Presented by: University of Ottawa iGEM 2008 Nations Capital - PowerPoint PPT Presentation

Presented by: University of Ottawa iGEM 2008

Nation’s Capital

University of Ottawa • Research intensive University • First participation in iGEM competition • Team exclusively composed and run by undergraduates

Recombinant Proteins

Expression System Bacteria Yeast Plant Mammalian Cells

I’m Limitations Tired Over-expression of recombinant protein • High metabolic burden + stress • Reduced cell growth and productivity • Selective advantage of non-producers

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

Our Project: The Pulsilator

Chen M.-T., and Weiss R. (2005). Chassis Nature Biotech. 23(12): 1551-1555.

Chen M.-T., and Weiss R. (2005). Chassis Nature Biotech. 23(12): 1551-1555.

Chen M.-T., and Weiss R. (2005). Chassis Nature Biotech. 23(12): 1551-1555. Receiver cells Sender cells

Chen M.-T., and Weiss R. (2005). Chassis Nature Biotech. 23(12): 1551-1555. Receiver cells Incorporate: (1) tet repressor feedforward Sender cells (2) Inducer degradation – Ckx Objectives: (1) Enable pulse-generation (2) Design network reset

Pulse Generator Network Motif Incoherent Type 1 Feedforward loop IP SKN7 TetR IT1 FFL GFP

Pulse Generator Reset CKX Neg. Feedback IT1 FFL IP SKN7 TetR IT1 FFL GFP

The Design

The Design

Non-Genetic Knobs and Dials CKX E A gal/gluc IP SKN7 TetR aTc GFP

Modeling

Chen & Weiss, 2005

Chen & Weiss, 2004 Population dynamics Inducer Pulse Generator Network

Pulse Characteristics c 1.5 Concentration (a.u.) Amp. 1.3 Gain = (c-b)/b Resp. Width Time 1.1 b Offset = b-a a 0 10 20 30 Time (h)

Mixed Sender-Receiver Population

Mixed Sender-Receiver Population 0.050 k deg = 0.5 min -1 0.025 Concentration (a.u.) Pulsilator oscillations 0 somewhat fragile to 0 25 50 75 variation in Ckx 0.050 k deg = 50 min -1 parameters 0.025 0 0 25 50 75 Time (hours) K M = 40 K M = 0.15 K M = 1.5 K M = 0.015

Periodic Induction Pulsilation!!!

Periodic Induction

Periodic Induction

Periodic Induction offset offset …Low-pass filter!!!

Fine Tuning E A CKX gal/gluc IP aTc SKN7 TetR GFP

Fine Tuning Concentration (a.u.) 1.0 0.5 0 0 40 80 Time (min) No aTc Medium aTc Low aTc High aTc

Modeling Summary • 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

Lab Work Coarse tuning (genetic)

SSRE Induction by IP Flow cytometry – single SSRE Cell Count Fluorescence Intensity at 530nm

SSRE Induction by IP Flow cytometry – tandem SSRE 900 With IP Without IP Cell Count 10 0 10 1 10 2 10 3 Fluorescence Intensity (530nm)

SSRE Induction by IP IP dose response curve Mean Fluorescence (530nm) IP concentration (µM)

Future Directions • Maximize induction by IP • Integrate Ckx/TetR elements • Monitor IP decay by MS • Fine tuning • Adapt protocol for bioreactor production of different recombinant proteins

Acknowledgements • 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

Thank you to our sponsors