By Terry J. McGenity, Kenneth N. Timmis, Balbina Nogales
This quantity offers suitable single-cell and single-molecule techniques within the learn of microbes generating and using hydrocarbons and lipids. whereas generically acceptable for all microorganisms, the ways defined are, anywhere attainable, tailored to the sphere of analysis of hydrocarbon and lipid microbiology. The equipment contain uncomplicated approaches for separating unmarried cells through microfluidics and circulation cytometry, and their cultivation in arrays as natural clones; for setting apart, amplifying and sequencing single-cell genomes and transcriptomes; and for analysing single-cell metabolomes via Raman spectroscopy. Single-molecule methods contain using protein:fluorescent dye fusions for protein localization and strategies for the construction of cellphone department protostructures and lipid monolayers. equipment for the useful research of unmarried cells comprise detection of metabolically energetic (protein-synthesizing) cells in environmental samples by way of bioorthogonal non-canonical amino acid tagging, Raman spectroscopy mixed with reliable isotope labelling and fluorescent in situ hybridisation, and visualization of unmarried cells engaging in gene move task. finally, protocols are offered for single-cell biotechnological functions, together with biofuel production.
Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of hundreds of thousands of structurally various hydrocarbons, hydrocarbon derivatives and lipids, and a wide range of those molecules are required for cells to operate. the worldwide hydrocarbon cycle, that's mostly pushed via microorganisms, has an incredible influence on the environment and weather. Microbes are chargeable for cleansing up the environmental toxins as a result of the exploitation of hydrocarbon reservoirs and also will be pivotal in decreasing our reliance on fossil fuels via supplying biofuels, plastics and commercial chemical compounds. Gaining an realizing of the correct capabilities of the wide variety of microbes that produce, devour and adjust hydrocarbons and similar compounds might be key to responding to those demanding situations. This finished selection of present and rising protocols will facilitate acquisition of this realizing and exploitation of necessary actions of such microbes.
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Extra resources for Hydrocarbon and Lipid Microbiology Protocols: Single-Cell and Single-Molecule Methods
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That is, the fluorescent intensity in a cell resulting from the experiment implies accumulation of multiple mRNAs. In this chapter, we describe the protocol to visualize specific mRNA, expressed from the TOL plasmid pWW0 of the Pseudomonas putida mt-2. The procedure is demonstrated below using the Stellaris FISH probe set including 48 oligos labeled with a fluorophore corresponding to the xylX gene that is transcribed in the presence of aromatic compound such asm-xylene . This method can be adjusted to other RNAs in other bacterial species with some modifications.
Good aseptic techniques and the use of antibiotics for selection are important, and they can be complemented with genetic verification (for instance, plasmid isolation or PCR). When the reporter strain carries a plasmid, it is recommended to test for plasmid loss by plating a sample of the bioreporter cell suspension on agar plates with or without antibiotic selection. The ratio of colony-forming units in the presence or absence of antibiotics thus gives the percentage of cells that have maintained the plasmid; this percentage should be 100% or close.