(4t) Systems and Synthetic Biology in Bacterial and Human T-Cells

The main focus of my dissertation work is to uncover operating principles in Escherichia coli metabolism and regulation. Due to the highly complex nature of biological systems, fundamental principles are often masked by secondary effects (1-2). To uncover various design principles, different research approaches were taken: systems and synthetic.

Using the systems approach, I uncovered a relationship where the growth rate is less than or equal to the square-root of the product among the biomass yield, the protein synthesis rate of the carbon source transporter and its turnover number. Through theoretical analysis, this relationship appears to be an evolutionary optimal. I further demonstrated that adaptively evolved strain, knockout mutants, and ethanol-challenged strains also follow the square-root relationship. Moreover, this relationship provides an explanation as to why the maximal growth rate does not occur at the maximal yield, and a design principle for engineering product formation strains (3-4).

Through the synthetic approach, a gene-metabolic oscillator, termed ?metabolator?, was designed and constructed. Autonomous oscillations found in neuronal, cardiac, metabolic, and gene expression systems have attracted significant attention because of their biological significance and their intriguing dynamics. We constructed a synthetic gene-metabolic oscillator in Escherichia coli K12 using the glycolytic flux to drive transcriptional and metabolic oscillation through a signaling metabolite, acetyl phosphate. We showed that metabolic oscillation can be generated from metabolic flux imbalance (5-7).

Furthermore, single-cell characterization of the proteolysis system used in the metabolator showed zeroth-order kinetics. In addition, we showed that zeroth-order degradation kinetics, coupled with long-tailed initial protein distribution, will generate first-order population degradation kinetics. Through simulation, I demonstrated that zeroth-order kinetics can significantly enhance the robustness of the metabolator. This result highlights the importance of single-cell measurements in determining the dynamics of a process (8).

Currently at UCSF, I am employing the systems and synthetic approach to understand human T-cells signaling and create T-cells with novel function for therapeutic purpose. Adoptive transfer of T-cells expressing chimeric antigen receptors (a fusion of tumor specific single-chain antibodies and intracellular signaling domains from the T-cell receptor (TCR) signaling pathway) has shown promise as an anti-tumor therapy. However, their efficacy is limited because of limited in vivo activity, T-cell exhaustion after ex vivo expansion, low persistence inside the host, and potential safety concerns. In the Cell Propulsion Lab, we are applying synthetic biology approaches to engineer and optimize the behaviors of these tumor targeted T-cells. By engineering synthetic positive and negative feedback loops in T-cells, we show that we can precisely tune the amplitude and dynamics of the T-cell response. We have demonstrated that expression of bacterial effector proteins can be used to shut down and attenuate the response. Conversely, we have also shown that we can amplify output by expressing fusions of signaling proteins that mimic an activated signaling complex. The modularity of these circuits should allow us to construct and search for altered response profiles that improve the persistence and anti-tumor activity of engineered T-cells. Our approaches complement ongoing work on chimeric antigen receptor engineering. Furthermore, this work has yielded a new set of genetic tools to control the T-cell signaling dynamics.

1 Wong, W.W., Liao, J.C. The Synthetic Approach for Regulatory Circuits and Metabolism. (Systems Biology and Synthetic Biology, John Wiley & Sons)

2 Wong, W.W., Liao, J.C. A Synthetic Approach to Transcriptional Regulatory Engineering. (In Press, The Metabolic Pathway Engineering Handbook: Fundamentals, CRC Press)

3 Wong, W.W., Tran, L.M., Liao, J.C. 2009 A hidden square-root boundary between growth rate and biomass yield. Biotechnology and Bioengineering. 1(102): 73-80.

4 Wong, W.W., Tran, L.M., Liao, J.C. 2009 Microbial maximal specific growth rate as a square-root function of biomass yield and two kinetic parameters. Metabolic Engineering 11(6):409-14

5 Fung, E., Wong, W.W., Suen, J.K., Bulter, T., Lee, S., Liao, J.C. 2005. A synthetic gene-metabolic oscillator. Nature. 435(5): 118-122.

6 Bulter, T., Lee, S., Wong, W.W., Fung, E., Connor, M. and Liao, J.C. 2004. Design of artificial cell-cell communication for gene and metabolic circuits. Proceedings of the National Academy of Science, USA. 101(8): 2299-2304.

7 Wong, W.W., Liao, J.C. 2006. The design of intracellular oscillators that interact with metabolism. Cellular and Molecular Life Science. 63(11): 1215-1220.

8 Wong, W.W., Tsai T.Y., Liao, J.C. 2007. Single-cell zeroth-order protein degradation enhances the robustness of synthetic oscillator. Molecular Systems Biology. 3(130): 1-8.

9. Xie, X., Wong, W.W., Tang, Y. 2007. ?Improving simvastatin bioconversion in Escherichia coli by deletion of bioH. Metabolic Engineering. 9(4): 379-386. 10. Wong, W.W., and Newman, J.S. 2002. Monte Carlo Simulation of the Open-Circuit Potential and the Entropy of Reaction in Lithium Manganese Oxide. Journal of the Electrochemical Society. 149(4):A493-A498.

11. Wang, J., Chun, H.J., Wong, W.W., Spencer, D.M., and Lenardo, M.J. 2001. Caspase-10 is an initiator caspase in death receptor signaling. Proceedings of the National Academy of Science, USA. 98(24): 13884-13888.

12. Brynlidsen, M.P., Wong, W.W., Liao, J.C. 2005. Transcriptional regulation and metabolism. Biochemical Society Transactions. 33(6): 1423-1426.