was built and characterized inS. aureus, which showed a 380-fold activity range. This promoter library was also assayed inBacillus subtilis(122-fold activity range) to demonstrate the transferability of the constitutive promoters between these Gram-positive bacteria. By applying an iterative design-build-test-learn cycle, we demonstrated the use of our toolbox for the rational design and engineering of a tetracycline sensor inS. aureususing the PXyl-TetOaTc-inducible promoter that achieved 25.8-fold induction. This toolbox greatly expands the growing number of genetic parts for Gram-positive bacteria and will allow researchers to leverage synthetic biology approaches to study and engineer cellular processes inS. aureus.