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MIT7_13f08_lab01_ProjectSummaries

MIT7_13f08_lab01_ProjectSummaries - MIT OpenCourseWare...

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MIT OpenCourseWare http://ocw.mit.edu 7.13 Experimental Microbial Genetics Fall 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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Project List (1) Determining whether phenazines can support intracellular iron acquisition (2) Selecting and screening for mutations in Fur (3) Determining the mechanism of wrinking in response to phenazines (4) Screening different CF-lung isolates for phenazine production and identifying their SoxR regulon (5) Identifying genes required for swimming motility (6) Identifying genes required for twitching motility and pili biogenesis (7) Identifying genes required for swarming motility (8) Identifying genes that contribute to antibiotic resistance and phenotypic variation
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Project 1 : Determining whether phenazines can support intracellular iron acquisition One of the physiological functions that may be important for phenazines is their role in iron acquisition. Through redox reactions with ferric iron, phenazines reduce ferric iron to ferrous iron, which is more bioavailable. In strain PAO1, it has been shown that the ability of P. aeruginosa to acquire iron is critical for biofilm formation. In PAO1 (which is compromised in phenazine production), iron acquisition is thought to be achieved mainly by siderophores (high-affinity iron-binding compounds), however, in strain PA14, both siderophores and phenazines appear to be important. One way to test this is to determine whether phenazines affect the intracellular iron pool in PA14. pvdS (a gene involved in the biosynthesis of the siderophore, pyoverdine) is activated by a transcription factor that senses intracellular Fe. Students will make a transcriptional gfp fusion to pvdS , and express it both on a plasmid or in single-copy on the chromosome in different PA14 strains (e.g. wild-type, phenazine mutant, siderophore mutant, and phz/sid mutant). Fluorescence spectroscopy and microscopy will be used to monitor gene expression under different conditions. Key reference: E. Banin, M.L. Vasil, E.P. Greenberg (2006) Iron and Pseudomonas aeruginosa biofilm formation, PNAS , 102:11076-11081.
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Project 1 – Flow Chart Construct a transcriptional gfp fusion to the promoter of the pvdS gene Move this construct onto the chromosome and/or a plasmid Check for expression using spectrofluorimetry under different conditions in liquid medium for WT cells Move construct into different strain backgrounds Compare fluorescence in different strains using spectrofluorimetry in liquid and microscopic observation Compare biofilm development of different mutant constructs under different conditions
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Project 2: Selecting and screening for mutations in Fur Fur is an iron-dependent transcription factor responsible for turning on the expression of genes involved in iron acquisition (including pvdS , see project 1). Its activity appears to be influenced by phenazine production (i.e. in the presence of phenazines, Fur is silenced). In Pseudomonas aeruginosa , fur appears to be an essential gene (i.e. mutants that cannot make Fur die).
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