Stephen N Hyland, Sreedevi Chinthamani, Sushanta Ratna, Kimberly A Wodzanowski, Liam-Michael D Sandles, Kiyonobu Honma, Catherine Leimkuhler-Grimes, Ashu Sharma
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引用次数: 0
Abstract
The objective of this chapter is to provide a detailed protocol for the peptidoglycan (cell wall) labeling of the periodontal pathogen Tannerella forsythia and the development of a laboratory-safe Escherichia coli strain utilizing the N-acetylmuramic acid recycling enzymes AmgK, N-acetylmuramate/N-acetylglucosamine kinase, and MurU, N-acetylmuramate alpha-1-phosphate uridylyltransferase, from T. forsythia. The procedure involves bioorthogonal labeling of bacterial cells with an azido-modified analog of the amino sugar, N-acetylmuramic acid, through "click chemistry" with a fluorescent dye. The protocol is suitable for the generation of fluorescently labeled peptidoglycan molecules for applications in the study of bacterial and peptidoglycan trafficking in the host cells and cell wall recycling in complex microbiomes.
期刊介绍：
For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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