Engineering plasmid copy number heterogeneity for dynamic microbial adaptation.

Natural microbial populations exploit phenotypic heterogeneity for survival and adaptation. However, in engineering biology, limiting the sources of variability is a major focus. Here we show that intentionally coupling distinct plasmids via shared replication mechanisms enables bacterial populations to adapt to their environment. We demonstrate that plasmid coupling of carbon-metabolizing operons facilitates copy number tuning of an essential but burdensome construct through the action of a stably maintained, non-essential plasmid. For specific cost-benefit situations, incompatible two-plasmid systems can stably persist longer than compatible ones. We also show using microfluidics that plasmid coupling of synthetic constructs generates population-state memory of previous environmental adaptation without additional regulatory control. This work should help to improve the design of synthetic populations by enabling adaptive engineered strains to function under changing growth conditions without strict fine-tuning of the genetic circuitry.

A Standardized Set of MoClo-Compatible Inducible Promoter Systems for Tunable Gene Expression in Yeast.

Small-molecule control of gene expression underlies the function of numerous engineered gene circuits that are capable of environmental sensing, computation, and memory. While many recently developed inducible promoters have been tailor-made for bacteria or mammalian cells, relatively few new systems have been built for Saccharomyces cerevisiae, limiting the scale of synthetic biology work that can be done in yeast. To address this, we created the yeast Tunable Expression Systems Toolkit (yTEST), which contains a set of five extensively characterized inducible promoter systems regulated by the small-molecules doxycycline (Dox), abscisic acid (ABA), danoprevir (DNV), 1-naphthaleneacetic acid (NAA), and 5-phenyl-indole-3-acetic acid (5-Ph-IAA). Assembly was made to be compatible with the modular cloning yeast toolkit (MoClo-YTK) to enhance the ease of use and provide a framework to benchmark and standardize each system. Using this approach, we built multiple systems with maximal expression levels greater than those of the strong constitutive TDH3 promoter. Furthermore, each of the five classes of systems could be induced at least 60-fold after a 6 h induction and the highest fold change observed was approximately 300. Thus, yTEST provides a reliable, diverse, and customizable set of inducible promoters to modulate gene expression in yeast for applications in synthetic biology, metabolic engineering, and basic research.

High-resolution temporal profiling of E. coli transcriptional response.

Understanding how cells dynamically adapt to their environment is a primary focus of biology research. Temporal information about cellular behavior is often limited by both small numbers of data time-points and the methods used to analyze this data. Here, we apply unsupervised machine learning to a data set containing the activity of 1805 native promoters in E. coli measured every 10 minutes in a high-throughput microfluidic device via fluorescence time-lapse microscopy. Specifically, this data set reveals E. coli transcriptome dynamics when exposed to different heavy metal ions. We use a bioinformatics pipeline based on Independent Component Analysis (ICA) to generate insights and hypotheses from this data. We discovered three primary, time-dependent stages of promoter activation to heavy metal stress (fast, intermediate, and steady). Furthermore, we uncovered a global strategy E. coli uses to reallocate resources from stress-related promoters to growth-related promoters following exposure to heavy metal stress.

Design, mutate, screen: Multiplexed creation and arrayed screening of synchronized genetic clocks.

A major goal in synthetic biology is coordinating cellular behavior using cell-cell interactions; however, designing and testing complex genetic circuits that function only in large populations remains challenging. Although directed evolution has commonly supplemented rational design methods for synthetic gene circuits, this method relies on the efficient screening of mutant libraries for desired phenotypes. Recently, multiple techniques have been developed for identifying dynamic phenotypes from large, pooled libraries. These technologies have advanced library screening for single-cell, time-varying phenotypes but are currently incompatible with population-level phenotypes dependent on cell-cell communication. Here, we utilize directed mutagenesis and multiplexed microfluidics to develop an arrayed-screening workflow for dynamic, population-level genetic circuits. Specifically, we create a mutant library of an existing oscillator, the synchronized lysis circuit, and discover variants with different period-amplitude characteristics. Lastly, we utilize our screening workflow to construct a transcriptionally regulated synchronized oscillator that functions over long timescales. A record of this paper's transparent peer review process is included in the supplemental information.

Tumor-on-a-chip platform to investigate progression and drug sensitivity in cell lines and patient-derived organoids.

Most cancer treatment strategies target cell proliferation, angiogenesis, migration, and intravasation of tumor cells in an attempt to limit tumor growth and metastasis. An in vitro platform to assess tumor progression and drug sensitivity could provide avenues to enhance our understanding of tumor metastasis as well as precision medicine. We present a microfluidic platform that mimics biological mass transport near the arterial end of a capillary in the tumor microenvironment. A central feature is a quiescent perfused 3D microvascular network created prior to loading tumor cells or patient-derived tumor organoids in an adjacent compartment. The physiological delivery of nutrients and/or drugs to the tumor then occurs through the vascular network. We demonstrate the culture, growth, and treatment of tumor cell lines and patient-derived breast cancer organoids. The platform provides the opportunity to simultaneously and dynamically observe hallmark features of tumor progression including cell proliferation, angiogenesis, cell migration, and tumor cell intravasation. Additionally, primary breast tumor organoids are viable in the device for several weeks and induce robust sprouting angiogenesis. Finally, we demonstrate the feasibility of our platform for drug discovery and personalized medicine by analyzing the response to chemo- and anti-angiogenic therapy. Precision medicine-based cancer treatments can only be realized if individual tumors can be rapidly assessed for therapeutic sensitivity in a clinically relevant timeframe (⪅14 days). Our platform indicates that this goal can be achieved and provides compelling opportunities to advance precision medicine for cancer.

Low levels of physiological interstitial flow eliminate morphogen gradients and guide angiogenesis.

Convective transport can significantly distort spatial concentration gradients. Interstitial flow is ubiquitous throughout living tissue, but our understanding of how interstitial flow affects concentration gradients in biological processes is limited. Interstitial flow is of particular interest for angiogenesis because pathological and physiological angiogenesis is associated with altered interstitial flow, and both interstitial flow and morphogen gradients (e.g., vascular endothelial growth factor, VEGF) can potentially stimulate and guide new blood vessel growth. We designed an in vitro microfluidic platform to simulate 3D angiogenesis in a tissue microenvironment that precisely controls interstitial flow and spatial morphogen gradients. The microvascular tissue was developed from endothelial colony forming cell-derived endothelial cells extracted from cord blood and stromal fibroblasts in a fibrin extracellular matrix. Pressure in the microfluidic lines was manipulated to control the interstitial flow. A mathematical model of mass and momentum transport, and experimental studies with fluorescently labeled dextran were performed to validate the platform. Our data demonstrate that at physiological interstitial flow (0.1-10 µm/s), morphogen gradients were eliminated within hours, and angiogenesis demonstrated a striking bias in the opposite direction of interstitial flow. The interstitial flow-directed angiogenesis was dependent on the presence of VEGF, and the effect was mediated by αvß3 integrin. We conclude that under physiological conditions, growth factors such as VEGF and fluid forces work together to initiate and spatially guide angiogenesis.