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While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into

Droplet interface bilayers (DIBs) mimic the cell membrane and provide a model membrane platform for studying basic biophysical processes. This paper demonstrates a pressure-driven microfluidic system for the rapid and automated generation of alternating DIB networks, each comprised of four aqueous nanoliter droplets. The microfluidic device features five inlets, one for the continuous oil phase and four independent aqueous channels for T-junction droplet generation. Droplet production rates are controlled by adjusting the applied pressure of each inlet; therefore, controlling the pattern of droplets produced in the main channel and further stored in a downstream hydrodynamic trapping array. Each trap is designed to capture and hold in place one row of four droplets, forming three interfacial lipid bilayers per network. The potential for greater combinations of droplets in a network enables an increased complexity necessary for performing parallel multiplexed biological assays. We further examined flow behavior in response to changes in resistance of the microfluidic device when using a pressure driven source. This microfluidic system provides a high-throughput method for generating DIB networks of complex droplet patterning.

Summary Reverse genetics approaches have revolutionized plant biology and agriculture. Phenomics has the prospect of bridging plant phenotypes with genes, including transgenes, to transform agricultural fields. Genetically encoded fluorescent proteins (FPs) have revolutionized plant biology paradigms in gene expression, protein trafficking and plant physiology. While the first instance of plant canopy imaging of green fluorescent protein (GFP) was performed over 25 years ago, modern phenomics has largely ignored fluorescence as a transgene expression device despite the burgeoning FP colour palette available to plant biologists. Here, we show a new platform for stand‐off imaging of plant canopies expressing a wide variety of FP genes. The platform—the fluorescence‐inducing laser projector (FILP)—uses an ultra‐low‐noise camera to image a scene illuminated by compact diode lasers of various colours, coupled with emission filters to resolve individual FPs, to phenotype transgenic plants expressing FP genes. Each of the 20 FPs screened in plants were imaged at >3 m using FILP in a laboratory‐based laser range. We also show that pairs of co‐expressed fluorescence proteins can be imaged in canopies. The FILP system enabled a rapid synthetic promoter screen: starting from 2000 synthetic promoters transfected into protoplasts to FILP‐imaged agroinfiltrated Nicotiana benthamiana plants in a matter of weeks, which was useful to characterize a water stress‐inducible synthetic promoter. FILP canopy imaging was also accomplished for stably transformed GFP potato and in a split‐GFP assay, which illustrates the flexibility of the instrument for analysing fluorescence signals in plant canopies.

SummaryReverse genetics approaches have revolutionized plant biology and agriculture. Phenomics has the prospect of bridging plant phenotypes with genes, including transgenes, to transform agricultural fields1. Genetically-encoded fluorescent proteins (FPs) have transformed studies in gene expression, protein trafficking, and plant physiology. While the first instance of plant canopy imaging of green fluorescent protein (GFP) was performed over 20 years ago2, modern phenomics has largely ignored fluorescence as a transgene indicator despite the burgeoning FP color palette currently available to biologists3–5. Here we show a new platform for standoff imaging of plant canopies expressing a wide variety of FP genes in leaves. The platform, the fluorescence-inducing laser projector (FILP), uses a low-noise camera to image a scene illuminated by compact diode lasers of various colors and emission filters to phenotype transgenic plants expressing multiple constitutive or inducible FPs. Of the 20 FPs screened, we selected the top performing candidates for standoff phenomics at ≥ 3 m using FILP in a laboratory-based laser range. Included in demonstrated applications is the performance of an osmotic stress-inducible synthetic promoter selected from a high throughput library screen. While FILP has unprecedented versatility as a laboratory platform, we envisage future iterations of the system for use in automated greenhouse or even drone-fielded versions of the platform for crop screening.

This work draws inspiration from totipotent cellular systems to design smart materials whose compositions and properties can be learned or evolved. Totipotency refers to the inherent genetic potential of a single cell to adapt and produce all types of differentiated cells within an organism. To study this principal and apply it synthetically, tissue-like compartmentalized assemblies are constructed via lipid membrane-separated aqueous droplets in a hydrophobic medium through the droplet interface bilayer (DIB) method. Within our droplets, we explore synthetic totipotency via cell-free reactions including actin polymerization and cell free protein synthesis (CFPS). The transcription and translation of our CFPS reactions are controlled by stimuli-responsive riboswitches (RS). Via this scheme, adaptable material properties and functions are achieved in vitro via protein production from cell-free machinery administered through RS governance. Here, we present thermally or chemically-triggered riboswitches for orthogonal production of representative fluorescent protein products, as well functional proteins. To characterize the material properties of target proteins, we study the formation of polymerized actin shells to stabilize organically-encased droplets and span DIBs. We present a modified protocol for chemically-triggered actin polymerization as well as a thermally triggered actin RS. We characterize theophylline (TP)-triggered production of alpha hemolysin (α-HL) through CFPS and synthesized an organic-soluble trigger that can be sensed from the oil phase by a RS in an aqueous bioreactor droplet. We also demonstrate increased droplet conductivity when CFPS α-HL products are incorporated in DIBs. This interdisciplinary work involves cell culture, gene expression, organic synthesis, vesicle formation, protein quantification, tensiometry, droplet aspiration, microplate fluorescence/absorption experiments, fluorescent microscopy, and electrophysiology. This project is an essential design analysis for creating smart, soft materials using synthetic biology and provides motivation for artificial tissues capable of adapting in response to external stimuli.