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This volume provides the most widely used protocols and techniques employed in lipopolysaccharide( LPS) transport studies. Chapters detail identification and analysis of essential genes and crucial mutations in LPS transport-related genes, LPS profile determination, LPS chemical analysis, protein-LPS interaction, and LPS visualization. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and cutting-edge, Lipopolysaccharide Transport: Methods and Protocols aims to deliver an overview on the multidisciplinary study of LPS transport and to identify new and innovative antibacterial strategies.
List of contents
Use of Mutagenesis and Functional Screens to Characterize Essential Genes Involved in Lipopolysaccharide Transport.- Generation of Stable and Unmarked Conditional Mutants in Pseudomonas aeruginosa.- Analysing the Function of Essential Genes by Plasmid Shuffling.- Membrane Fractionation by Isopycnic Sucrose Density Gradient Centrifugation for Qualitative Analysis of LPS in Escherichia Coli.- Exploring The Topology of Cytoplasmic Membrane Proteins Involved in Lipopolysaccharide Biosynthesis by in silico and Biochemical Analyses.- Use of Site-Directed Spin Labeling EPR Spectroscopy to Study Protein-LPS Interactions.- A Photo-Crosslinking Approach to Monitor Assembly of an LptD Intermediate with LptE in a Living Cell.- Mass Spectrometry Analysis of Dynamics and Interactions of the LPS translocon LptDE.- Affinity Purification and Co-Immunoprecipitation of Trans-envelope Protein Complexes in Gram-negative Bacteria.- A Bacterial Two-Hybrid System for in vivo assays of Protein-Protein Interactions and Drug Discovery.- Detecting Lipoproteins Sneaking out of the Lipopolysaccharide Leaflet.- Dissecting Lipopolysaccharide Composition and Structure by GC-MS and MALDI Spectrometry.- Probing the Functional Interaction Interface of Lipopolysaccharide and Antimicrobial Peptides: A Solution-State NMR Perspective.- Cryo-EM Analysis of the Lipopolysaccharide Flippase MsbA.- Protein Crystallization of Two Recombinant Lpt Proteins.- Metabolic Incorporation of Azido-Sugars into LPS to Enable Live Cell Fluorescence Imaging.- Use of Atomic Force Microscopy to Characterize LPS Perturbations.
