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Progress Report

Progress Report. Wu, Kuan-Yi. 921. Why parA ?. deconvoluted parA. wt-J3310. ΔparA-J3318. Dagmara Jakimowicz et al (2007). Undergoing projects. Localization of ParA scoe in E.coli Strains defect in other cytoskeleton proteins In the presence of ParB scoe

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Progress Report

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  1. Progress Report Wu, Kuan-Yi 921

  2. Why parA?

  3. deconvoluted parA wt-J3310 ΔparA-J3318 Dagmara Jakimowicz et al (2007)

  4. Undergoing projects • Localization of ParAscoe in E.coli • Strains defect in other cytoskeleton proteins • In the presence of ParBscoe • Correlations with chromosome • ParAscoe in vitro studies • Plasmid construction for fusion proteins expression

  5. pMLB1113 in E.coli

  6. constructs • Plac-yfp (pSOT7) • Plac-yfp::parA (pSOT27) • Plac-parA1::parB (pSOT1013) • Plac-yfp :: parA1-parB (pSOT1014) • Plac-parB (pSOT1005) • Plac-parB::yfp (pSOT1016) • Plac-yfp::parB (pSOT1015)

  7. ParA localization • yfptagged on N-terminus • Fixed and live cell ovservation • Combination plasmids/ strains ☆ ☆

  8. pSOT27/MC1000 pSOT7/MC1000 pMLB1113/MC1000 Helical structure? Interacting with chromosome? Par A movement in the live cell?

  9. pSOT27/YLS1 pSOT7/YLS1 pMLB1113/YLS1

  10. pSOT27/RC1 pSOT7/RC1 pMLB1113/RC1

  11. yfp-ParA- B/MC1000 Real spots or only collection of inclusion bodies? How ParB affect the ParA distribution pattern in E. coli?

  12. Yfp-ParAlive cell observation

  13. Could ParA affect chromosome localization and package?

  14. parA- B/MC1000 pSOT26/MC1000 pMLB1113/MC1000 Chromosome length/package correspond to cell lenth?

  15. yfp::parB/MC1000 Localization correspond to the cell length?

  16. Yfp-ParBlive cell observation

  17. Still far from any conclusion…

  18. cloning for protein expression pHTPP10-His*6-CBP-parA1scoe pHTPP11-GST-His*6-parA1scoe pHTPP12-NUS-His*6-parA1scoe pHTPP13-His*6-parA1scoe pHTPP14-malE-2XHis*6-parA1scoe pHTPP15-Trx-His*6-parA1scoe (pSOT29) Which gives us most solubeParA?

  19. NEXT STEP • ParAscoe localization in the absence of mreB • Whole par system(Par A, B and S) in E. coli • Dynamic ParAscoe in S. coelicolor • ParAscoe in vitro studies • Protein purification • Functional assays • polymerization

  20. A22 pSOT27/YLS2 • pLE7(Plac::yfp-mreB) • Time required for A22 to deplete mreB before morphology changed • pMLB1113/MC1000(Plac) • Morphology change • MeOH treatment • Solvent control A22 treatment

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