Abstract：
Bacterial biofilms consist of surface-attached communities that secrete polymeric substances to form a biofilm matrix, generating a local microenvironment which helps protect from external factors. One such matrix component produced by a diverse list of microorganisms is the polysaccharide poly-β-1,6-N-acetylglucosamine (PNAG). Dispersin B is a PNAG-specific glycosyl hydrolase, which by leveraging its unique specificity, can be used to design a macromolecular fluorescent PNAG binding probe. An active site mutant of Dispersin B was fused to a fluorescent protein, to generate a probe that bound PNAG but did not hydrolyze its polysaccharide target. The ease and versatility of this strategy has made it possible to study PNAG in the context of maturing biofilms, as the probe tends to sequester in regions of high PNAG density. In this chapter, typical workflows from probe construction to cell-binding and imaging experiments are described.
摘要：
细菌生物膜由表面附着的群落组成，分泌聚合物形成生物膜基质，生成有助于保护免受外部因素影响的局部微环境。由多种微生物产生的一种这样的基质组分是多糖聚-β-1,6-N-乙酰葡糖胺(PNAG)。分散素B是一种PNAG特异性糖基水解酶，通过利用其独特的特殊性，可用于设计大分子荧光PNAG结合探针。分散蛋白B的活性位点突变体与荧光蛋白融合，以产生结合PNAG但不水解其多糖靶标的探针。这种策略的易用性和多功能性使得在成熟生物膜的背景下研究PNAG成为可能，因为探针倾向于在高PNAG密度的区域中隔离。在这一章中,描述了从探针构建到细胞结合和成像实验的典型工作流程。
