We will continue to design, construct and characterize genetic circuits. We will use micro fluidic tools to grow and observe single cells and colonies in precisely controlled environmental conditions, and we will test a subset of the engineered bacterial strains as therapies in animal models. Single cell and colony dynamics will inform mathematical models that will be used to identify key design characteristics, which will then be rigorously tested using previously established molecular biology techniques. Eight graduate students and postdocs will work on multiple aspects of the project, while maintaining a particular focus on modeling or technology development for monitoring bacteria or in vivo characterization. Our track record demonstrates our ability to train personnel in a multi-disciplinary approach that has led to new tools for Synthetic Biology, along with an increased understanding of gene and signaling networks generally. Our recent characterization of bacterial circuits in animal models has served to highlight the need for beneficial strains that are stable and safe over therapeutically relevant timescales. Accordingly, our Specific Aims focus on stability (Aim 1), delivery (Aim 2), safety (Aim 3), and in vivo testing (Aim 4). Gene circuits inevitably generate mutations that are selected to decrease the additional burden created by the inserted genetic machinery.
Our first aim will develop strategies for extending the lifetime of gene circuits in bacteria before selective pressure disables their desired functionality. We will develop computational models and experimentally quantify how circuit redundancy increases circuit lifetime. We will use our experimental platform to monitor functionality across scales from single-cell to batch culture environments.
Our second aim will primarily focus on engineering small bacterial ecologies. Here we will use modeling to guide the design of up to three interacting strains that can deliver therapies in a predetermined sequential order. In the third aim, we will build a safety circuit that triggers the death of all bacteria at a given threshold population density. The goal is to create an irreversible intracellular switch that rapidly and efficiently kills all cells before mutations can compromise the safety strategy. In the final aim, we will test the circuits designed in the first three aims in animal models. We will engineer optical markers that enable characterization of the dynamics of bacterial colonies and tumor size in vivo. Importantly, the relative ease and low cost of bacterial cloning will inevitably lead to a bottleneck for the field of Synthetic Biology, as therapeutic strains can be created at a rate that will far exceed the ability to test them. This highlights an acute need for quantitative models that have been thoroughly validated using in vitro technologies. Consequently, only a fraction of the circuits built in Aims 1-3 will be deemed worthy of in vivo testing. More generally, we anticipate that the computational models arising from these studies will be generally applicable across a wide range of emerging applications that employ bacteria.

Public Health Relevance

The long-held view of bacteria as strictly pathogens has given way to an appreciation of the widespread prevalence of beneficial microbes within the human body. Given this vast diversity, it is perhaps inevitable that some bacteria would evolve to preferentially grow in tumors, providing a natural platform for developing engineered therapies. This project combines computational modeling, microfluidic technology, and molecular biology to engineer bacteria to produce and release therapies after safely migrating to solid tumors within the human body.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069811-16
Application #
9751306
Study Section
Modeling and Analysis of Biological Systems Study Section (MABS)
Program Officer
Resat, Haluk
Project Start
2004-08-01
Project End
2020-07-31
Budget Start
2019-08-01
Budget End
2020-07-31
Support Year
16
Fiscal Year
2019
Total Cost
Indirect Cost
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
Bittihn, Philip; Din, M Omar; Tsimring, Lev S et al. (2018) Rational engineering of synthetic microbial systems: from single cells to consortia. Curr Opin Microbiol 45:92-99
Xiong, Liyang; Cooper, Robert; Tsimring, Lev S (2018) Coexistence and Pattern Formation in Bacterial Mixtures with Contact-Dependent Killing. Biophys J 114:1741-1750
Didovyk, Andriy; Tonooka, Taishi; Tsimring, Lev et al. (2017) Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. ACS Synth Biol 6:2198-2208
Scott, Spencer R; Din, M Omar; Bittihn, Philip et al. (2017) A stabilized microbial ecosystem of self-limiting bacteria using synthetic quorum-regulated lysis. Nat Microbiol 2:17083
Bittihn, Philip; Hasty, Jeff; Tsimring, Lev S (2017) Suppression of Beneficial Mutations in Dynamic Microbial Populations. Phys Rev Lett 118:028102
Cooper, Robert M; Tsimring, Lev; Hasty, Jeff (2017) Inter-species population dynamics enhance microbial horizontal gene transfer and spread of antibiotic resistance. Elife 6:
Din, M Omar; Danino, Tal; Prindle, Arthur et al. (2016) Synchronized cycles of bacterial lysis for in vivo delivery. Nature 536:81-85
Borek, Bart?omiej; Hasty, Jeff; Tsimring, Lev (2016) Turing Patterning Using Gene Circuits with Gas-Induced Degradation of Quorum Sensing Molecules. PLoS One 11:e0153679
Didovyk, Andriy; Borek, Bart?omiej; Hasty, Jeff et al. (2016) Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9. ACS Synth Biol 5:81-8
Didovyk, Andriy; Borek, Bart?omiej; Tsimring, Lev et al. (2016) Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications. Curr Opin Biotechnol 40:177-184

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