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Presented by: University of Ottawa iGEM 2008 Nations Capital - - PowerPoint PPT Presentation
Presented by: University of Ottawa iGEM 2008 Nations Capital - - PowerPoint PPT Presentation
Presented by: University of Ottawa iGEM 2008 Nations Capital University of Ottawa Research intensive University First participation in iGEM competition Team exclusively composed and run by undergraduates Recombinant Proteins
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University of Ottawa
- Research intensive University
- First participation in iGEM competition
- Team exclusively composed and run by undergraduates
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Recombinant Proteins
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Expression System
Bacteria Plant Mammalian Cells Yeast
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Over-expression of recombinant protein
- High metabolic burden + stress
- Reduced cell growth and productivity
- Selective advantage of non-producers
I’m Tired
Limitations
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Our Project: The Pulsilator
Synthesize recombinant proteins in short, controlled pulses, rather than continuously
- Reduce average metabolic load and cellular stress
- Allow cells time to recuperate
- Maintain productivity of culture over long periods of time
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Our Project: The Pulsilator
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Chen M.-T., and Weiss R. (2005). Nature Biotech. 23(12): 1551-1555.
Chassis
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Chen M.-T., and Weiss R. (2005). Nature Biotech. 23(12): 1551-1555.
Chassis
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Sender cells Receiver cells
Chen M.-T., and Weiss R. (2005). Nature Biotech. 23(12): 1551-1555.
Chassis
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Sender cells Receiver cells Objectives: (1) Enable pulse-generation (2) Design network reset Incorporate: (1) tet repressor feedforward (2) Inducer degradation – Ckx
Chen M.-T., and Weiss R. (2005). Nature Biotech. 23(12): 1551-1555.
Chassis
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SKN7 TetR GFP IP
IT1 FFL
Incoherent Type 1 Feedforward loop
Pulse Generator Network Motif
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SKN7 TetR GFP IP CKX
IT1 FFL Neg. Feedback IT1 FFL
Pulse Generator Reset
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The Design
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The Design
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SKN7 TetR GFP IP CKX
aTc EA
gal/gluc
Non-Genetic Knobs and Dials
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Modeling
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Chen & Weiss, 2005
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Chen & Weiss, 2004 Pulse Generator Network Population dynamics Inducer
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Pulse Characteristics
Time (h) 20 30 10 Resp. Time Width Gain = (c-b)/b Offset = b-a 1.1 1.3 1.5 Amp. Concentration (a.u.) a b c
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Mixed Sender-Receiver Population
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Pulsilator oscillations somewhat fragile to variation in Ckx parameters
KM = 40 KM = 1.5 KM = 0.15 Time (hours)
Concentration (a.u.)
0.050 25 50 75 0.025 0.050 0.025 25 50 75
kdeg= 0.5 min-1 kdeg= 50 min-1 KM = 0.015
Mixed Sender-Receiver Population
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Pulsilation!!! Periodic Induction
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Periodic Induction
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Periodic Induction
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- ffset
- ffset
…Low-pass filter!!! Periodic Induction
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SKN7 TetR GFP IP CKX
aTc EA
gal/gluc
Fine Tuning
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0.5 1.0 No aTc Medium aTc Low aTc High aTc 40 Time (min) 80 Concentration (a.u.)
Fine Tuning
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- Pulse generation
– Pulse characteristics sensitive to transcriptional kinetic parameters
- Pulsilation
– Sender-receiver configuration
- Highly sensitive to enzyme kinetics of IP degradation
– Periodic induction configuration
- Robust
- Low-pass filter
- Tunable
Modeling Summary
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Lab Work
Coarse tuning (genetic)
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SSRE Induction by IP
Fluorescence Intensity at 530nm Cell Count
Flow cytometry – single SSRE
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SSRE Induction by IP
100
Fluorescence Intensity (530nm)
101 102 103
Cell Count
Without IP With IP
900
Flow cytometry – tandem SSRE
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IP dose response curve
Mean Fluorescence (530nm) IP concentration (µM)
SSRE Induction by IP
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- Maximize induction by IP
- Integrate Ckx/TetR elements
- Monitor IP decay by MS
- Fine tuning
- Adapt protocol for bioreactor production of
different recombinant proteins
Future Directions
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- Mila Tepliakova
- Simon St. Pierre
- Dawn Fraser
- Dr. Ron Weiss
- Dr. Ernesto Andrianantoandro
- Dr. James Collins
- Dr. Thomas Schmülling
- Dr. Jikta Frébortová
- Dr. André Lalonde
Acknowledgements
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