Precise control over microbial cell growth conditions could enable detection of minute phenotypic changes, which would improve our understanding of how genotypes are shaped by adaptive selection. Although automated cell-culture systems such as bioreactors offer strict control over liquid culture conditions, they often do not scale to high-throughput or require cumbersome redesign to alter growth conditions. We report the design and validation of eVOLVER, a scalable DIY framework that can be configured to carry out high-throughput growth experiments in molecular evolution, systems biology, and microbiology. We perform high-throughput evolution of yeast across systematically varied population density niches to show how eVOLVER can precisely characterize adaptive niches. We describe growth selection using time-varying temperature programs on a genome-wide yeast knockout library to identify strains with altered sensitivity to changes in temperature magnitude or frequency. Inspired by large-scale integration of electronics and microfluidics, we also demonstrate millifluidic multiplexing modules that enable multiplexed media routing, cleaning, vial-to-vial transfers and automated yeast mating.

The transcription of genomic information in eukaryotes is regulated in large part by chromatin. How a diverse array of chromatin regulator (CR) proteins with different functions and genomic localization patterns coordinates chromatin activity to control transcription remains unclear. Here we take a synthetic biology approach to decipher the complexity of chromatin regulation by studying emergent transcriptional behaviors from engineered combinatorial, spatial, and temporal patterns of individual CRs. We fuse 223 yeast CRs to programmable zinc finger proteins. Site-specific and combinatorial recruitment of CRs to distinct intra-locus locations reveals a range of transcriptional logic and behaviors, including synergistic activation, long-range and spatial regulation, and gene expression memory. Comparing these transcriptional behaviors with annotated CR complex and function terms provides design principles for the engineering of transcriptional regulation. This work presents a bottom-up approach to investigating chromatin-mediated transcriptional regulation, and introduces new, chromatin-based components and systems for synthetic biology and cellular engineering.

SUMMARY Protein aggregation is a hallmark of many diseases, but also underlies a wide range of positive cellular functions. These phenomena have been difficult to study due to a lack of quantitative and high-throughput cellular tools. Here we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer “anti-prion drives” that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.

The traditional view of protein aggregation as being strictly disease-related has been challenged by many examples of cellular aggregates that regulate beneficial biological functions. When coupled with the emerging view that many regulatory proteins undergo phase separation to form dynamic cellular compartments, it has become clear that supramolecular assembly plays wide-ranging and critical roles in cellular regulation. This presents opportunities to develop new tools to probe and illuminate this biology, and to harness the unique properties of these selfassembling systems for synthetic biology for the purposeful manipulation of biological function.

Systems that allow researchers to precisely control the expression of genes are fundamental to biological research, biotechnology, and synthetic biology. However, few inducible gene expression systems exist that can enable simultaneous multigene control under common nutritionally favorable conditions in the important model organism and chassis Saccharomyces cerevisiae. Here we repurposed ligand binding domains from mammalian type I nuclear receptors to establish a family of up to five orthogonal synthetic gene expression systems in yeast. Our systems enable tight, independent, multigene control through addition of inert hormones and are capable of driving robust and rapid gene expression outputs, in some cases achieving up to 600-fold induction. As a proof of principle, we placed expression of four enzymes from the violacein biosynthetic pathway under independent expression control to selectively route pathway flux by addition of specific inducer combinations. Our results establish a modular, versatile, and potentially expandable toolkit for multidimensional control of gene expression in yeast that can be used to construct and control naturally occurring and synthetic gene networks.