Tools and reference standards supporting the engineering and evolution of synthetic biological systems

Abstract

Biological engineers have constructed a number of multi-part synthetic biological systems that conduct logical operations on input signals, produce oscillatory output signals, store memory, or produce desired products. However, very few of these genetically-encoded systems worked as originally designed. The typical process of constructing a functional system involves a period of tuning the system properties to find a functional variant. This tuning process has been optimized and applied with great success to the engineering of individual biological parts by directed evolution. For instance, researchers developing improved enzymes, transcriptional promoters, and fluorescent proteins have generated large libraries of variants and screened these libraries to find individual mutants that met desired performance specifications. In this thesis, I address some of the bottlenecks preventing the application of directed evolution to more complex devices and systems. First, I describe an input / output screening plasmid that was designed to enable screening of higher-order genetic devices based on the equilibrium response of the device. This plasmid includes two fluorescent reporters and an inducible promoter to enable screening of device libraries across a range of inputs. Second, I describe measurement kits and reference standards designed to improve the characterization of promoter and RBS parts that are used as input substrates for device evolution. By using the kits, researchers are able to report promoter and RBS activities in standard units (Standard Promoter Units, SPUs, and Standard RBS Units, SRUs) enabling the growth of a collection of well-characterized parts to draw on for assembling device variants. Finally, I describe a new microfluidic device, the Sortostat, that integrates a cell sorting chamber with a previously published microscope-mounted microfluidic chemostat.

(cont.) Researchers can use the Sortostat to apply morphological, time-varying, or other complex selective pressures to cells in continuous culture.