PhoU localization Experiment

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