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Team: Paul Bourgine, Sandra Dittmann, Katja Karstens, Manuel Gersbacher, Marius Müller, Robin Sorg, Michael Wild
Cell division counter Katja Karstens, Manuel Gersbacher, Marius - - PowerPoint PPT Presentation
Cell division counter Katja Karstens, Manuel Gersbacher, Marius - - PowerPoint PPT Presentation
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
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Cell division counter
- Binary system
- Extendable
⇒ Cell cycle dependent toggle switch
- Chassis: Yeast
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Applications
- Detection of abnormal growth (cancer)
- Coupling to cell cycle specific factors
- Age determination in cell culture
- ...
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The Project
In Detail ...
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external signal
counting device
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inducer
0 – module 1 – module
external signal
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sA1 R1 cA1
* *
1 – module
*
sA0 R0* cA0
external signal
inducer
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sA1 R1 cA1
* *
APC
internal signal
*
1 – module
*
sA0 R0* cA0
external signal
inducer
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sA1 R1 cA1
* *
APC
internal signal
*
1 – module
*
sA0 R0* cA0
external signal
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sA1 R1 cA1
* *
APC
internal signal
*
1 – module
*
sA0 R0* cA0
external signal
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sA1 sA0 R1 cA1 cA0 R0*
* * *
external signal
APC
internal signal
*
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sA1 sA0 R1 cA1 cA0 R0*
* * *
external signal
APC
internal signal
*
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Modeling
Characterization of parts ...
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Modeling: questions to be solved
I. Protein half life time
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?
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Modeling: promoter construction
TATA
One-operator system:
TATA
Two-operator system:
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
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Modeling: cell cycle dependent toggle switch
binary expression pattern
0 – specific expression 1 – specific expression
TF concentrations
Repressor0 Repressor1 cActivator0 cActivator1
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Outcome
What we reached to do ...
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sA1 sA0 R1 cA1 cA0 R0*
* * *
external signal
APC
internal signal
*
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- 3-7 operator copies
- Mutated Cyc1 promoter
Modular promoter
Kozak TATA
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sA1 sA0 R1 cA1 cA0 R0*
* * *
external signal
APC
internal signal
*
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Parts construction
BD AD/RD XFP APC NLS PEST DNA-BS TATA ADH1T
Linker Linker Linker Linker Linker
AD/RD
BD
XFP
NLS PEST APC
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BioBrick
TCTAGA AGATCT G C ACTAGT TGATCA T A
New fusion vector
Phillips and Silver (2006)
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BioBrick I BioBrick II BioBrick I BioBrick II
in frame
TCTAGA AGATCT
A
TGATC ACTAGT TGATCA CTAGA
T
TGATCT ACTAGA
New fusion vector
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- 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
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Measurement
- Optimal arrangement of domains
- Strength of the promoters
- Functioning of the tags
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Optimal arrangement of domains
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Parts submitted
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Acknowledgments
- Dr. Mariel Donzeau
- Michael Zimmermann &
Martin Dressler
- Prof. Peter Philippsen
- Prof. Bruno Chatton & Lab
- Dr. Vincent Phalipp & Lab
- Prof. Haiech & Maria
- Prof. Claude Kedinger
- The iGEM Freiburg Team
- Martin Cerff, Flo Rau & Tony Wiciok
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