INCORPORATION OF FLUORESCENT ANALOGUES IN ISOPRENOID PATHWAYS IN ESCHERICHIA COLI
1 online resource (96 pages) : PDF
University of North Carolina at Charlotte
Bacterial glycans and glycoconjugates have wide diversity compared to their eukaryotic counterparts. While methods have been developed to monitor and track glycans in eukaryotic systems with some success, there are limited methods with which to do so in prokaryotes. Work conducted by Bertozzi has utilized alkyne-tagged sugars that, once incorporated into enzymatic pathways, can be clicked to a fluorophore for characterization.1 However, due to the increased number of available sugars and potential combinations, this approach this approach becomes challenging in bacteria. Isoprenoids are one of the largest groups of naturally occurring compounds and are essential in all living organisms.2 They are constituted of the building block isopentenyl diphosphate (IPP) and, in prokaryotes, are vital for nearly all polysaccharide biosynthetic pathways via the formation of the anchor bactoprenyl phosphate (BP).3 This anchor is utilized in the cell membrane for a variety of biosynthetic pathways including those related to peptidoglycan (PG), capsular polysaccharides (CPS), exopolysaccharides (EPS), and others.4 This work examines utilizing an exogenously introduced fluorescent isoprenoid to tag and monitor the formation of glycan intermediates in Escherichia coli. This approach has applications in identifying the functions of pertinent enzymes in previously unexamined pathways as well as better understanding associated glycan intermediates. The presented research discusses preliminary methods for incorporation of fluorescent compounds as well as several applications.
Afonin, KirillVivero-Escoto, JuanSteck, Todd
Thesis (M.S.)--University of North Carolina at Charlotte, 2018.
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