Team : Paul Bourgine, Sandra Dittmann, Cell division counter Katja Karstens, Manuel Gersbacher, Marius Müller, Robin Sorg, Michael Wild
ESBS Strasbourg
Cell division counter - Binary system - Extendable ⇒ Cell cycle dependent toggle switch - Chassis: Yeast
Applications - Detection of abnormal growth (cancer) - Coupling to cell cycle specific factors - Age determination in cell culture - ...
The Project In Detail ...
external signal counting device
external signal inducer 0 – module 1 – module
external signal * R0 * inducer sA0 cA0 * * sA1 R1 cA1 1 – module
external signal * R0 * inducer sA0 cA0 internal signal * APC * * sA1 R1 cA1 1 – module
external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1 1 – module
external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1 1 – module
external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1
external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1
Modeling Characterization of parts ...
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?
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
Modeling: cell cycle dependent toggle switch binary expression pattern TF concentrations 0 – specific expression 1 – specific expression Repressor0 Repressor1 cActivator0 cActivator1
Outcome What we reached to do ...
external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1
Modular promoter Kozak TATA • 3-7 operator copies • Mutated Cyc1 promoter
external signal * R0 * sA0 cA0 internal signal * APC * * sA1 R1 cA1
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
New fusion vector TCTAGA T G ACTAGT BioBrick A TGATCA AGATCT C Phillips and Silver (2006)
New fusion vector A TCTAGA BioBrick I TGATC AGATCT ACTAGT CTAGA BioBrick II TGATCA T in frame ACTAGA BioBrick I BioBrick II TGATCT
‣ 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
Measurement • Optimal arrangement of domains • Strength of the promoters • Functioning of the tags
Optimal arrangement of domains
Parts submitted
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
Thank you for your attention! Merci ! Danke !
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