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Update on RelA Project Cloning of amplified relA

pGEM-T Easy. T. T. Update on RelA Project Cloning of amplified relA. PCR. Insert amplified product in both orientations. Update on RelA Project Cloning of amplified relA. pUR351a. pUR351b. Update on RelA Project Controlled overexpression of P tac - relA. ScaI. pUR351a. SalI. SalI.

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Update on RelA Project Cloning of amplified relA

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  1. pGEM-T Easy T T Update on RelA ProjectCloning of amplified relA PCR Insert amplified product in both orientations

  2. Update on RelA ProjectCloning of amplified relA pUR351a pUR351b

  3. Update on RelA ProjectControlled overexpression of Ptac-relA ScaI pUR351a SalI SalI SmaI

  4. Update on RelA ProjectControlled overexpression of Ptac-relA pUR354 (KT) Status • relA fragment in agarose • pRL502a digestion completed relA

  5. Update on RelA ProjectControlled overexpression of PEm-relA ScaI pUR351a ClaI SacI NsiI

  6. pBluescriptKS Update on RelA ProjectControlled overexpression of Ptac-relA ScaI SacI pUR351a EcoRV PstI SacI

  7. pBluescriptKS Update on RelA ProjectControlled overexpression of Ptac-relA PstI pUR355 (Karen) Status • First attempt failed • Second attempt transformed ClaI

  8. Update on RelA ProjectControlled production of antisense relA pUR351a DpnI DpnI SacI SmaI SacI (1072 bp) (93 bp)

  9. Update on RelA ProjectControlled production of antisense relA pUR357 (Julie) Status • DpnI fragment isolated • pRL502a digestion completed DpnI SacI DpnI

  10. Update on RelA ProjectConstruction of relA insertional mutant PvuII pUR351b HpyCH4IV NsiI ClaI SacI

  11. SacI pBluescriptKS SmaI Update on RelA ProjectConstruction of relA insertional mutant PvuII BglI Apr Apr PvuII pUR351b PvuII HpyCH4IV (2476 bp) (2597 bp) SacI

  12. Update on RelA ProjectConstruction of relA insertional mutant pUR352 (Carrie) HpyCH4IVmatches ClaI Status Apr pBluescriptKS • First try failed • Second try transformed PstImatches NsiI

  13. Update on E • Background (Trempy et al; Stragier et al) • Emade first as a precursor protein, P30 • spoIIGA + sigE expression in vegetative cells gives E • Sporulation-dependent expression requires SpoIIE • SpoIIE also required for sporulation-dependent division • Questions • Is SpoIIGA the processing protease? • Does processing require cell division?

  14. Update on ENews fromF • Margolis et al (1991). Science 254:562-565 • F required for expression of forespore genes • But spoIIAC (encodes F) expressed prior to septation • Is F active only in forespore? In both cells? • Method • Fuse F-dependent gene to lacZ • Use antibody to ß-gal and fluorescence microscopy

  15. Update on ENews fromF • Result • F-dependent genes turned on only in forespore • True even in case where nonsporulation gene used. • Conclusion • F active only in forespore • BUT F required for activity of E in mother cell! See Figure 5 of Losick & Stragier

  16. Update on all sporulation ’s Question What controls the activity of the five sporulation ’s? • Sigma Factors • H (spo0H/sigH) Karen • E (spoIIG/sigE) -- • F (spoIIA/sigF) Julie • G (spoIIIG/sigG) Carrie • K (spoIIIC/sigK)KT Presentations Wed, March 28 and Fri, March 30(article by Friday, March 23; meetings to be scheduled)

  17. Caulobacter crescentusCell cycle-regulated differentiation

  18. Caulobacter crescentusCell cycle-regulated differentiation The phenomenon: Sheffery and Newton (1981) Cell 24:49-57 Regulation of periodic protein synthesis in the cell cycle:Control of initiation and termination of flagellar gene expression To be discussed Wednesday, March 21 Focus: Fig 2 (Carrie), Fig 3 (KT), Fig 4 (Julie), Fig 5 (Karen)

  19. Coming Attractions Wed Mar 21 Sheffery & Newton (Caulobacter periodicity) Continue constructs; ligate Fri Mar 23 Quon et al (Caulobacter master regulator) Mon Mar 26 Continue Quon et al; Organismal databases Wed Mar 28 Two presentations Fri Mar 30 Two presentations

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