Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917.
Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917.
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DOI：
10.1021/acssynbio.0c00466
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发表时间：
2021-01-15
影响因子：
4.7
通讯作者：
Joshi NS
中科院分区：
文献类型：
--
作者：
Kan A;Gelfat I;Emani S;Praveschotinunt P;Joshi NS
大肠杆菌Nissle 1917(ECN)是一种益生菌，通常用于治疗某些胃肠道疾病。得益于丰富的大肠杆菌生物学知识和易于操作，它正迅速成为开发治疗性工程菌的重要目标。细菌合成生物学项目通常使用工程质粒载体，这种载体工程简单，可以可靠地实现高水平的蛋白质表达。然而，质粒的维护通常需要抗生素，而抗生素的使用往往与体内实验或治疗不相容。ECN本身含有质粒pMUT1和pMUT2，它们没有已知的功能，但在细菌中稳定。在这里，我们描述了pMUT质粒的发展成为一个强大的平台，用于体内实验的ECN工程，并与CRISPR-Cas9系统一起去除天然质粒。我们系统地设计了这两个pMUT质粒，使其包含选择标记、荧光标记、温度敏感表达和卷曲分泌系统，以将可定制的功能材料输出到细胞外空间。然后，我们证明了当工程菌未经选择地通过小鼠胃肠道时，工程质粒在细菌中保持不变，并且分泌系统保持功能，将功能化的卷曲蛋白输出到肠道中。我们的质粒系统为治疗性ECN细菌的快速发展提供了一个平台。
Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium, commonly employed to treat certain gastrointestinal disorders. It is fast emerging as an important target for the development of therapeutic engineered bacteria, benefiting from the wealth of knowledge of E. coli biology and ease of manipulation. Bacterial synthetic biology projects commonly utilize engineered plasmid vectors, which are simple to engineer and can reliably achieve high levels of protein expression. However, plasmids typically require antibiotics for maintenance, and the administration of an antibiotic is often incompatible with in vivo experimentation or treatment. EcN natively contains plasmids pMUT1 and pMUT2, which have no known function but are stable within the bacteria. Here, we describe the development of the pMUT plasmids into a robust platform for engineering EcN for in vivo experimentation, alongside a CRISPR-Cas9 system to remove the native plasmids. We systematically engineered both pMUT plasmids to contain selection markers, fluorescent markers, temperature sensitive expression, and curli secretion systems to export a customizable functional material into the extracellular space. We then demonstrate that the engineered plasmids were maintained in bacteria as the engineered bacteria pass through the mouse GI tract without selection, and that the secretion system remains functional, exporting functionalized curli proteins into the gut. Our plasmid system presents a platform for the rapid development of therapeutic EcN bacteria.
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影响因子：
8.4
作者：
Kang, Chae Won;Lim, Hyun Gyu;Jung, Gyoo Yeol
通讯作者：
Jung, Gyoo Yeol
影响因子：
7.3
作者：
Lerner A;Matthias T;Aminov R
通讯作者：
Aminov R
DOI：
10.1073/pnas.94.3.934
发表时间：
1997-02-04
影响因子：
11.1
作者：
Heithoff, DM;Conner, CP;Mahan, MJ
通讯作者：
Mahan, MJ
影响因子：
2.6
作者：
Blum-Oehler, G;Oswald, S;Hacker, J
通讯作者：
Hacker, J
影响因子：
3.2
作者：
COLLINSON, SK;EMODY, L;KAY, WW
通讯作者：
KAY, WW