Engineered Gene Circuits for Basic Science and Biotechnology

University Of California, San Diego, La Jolla CA

Investigators

Linked publications, trials & patents

Abstract

Project Summary We will continue to refine our computational modeling approach to the design, construction and char- acterization of genetic circuits. We will develop new microfluidic tools to grow and observe how single cells and bacterial colonies respond to heterogeneous environmental conditions (in Aims 1 and 2) and we will test engineered adaptive strains in tumor spheroids (in Aim 3). The quantitative analysis of cellular behavior across multiple experimental platforms will inform mathematical models that will be used to identify key design characteristics, which will then be rigorously tested using previously established tech- niques. Two Postdocs, a Staff Research Scientist, and two Graduate Student Researchers will work with Drs. Hasty and Tsimring on multiple aspects of the project in an integrated manner. 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 past characterization of bacterial circuits in animal tumor models highlighted the need for a pre- dictive understanding of how engineered bacteria function in heterogeneous environments. Accordingly, our Specific Aims focus on computationally-driven engineering of (i) adaptive synthetic clonal populations, (ii) spatially organizing colonies that respond to heterogeneous environments, and (iii) thermally-activated gene circuitry that enables low density population capping during initial strain differentiation. Specifi- cally, our first aim will generate phenotypic heterogeneity and thereby rapid adaptation to environmental condition within a clonal population. We will co-transform two distinct plasmids with shared replication machinery to couple their copy numbers and generate copy number heterogeneity. Our prelminary mod- eling predicts that single strains carrying coupled plasmids are not only capable of phenotypic adaptation to environmental stimuli, but also can retain their divergent state in the absence of continued stimuli. We propose that this suggested memory property of our populations can be engineered to be either reversible using plasmid stabilization or permanent using inducible copy number repression. Our second aim will center on the development of a new multi-dimensional microfluidic system to investigate engineered popu- lation patterning within tumor-relevant small molecule gradients. Preliminary modeling presented in the proposal characterizes the conditions when single strains with coupled plasmids can differentially colonize across tumor-relevant heterogeneous environments. Lastly, we will engineer an ultrasound-activated liv- ing therapeutic within nonuniform tumor environments. Following spatial differentiation of two adaptive strains, we will use ultrasound to “flip” a toggle switch from low density growth to high population-cycling growth. We will test this system in colo-rectal tumor spheroids, using methods developed in the previous cycle of this grant.