Optogenetic Control of B. subtilis Gene Expression Using the CcaSR System.

Optogenetics enables precise control of gene expression in a variety of organisms. We recently developed the first system for optogenetic control of transcription in Bacillus subtilis. This system is based on CcaSR, a light-responsive two-component regulatory system originally derived from Synechocystis PCC 6803.

Dissecting the regulatory logic of specification and differentiation during vertebrate embryogenesis.

The interplay between transcription factors and chromatin accessibility regulates cell type diversification during vertebrate embryogenesis. To systematically decipher the gene regulatory logic guiding this process, we generated a single-cell multi-omics atlas of RNA expression and chromatin accessibility during early zebrafish embryogenesis. We developed a deep learning model to predict chromatin accessibility based on DNA sequence and found that a small number of transcription factors underlie cell-type-specific chromatin landscapes.

Iterative deep learning-design of human enhancers exploits condensed sequence grammar to achieve cell type-specificity.

An important and largely unsolved problem in synthetic biology is how to target gene expression to specific cell types. Here, we apply iterative deep learning to design synthetic enhancers with strong differential activity between two human cell lines. We initially train models on published datasets of enhancer activity and chromatin accessibility and use them to guide the design of synthetic enhancers that maximize predicted specificity.

The regulatory landscape of 5' UTRs in translational control during zebrafish embryogenesis.

The 5' UTRs of mRNAs are critical for translation regulation, but their regulatory features are poorly characterized. Here, we report the regulatory landscape of 5' UTRs during early zebrafish embryogenesis using a massively parallel reporter assay of 18,154 sequences coupled to polysome profiling. We found that the 5' UTR is sufficient to confer temporal dynamics to translation initiation, and identified 86 motifs enriched in 5' UTRs with distinct ribosome recruitment capabilities.

Meeting Measurement Precision Requirements for Effective Engineering of Genetic Regulatory Networks.

Reliable, predictable engineering of cellular behavior is one of the key goals of synthetic biology. As the field matures, biological engineers will become increasingly reliant on computer models that allow for the rapid exploration of design space prior to the more costly construction and characterization of candidate designs. The efficacy of such models, however, depends on the accuracy of their predictions, the precision of the measurements used to parametrize the models, and the tolerance of biological devices for imperfections in modeling and measurement.

Machine Learning for Designing Next-Generation mRNA Therapeutics.

Over just the last 2 years, mRNA therapeutics and vaccines have undergone a rapid transition from an intriguing concept to real-world impact. However, whereas some aspects of mRNA therapeutics, such as the use of chemical modifications to increase stability and reduce immunogenicity, have been extensively optimized for over two decades, other aspects, particularly the selection and design of the noncoding leader and trailer sequences which control translation efficiency and stability, have received comparably less attention. In practice, such 5' and 3' untranslated regions (UTRs) are often borrowed from highly expressed human genes with few or no modifications, as in the case for the Pfizer/BioNTech Covid vaccine.

DIY optogenetics: Building, programming, and using the Light Plate Apparatus.

Optogenetic systems enable unmatched precision for controlling molecular biological processes but require the use of specialized electrical and optical hardware. In an effort to make working with optogenetic systems more accessible, we recently described an open-source hardware platform called the Light Plate Apparatus (LPA). The LPA is a device capable of delivering two independent light signals from standard LEDs to wells of a 24-well culture plate.

Communicating Structure and Function in Synthetic Biology Diagrams.

Biological engineers often find it useful to communicate using diagrams. These diagrams can include information both about the structure of the nucleic acid sequences they are engineering and about the functional relationships between features of these sequences and/or other molecular species. A number of conventions and practices have begun to emerge within synthetic biology for creating such diagrams, and the Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard to organize, systematize, and extend such conventions in order to produce a coherent visual language.

Optogenetic control of Bacillus subtilis gene expression.

The Gram-positive bacterium Bacillus subtilis exhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest into B.

An Engineered Inducible Promoter System with over 10 000-Fold Dynamic Range.

is the leading model Gram-positive bacterium, and a widely used chassis for industrial protein production. However, research is limited by a lack of inducible promoter systems with low leakiness and high dynamic range. Here, we engineer an inducible promoter system based on the T7 RNA Polymerase (T7 RNAP), the lactose repressor LacI, and the chimeric promoter P, integrated as a single copy in the genome.

An open-hardware platform for optogenetics and photobiology.

In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution.

FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary to Calibrated Units.

Flow cytometry is widely used to measure gene expression and other molecular biological processes with single cell resolution via fluorescent probes. Flow cytometers output data in arbitrary units (a.u.

Design principles for robust oscillatory behavior.

Oscillatory responses are ubiquitous in regulatory networks of living organisms, a fact that has led to extensive efforts to study and replicate the circuits involved. However, to date, design principles that underlie the robustness of natural oscillators are not completely known. Here we study a three-component enzymatic network model in order to determine the topological requirements for robust oscillation.

How to train your microbe: methods for dynamically characterizing gene networks.

Gene networks regulate biological processes dynamically. However, researchers have largely relied upon static perturbations, such as growth media variations and gene knockouts, to elucidate gene network structure and function. Thus, much of the regulation on the path from DNA to phenotype remains poorly understood.