BACKGROUND Bacterial surface display libraries are a popular tool for novel ligand discovery due to their ease of manipulation and rapid growth rates. These libraries typically express a scaffold protein embedded within the outer membrane with a short, surface-exposed peptide that is either terminal or is incorporated into an outer loop, and can therefore interact with and bind to substrates of interest. RESULTS In this study, we employed a novel bacterial peptide display library which incorporates short 15-mer peptides on the surface of E. coli, co-expressed with the inducible red fluorescent protein DsRed in the cytosol, to investigate population diversity over two rounds of biopanning. The naive library was used in panning trials to select for binding affinity against 3D printing plastic coupons made from polylactic acid (PLA). Resulting libraries were then deep-sequenced using next generation sequencing (NGS) to investigate selection and diversity. CONCLUSIONS We demonstrated enrichment for PLA binding versus a sapphire control surface, analyzed population composition, and compared sorting rounds using a binding assay and fluorescence microscopy. The capability to produce and describe display libraries through NGS across rounds of selection allows a deeper understanding of population dynamics that can be better directed towards peptide discovery.

中文翻译：

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下一代生物淘选：下一代测序改进了细菌展示文库的分析。

背景技术细菌表面展示文库由于其易于操作和快速生长速率而成为新配体发现的流行工具。这些文库通常表达嵌入外膜内的支架蛋白，该支架蛋白具有短的、表面暴露的肽，该肽或者是末端的或者被掺入到外环中，因此可以与感兴趣的底物相互作用并结合。结果在这项研究中，我们采用了一种新型细菌肽展示文库，该文库在大肠杆菌表面整合了 15 聚体短肽，并与胞质溶胶中的诱导型红色荧光蛋白 DsRed 共表达，以研究两轮展示中的群体多样性。生物淘选。该原始库用于淘选试验，以选择对由聚乳酸 (PLA) 制成的 3D 打印塑料优惠券的结合亲和力。然后使用下一代测序 (NGS) 对所得文库进行深度测序，以研究选择和多样性。结论 我们证明了 PLA 结合与蓝宝石对照表面的富集，分析了群体组成，并使用结合测定和荧光显微镜比较了分选轮次。通过 NGS 跨轮选择生成和描述展示文库的能力可以更深入地了解群体动态，从而更好地指导肽发现。