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THE PULSILATOR C. Batenchuk, D. Jedrysiak, N. Abdennur, M. Orton, T. Tye, K. Jolin-Dahel, K. Morin, S. Adiga, C. Euler, R. Zinoviev, M. Kaern

Department of Cellular and Molecular Medicine, University of Ottawa

A synthetic pulse generator in yeast for sustainable expression of recombinant proteins

Objective

Network Design

Results

Conclusion

Modelling and Simulations

References

Biobrick Dawn Fraser Simon St-Pierre Mila Tepliakova

Dr. Ernesto Andrianantoandro Dr. Ron Weiss

Dr. Thomas Schmülling Dr. James Collins Dr. André Lalonde

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Fig 1. Biobrick characterization. The promoter driving PRP22 expression was investigated as a constitutive promoter.

(a) Basal GFP expression relative to the auto-fluorescence of the wild-type strain BY4741. (b) Dose response of mean fluorescence to the methylating agent methyl methane-sulfonate (MMS). As PRP22 is involved in RNA splicing, increased protein and RNA turnover may increase the activity at this promoter. (c) Dose response of mean fluorescence to the concentration of sodium cholride (NaCl). NaCl was selected to investigate whether activity at this promoter is invoked as a general stress response.

These results suggest that the PRP22 promoter is selectively responsive to increased turnover rates.

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Abstract

[1] Pinasch J., de Mas C., Lopez-Santin J. (2008). Induction strategies in fed-batch cultures for recombinant protein production in Escherichia coli: application to rhamnulose 1-phosphate aldolase. Biochem. Eng. J. 41: 181-187. [2] Weiss R., and Chen M.-T. (2005). Artificial cell-cell communication in yeast Saccharomyces cerevisiae using signaling elements from Arabidopsis thaliana. Nature Biotech. 23(12): 1551-1555. [3] Laskey, J.G., P. Patterson, et al. (2003). "Rate enhancement of cytokinin oxidase/dehydrogenase using 2,6-dichloroindophenol as an electron acceptor." Plant Growth Regulation 40: 189–196 [4] Schmulling T, Werner T, Riefler M, Krupkova E, Bartrina y Manns I (2003) Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. J Plant Res 116:241-252 [5] Alon U., and Mangan S. (2003). Structure and function of the feed-foward loop network motif. PNAS 100(21): 11980-11985.

Fig 2. To the model of the SKN7 signaling pathway we incorporate differential equations describing population dynamics, inducer concentration as well as our pulse generator network modeled using Hill-type kinetics.

1) Inducer (IP) is produced by a sender cell population.

2) IP triggers a signaling pathway in the receiver cell, resulting in activation of transcription factor SKN7.

3) SKN7 directly drives expression of GFP, as well as cytokinin dehydrogenase (CKX) via their SSRE promoters.

4) SKN7 also indirectly represses t h e s e g e n e s b y d r i v i n g expression of the tet repressor.

5) The system is reset by CKX

Today, multi-billion dollar industries depend on the production of recombinant proteins and enzymes. Large-scale production of recombinant proteins for pharmaceutical, industrial or research purposes typically involves growing genetically modified microorganisms in bioreactors. With the metabolic stress imposed by over-expression and the consequent selective pressure against producers, culture productivity tends to diminish rapidly over time1. To overcome this difficulty, we have designed a yeast strain capable of producing short bursts of gene expression in a controlled and inducible manner. Our system uses a feed-forward network motif to generate a pulse of our protein of interest (GFP) as well as an enzyme capable of degrading the inducer molecule, allowing the system to “reset” itself for subsequent induction events. Since this system can be induced repeatedly, co-culture with inducer-synthesizing sender cells can allow oscillatory expression dynamics. Such pulse trains or “pulsilations” of gene expression can increase the sustainability of bioreactor cultures by reducing metabolic load and allowing cells to recover from the stress of over-expression.

Coarse tuning of transcriptional kinetics can be achieved through modification of TATA box, SSRE, and tetO elements.

Pulse dynamics can be fine tuned by the use of anhydrotetracycline (aTc) to inhibit TetR activity, galactose to control IP production, and several electron acceptor compounds4 to modulate CKX activity.

Our objective is to implement a pulse generating gene network in a yeast strain that uses a synthetic signaling pathway which responds to the plant cytokinin isopentenyladenine (IP)..2 To enable production of multiple pulses by repeated stimulation, we incorporate a resetting mechanism whereby the enzyme cytokinin dehydrogenase (CKX) is transiently expressed to degrade the inducer signal3.

Modeling suggests that the pulsilator system is readily tunable and versatile. We are currently finalizing the construction of the complete network and intend to begin testing the production of different recombinant proteins in bio-reactor cultures this winter. We are also developing a general model of the effects of pulsatile protein expression on productivity and yield.

Fig 5. Fine tuning with aTc can modulate the amplitude and refractory period of the pulses, so that pulsilation dynamics can be optimized to the requirements of a particular protein of interest.

Fig 3. In order to map the effects of parameter variation on pulse dynamics, a set of pulse characteristics shown below were defined and measured over many simulations. We observed a general trade-off between short pulse width and high amplitude that occurs as a result of the competition between transcriptional activation by SKN7 and repression by TetR.

Fig 4. Pulsilation simulations. (a) Simulation of the mixed sender-receiver configuration revealed that when the senders produce IP at a constant rate, oscillatory dynamics are relatively fragile, depending highly on the kinetics of enzymatic degradation of IP. (b) However, periodic induction events can produce robust pulsilations, with the maximum frequency of induction depending on the pulse refractory period (time for the system to reset itself).

Acknowledgements

Sponsors

Fig 6. Characterization of the SSRE promoter driving GFP expression. (a) Flow cytometry results illustrating GFP expression driven by a single SSRE element pre (red) and post (blue) induction with 50µM of IP (sigma). (b) Double SSRE element. (c) Dose response curve measuring mean fluorescence as a function of IP concentration. (d) Impact of modulating galactose concentration on mean SSRE expression before optimization of the detection protocol .

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