In the proposed project, we will continue to design, construct and characterize genetic circuits. We will use microfluidic tools to grow and observe single cells in precisely controlled environmental conditions. Single cell data will inform a set o mathematical models that will be used to identify key design characteristics, which will then be rigorously, tested using previously established molecular biology techniques. This multi- disciplinary approach will increase our understanding of gene regulation and lead to new tools for the synthetic biology community.
Our first aim will be to explore the interaction of "nested clocks". We previously constructed a robust intracellular clock and an intercellularly synchronized colony of clocks. Characterization of these systems revealed that the native enzymatic machinery induces a coupling between destabilized proteins that are waiting" to be degraded.
In Aim 1, we will explore how such intracellular coupling can lead to clocks that are synchronized at multiple (intra- and intercellular) scales. In the next aim, we will explore the us of two intercellular coupling mechanisms to develop a new platform for synthetic biology. We have previously shown how quorum sensing and redox communication can be used to design a macroscopic (1cm) biosensor.
In Aim 2, we will show how these coupling mechanisms can lead to an extremely stable toggle switch with switching transitions that are highly uniform at the single cell level. In the next aim, we will engineer light-sensitive circuits that produce complex spatiotemporal dynamics. Optogenetic circuits have recently been developed by several other groups and we plan to couple light-sensitive elements to our circuits to explore the light-guided propagation of signals throughout a spatially extended population of cells.
In Aim 4, we will continue our work on a mammalian oscillator. Here, we will engineer a novel synthetic mammalian circuit that relies on a negative feedback mechanism that is mediated by a transrepressor that acts by inducing local chromatin remodeling upon binding the hybrid promoter. We will integrate our synthetic circuits into the cell genome in order to study how the molecular dynamics function within the chromosomal regulatory context. Finally, in Aim 5 we will develop bacterial minicells as a platform for delivering synthetic circuits to mammalian cells. To improve the functionality of minicells, we will construct and transfer to minicells an additional synthetic network that provides supplemental RNA polymerase, enabling independent gene expression long after minicell separation from parental bacteria. We will tailor our microuidic devices for housing and tracking minicells and will characterize circuit behavior using time-lapse uorescence microscopy. The successful completion of this project will lead to advances in our understanding of gene regulation and could ultimately result in the utilization of programmable logic in a gene-delivery context. 1

Public Health Relevance

Synthetic biology can be used to engineer novel gene-based therapies and to systematically characterize the relationship between regulatory networks and cellular behavior. This project combines computational modeling, microuidic technology and molecular biology to construct synthetic gene networks that function as biological clocks and switches. New methods of drug delivery will be developed in order to facilitate translation of the basic science of synthetic biology into therapeutic applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069811-10
Application #
8534145
Study Section
Modeling and Analysis of Biological Systems Study Section (MABS)
Program Officer
Lyster, Peter
Project Start
2003-12-01
Project End
2016-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
10
Fiscal Year
2013
Total Cost
$468,427
Indirect Cost
$154,106
Name
University of California San Diego
Department
Engineering (All Types)
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Mather, William; Hasty, Jeff; Tsimring, Lev S (2014) Synchronization of degrade-and-fire oscillations via a common activator. Phys Rev Lett 113:128102
Fernandez, Bastien; Tsimring, Lev S (2013) Typical trajectories of coupled degrade-and-fire oscillators: from dispersed populations to massive clustering. J Math Biol :
Prindle, Arthur; Samayoa, Phillip; Razinkov, Ivan et al. (2012) A sensing array of radically coupled genetic 'biopixels'. Nature 481:39-44
Selimkhanov, Jangir; Hasty, Jeff; Tsimring, Lev S (2012) Recent advances in single-cell studies of gene regulation. Curr Opin Biotechnol 23:34-40
Mather, William H; Hasty, Jeff; Tsimring, Lev S (2012) Fast stochastic algorithm for simulating evolutionary population dynamics. Bioinformatics 28:1230-8
Boyer, Denis; Mather, William; Mondragon-Palomino, Octavio et al. (2011) Buckling instability in ordered bacterial colonies. Phys Biol 8:026008
Danino, Tal; Volfson, Dmitri; Bhatia, Sangeeta N et al. (2011) In-silico patterning of vascular mesenchymal cells in three dimensions. PLoS One 6:e20182
Mondragon-Palomino, Octavio; Danino, Tal; Selimkhanov, Jangir et al. (2011) Entrainment of a population of synthetic genetic oscillators. Science 333:1315-9
Longo, Diane M; Hoffmann, Alexander; Tsimring, Lev S et al. (2010) Coherent activation of a synthetic mammalian gene network. Syst Synth Biol 4:15-23
Danino, Tal; Mondragon-Palomino, Octavio; Tsimring, Lev et al. (2010) A synchronized quorum of genetic clocks. Nature 463:326-30

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