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PhoU localization Experiment

This study explores the localization of PhoU, a 27kDa protein involved in the regulation of the phosphate transport (Pst) system in E. coli. Utilizing a GFP-tagging approach in various knockout mutant strains, we investigate the association of PhoU with old-pole-localized proteins. Preliminary findings indicate significant effects on localization with certain proteins, such as PldB, Ydik, and WzzB. The study addresses reliability issues in experimental conditions and suggests future experiments to better understand PhoU's role in Pi repression.

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PhoU localization Experiment

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  1. PhoU localization Experiment Andrew Christopher Wanner Lab

  2. Introduction: PhoU • PhoU: involved in Pst system regulation, • Associates with PhoRR, PstSCAB & PhoB to form the Pi repression complex

  3. Introduction: PhoU • 226 aa long, 27kDa • Cytoplasmic • PDB id: 1SUM

  4. Introduction: PhoU • Localized at the old pole • PyMol image: Old pole Division new pole

  5. Hypothesis • The localization of PhoU at the old pole is caused by an association with an old-pole-localized protein

  6. Materials • Media: TYE & TYE-Cm agar plates, SOB, SOC, QIAprep Miniprep kit • E. coli ASKA – GFP library • E. coli KO library (103 strains found to localize in old pole)

  7. ASKA-GFP library construction ORF phoU transformation E. Coli WT + p(phoU-GFP)

  8. The KO Library Gene Knockout Strategy

  9. The KO Library Primer Design and Construction of “KO Collection”

  10. Method • Extract the gfp-phoU plasmid (QIA Miniprep) • Revive KO mutants pGFP-PHOU + Cm TYE Plates

  11. Method • Transform phoU-GFP plasmid into KO mutants (cell-porator) E.Coli cell with 1 chromosomal KO gene & Gfp-tagged phoU expression

  12. Method • Observe transformed mutants under confocal microscope, look for GFP expression level outside the old pole • Repeat experiment again for the potential strains Delocalized Gfp

  13. Result • 3 old-pole proteins with most significant localization effect: • pldB: 50-50 (delocalized:localized) • ydik: 30-70 • wzzB: 25-75 • Compared to WT phoU-GFP strain, ~13 mutant strains have localized GFP but significantly increased delocalized [GFP]

  14. Problems • Reliability • Differing results after experiment was repeated • Due to equipment used, varying experimental condition • Future experiment: • repeating the localization experiment • blind condition • with random control strains. • Observe localization of similar old-pole proteins

  15. Acknowledgements • Dr. Barry Wanner • Yi-Ju Hsieh • Jie Shao • Yang • Howard Hughes Medical Institute

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