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Cell division counter Katja Karstens, Manuel Gersbacher, Marius - PowerPoint PPT Presentation

Jan 22, 2023 •151 likes •455 views

Team : Paul Bourgine, Sandra Dittmann, Cell division counter Katja Karstens, Manuel Gersbacher, Marius Mller, Robin Sorg, Michael Wild ESBS Strasbourg Cell division counter - Binary system - Extendable Cell cycle dependent toggle switch

  1. Team : Paul Bourgine, Sandra Dittmann, Cell division counter Katja Karstens, Manuel Gersbacher, Marius Müller, Robin Sorg, Michael Wild

  2. ESBS Strasbourg

  3. Cell division counter - Binary system - Extendable ⇒ Cell cycle dependent toggle switch - Chassis: Yeast

  4. Applications - Detection of abnormal growth (cancer) - Coupling to cell cycle specific factors - Age determination in cell culture - ...

  5. The Project In Detail ...

  6. external signal counting device

  7. external signal inducer 0 – module 1 – module

  8. external signal * R0 * inducer sA0 cA0 * * sA1 R1 cA1 1 – module

  9. external signal * R0 * inducer sA0 cA0 internal signal * APC * * sA1 R1 cA1 1 – module

  10. external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1 1 – module

  11. external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1 1 – module

  12. external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1

  13. external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1

  14. Modeling Characterization of parts ...

  15. Modeling : questions to be solved Protein half life time I. dilution sufficient? PEST? II. APC degradation motif time frame of activity? III. Promoter construction sA/cA/R expression strength? N° of operator repetitions? same operator for A and R?

  16. Modeling: promoter construction One-operator system: Two-operator system: TATA TATA A:R = 1:0 A:R = 1:1 A:R = 1:2 A:R = 1:3 A:R = 1:0 A:R = 1:1 A:R = 1:2 A:R = 1:3

  17. Modeling: cell cycle dependent toggle switch binary expression pattern TF concentrations 0 – specific expression 1 – specific expression Repressor0 Repressor1 cActivator0 cActivator1

  18. Outcome What we reached to do ...

  19. external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1

  20. Modular promoter Kozak TATA • 3-7 operator copies • Mutated Cyc1 promoter

  21. external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1

  22. Parts construction AD/RD NLS XFP APC PEST BD BD XFP AD/RD APC NLS PEST ADH1T DNA-BS TATA Linker Linker Linker Linker Linker

  23. New fusion vector TCTAGA T G ACTAGT BioBrick A TGATCA AGATCT C Phillips and Silver (2006)

  24. New fusion vector A TCTAGA BioBrick I TGATC AGATCT ACTAGT CTAGA BioBrick II TGATCA T in frame ACTAGA BioBrick I BioBrick II TGATCT

  25. ‣ VP16 activator domain for eucaryotic polymerase III from Herpes Simplex Virus ‣ repression domain of S. cerevisiae transcription factor TUP1 ‣ DNA binding domains with specific bait sequences (tetR, cI, lacI, lexA, Gal4) ‣ degradation motifs of hsl1 & cln2

  26. Measurement • Optimal arrangement of domains • Strength of the promoters • Functioning of the tags

  27. Optimal arrangement of domains

  28. Parts submitted

  29. Acknowledgments ‣ Dr. Vincent Phalipp & Lab ‣ Dr. Mariel Donzeau ‣ Prof. Haiech & Maria ‣ Michael Zimmermann & ‣ Prof. Claude Kedinger Martin Dressler ‣ The iGEM Freiburg Team ‣ Prof. Peter Philippsen ‣ Martin Cerff, Flo Rau & Tony Wiciok ‣ Prof. Bruno Chatton & Lab

  30. Thank you for your attention! Merci ! Danke !

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