Wu Jianfeng, Xi Chuanwu
Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI 48109, USA.
Cold Spring Harb Protoc. 2010 Jul 1;2010(7):pdb.prot5456. doi: 10.1101/pdb.prot5456.
Biofilm is an accumulation of bacterial cells encapsulated in a structurally assembled extracellular polymer matrix that adheres to surfaces. The extracellular polymer matrix is composed mainly of polysaccharides, proteins, and amphiphilic polymers, which are secreted by microorganisms into their environment. Extracellular DNA (eDNA) was recently identified as one of the major components of extracellular polymeric substances (EPSs) and plays an important role in the development of biofilm, especially the early stages, by facilitating initial bacterial attachment and maintaining biofilm structure. More research is needed to fully elucidate the role of eDNA in biofilm development, and such investigations require that extraction of eDNA from the complex biofilm matrix is achieved without any genomic DNA contamination. However, the physical and chemical association of eDNA with extracellular proteins, polysaccharides, and other polymers in the complex matrix of EPS may hinder the liberation of eDNA. Therefore, it is difficult to release eDNA and other materials from the biofilm matrix solely by vortexing or homogenizing, and degradation of certain components of EPSs in the biofilm matrix is necessary in order to release eDNAs that may bind to these compounds. In this protocol, N-glycanase (glycoprotein-degradation hydrolase), dispersin B (biofilm-dispersing glycoside hydrolase), and proteinase K (protein hydrolase) are used to break down biofilm matrix for more efficient and complete extraction of eDNA.
生物膜是包裹在结构有序的细胞外聚合物基质中的细菌细胞聚集体，该基质附着于表面。细胞外聚合物基质主要由多糖、蛋白质和两亲聚合物组成，这些物质由微生物分泌到其周围环境中。细胞外DNA（eDNA）最近被确定为细胞外聚合物（EPS）的主要成分之一，并且在生物膜形成过程中，尤其是在早期阶段，通过促进细菌的初始附着和维持生物膜结构发挥重要作用。需要更多的研究来充分阐明eDNA在生物膜形成中的作用，而此类研究要求从复杂的生物膜基质中提取eDNA时不受到任何基因组DNA的污染。然而，在EPS的复杂基质中，eDNA与细胞外蛋白质、多糖和其他聚合物之间的物理和化学结合可能会阻碍eDNA的释放。因此，仅通过涡旋或匀浆很难从生物膜基质中释放eDNA和其他物质，为了释放可能与这些化合物结合的eDNA，有必要降解生物膜基质中EPS的某些成分。在本方案中，N-聚糖酶（糖蛋白降解水解酶）、分散素B（生物膜分散糖苷水解酶）和蛋白酶K（蛋白水解酶）用于分解生物膜基质，以更高效、完整地提取eDNA。
