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首页> 外文期刊>Applied Microbiology >Congo Red Fluorescence for Rapid In Situ Characterization of Synthetic Curli Systems
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Congo Red Fluorescence for Rapid In Situ Characterization of Synthetic Curli Systems

机译：刚果红荧光用于合成Curli系统的快速原位表征

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Curli are amyloid proteins that are assembled into extracellular polymeric fibers by bacteria during biofilm formation. The beta-sheet-rich protein CsgA, the primary structural component of the fibers, is secreted through dedicated machinery and self-assembles into cell-anchored fibers many times longer than the cell. Here, we have developed an in situ fluorescence assay for curli production that exploits the fluorescent properties of Congo red (CR) dye when bound to amyloid, allowing for rapid and robust curli quantification. We initially evaluated three amyloid-binding dyes for the fluorescent detection of curli in bacterial culture and found only Congo red compatible with in situ quantification. We further characterized the fluorescent properties of the dye directly in bacterial culture and calibrated the fluorescence using purified CsgA protein. We then used the Congo red assay to rapidly develop and characterize inducible curli-producing constructs in both an MC4100-derived lab strain of Escherichia coli and a derivative of the probiotic strain E. coli Nissle. This technique can be used to evaluate curli production in a minimally invasive manner using a range of equipment, simplifying curli quantification and the development of novel engineered curli systems.IMPORTANCE Curli are proteins produced by many bacteria as a structural component of biofilms, and they have recently emerged as a platform for fabrication of biological materials. Curli fibers are very robust and resistant to degradation, and the curli subunits can tolerate many protein fusions, facilitating the biosynthesis of novel functional materials. A serious bottleneck in the development of more sophisticated engineered curli systems is the rapid quantification of curli production by the bacteria. In this work we address this issue by developing a technique to monitor curli production directly in bacterial cultures, allowing for rapid curli quantification in a manner compatible with many powerful high-throughput techniques that can be used to engineer complex biological material systems.
机译：Curli是淀粉样蛋白，在生物膜形成过程中被细菌组装成细胞外聚合物纤维。富含β-折叠的蛋白质CsgA是纤维的主要结构成分，是通过专用机器分泌的，并且自组装成细胞锚定的纤维，比细胞长很多倍。在这里，我们开发了一种用于冰壶生产的原位荧光测定法，该技术利用结合到淀粉样蛋白上的刚果红（CR）染料的荧光特性，实现了快速而稳健的冰壶量化。我们最初评估了三种淀粉样蛋白结合染料在细菌培养物中荧光检测卷曲素，发现只有刚果红与原位定量兼容。我们进一步表征了染料直接在细菌培养物中的荧光特性，并使用纯化的CsgA蛋白校准了荧光。然后，我们使用刚果红分析法快速开发并鉴定了MC4100衍生的大肠杆菌实验室菌株和益生菌菌株Nissle的衍生物中可诱导的产生卷曲的构建体。这项技术可用于使用多种设备以最小侵入性的方式评估卷发的产生，简化卷发的定量以及开发新型工程卷发系统的重要性.Curli是由许多细菌产生的蛋白质作为生物膜的结构成分，它们具有最近，它成为制造生物材料的平台。卷曲的纤维非常结实并且抗降解，并且卷曲的亚基可以耐受许多蛋白质融合，从而促进新型功能材料的生物合成。开发更复杂的工程卷发系统的一个严重瓶颈是细菌对卷发产量的快速定量。在这项工作中，我们通过开发一种技术来直接监测细菌培养物中的卷发产量，从而以与可用于工程化复杂生物材料系统的许多强大的高通量技术兼容的方式进行快速卷发定量，从而解决了这一问题。

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