Alker Amanda T, Aspiras Alpher E, Dunbar Tiffany L, Farrell Morgan V, Fedoriouk Andriy, Jones Jeffrey E, Mikhail Sama R, Salcedo Gabriella Y, Moore Bradley S, Shikuma Nicholas J
bioRxiv. 2023 Jan 31:2023.01.31.526474. doi: 10.1101/2023.01.31.526474.
A conspicuous roadblock to studying marine bacteria for fundamental research and biotechnology is a lack of modular synthetic biology tools for their genetic manipulation. Here, we applied, and generated new parts for, a modular plasmid toolkit to study marine bacteria in the context of symbioses and host-microbe interactions. To demonstrate the utility of this plasmid system, we genetically manipulated the marine bacterium , which stimulates the metamorphosis of the model tubeworm, . Using these tools, we quantified constitutive and native promoter expression, developed reporter strains that enable the imaging of host-bacteria interactions, and used CRISPR interference (CRISPRi) to knock down a secondary metabolite and a host-associated gene. We demonstrate the broader utility of this modular system for rapidly creating and iteratively testing genetic tractability by modifying marine bacteria that are known to be associated with diverse host-microbe symbioses. These efforts enabled the successful transformation of twelve marine strains across two Proteobacteria classes, four orders and ten genera. Altogether, the present study demonstrates how synthetic biology strategies enable the investigation of marine microbes and marine host-microbe symbioses with broader implications for environmental restoration and biotechnology.
对于基础研究和生物技术领域而言，研究海洋细菌存在一个明显的障碍，即缺乏用于其基因操作的模块化合成生物学工具。在此，我们应用了一种模块化质粒工具包，并为其生成了新的元件，以便在共生以及宿主 - 微生物相互作用的背景下研究海洋细菌。为了证明该质粒系统的实用性，我们对海洋细菌进行了基因操作，这种细菌能刺激模式管虫的变态。利用这些工具，我们对组成型启动子和天然启动子的表达进行了定量分析，构建了能够对宿主 - 细菌相互作用进行成像的报告菌株，并使用CRISPR干扰（CRISPRi）来敲低一种次级代谢产物和一个与宿主相关的基因。我们证明了这个模块化系统在通过改造已知与多种宿主 - 微生物共生相关的海洋细菌来快速创建并迭代测试遗传可操作性方面具有更广泛的实用性。这些努力使得成功转化了来自两个变形菌纲、四个目和十个属的十二种海洋菌株。总体而言，本研究展示了合成生物学策略如何能够用于研究海洋微生物以及海洋宿主 - 微生物共生关系，这对于环境修复和生物技术具有更广泛的意义。
