PhageBox: An Open Source Digital Microfluidic Extension With Applications for Phage Discovery.

OBJECTIVE: Recent advancements demonstrate the significant role of digital microfluidics in automating laboratory work with DNA and on-site viral testing. However, since commercially available instruments are limited to droplet manipulation, our work addresses the need for accelerated integration of other components, such as temperature control, that can expand the application domain. METHODS: We developed PhageBox-an accessible device that can be used as a biochip extension. At hardware level, PhageBox integrates temperature and electromagnetic control modules. At software level, PhageBox is controlled by embedded software containing a unique model for bio-protocol programming, and a graphical user interface for visual device feedback and operation. RESULTS: To evaluate PhageBox's efficacy for biomedical applications, we performed functional testing. Similarly, we validated the temperature control using thermography, obtaining a range of ±0.2[Formula: see text]. The electromagnets produced a magnetic force of 15 milliTesla, demonstrating precise immobilization of magnetic beads. We show the potential of PhageBox for bacteriophage research through three initial protocols: a universal framework for PCR, T7 bacteriophage restriction enzyme digestion, and concentrating ϕX174 RF genomic DNA. CONCLUSION: Our work presents an open-source hardware and software extension for digital microfluidics devices. This extension integrates temperature and electromagnetic modules, demonstrating efficacy in biomedical applications and potential for bacteriophage research. SIGNIFICANCE: We developed PhageBox to be accessible: the components are off-the-shelf at a low cost ( ≤ $200), and the hardware designs and software code are open-source. With the long aim of ensuring reproducibility and accelerating collaboration, we also provide a DIY-build document.

Ten simple rules for managing laboratory information.

Information is the cornerstone of research, from experimental (meta)data and computational processes to complex inventories of reagents and equipment. These 10 simple rules discuss best practices for leveraging laboratory information management systems to transform this large information load into useful scientific findings.

Investigating and Modeling the Factors That Affect Genetic Circuit Performance.

Over the past 2 decades, synthetic biology has yielded ever more complex genetic circuits that are able to perform sophisticated functions in response to specific signals. Yet, genetic circuits are not immediately transferable to an outside-the-lab setting where their performance is highly compromised. We propose introducing a broader test step to the design-build-test-learn workflow to include factors that might contribute to unexpected genetic circuit performance. As a proof of concept, we have designed and evaluated a genetic circuit in various temperatures, inducer concentrations, nonsterilized soil exposure, and bacterial growth stages. We determined that the circuit's performance is dramatically altered when these factors differ from the optimal lab conditions. We observed significant changes in the time for signal detection as well as signal intensity when the genetic circuit was tested under nonoptimal lab conditions. As a learning effort, we then proceeded to generate model predictions in untested conditions, which is currently lacking in synthetic biology application design. Furthermore, broader test and learn steps uncovered a negative correlation between the time it takes for a gate to turn ON and the bacterial growth phases. As the synthetic biology discipline transitions from proof-of-concept genetic programs to appropriate and safe application implementations, more emphasis on test and learn steps (i.e., characterizing parts and circuits for a broad range of conditions) will provide missing insights on genetic circuit behavior outside the lab.

Robustness and reproducibility of simple and complex synthetic logic circuit designs using a DBTL loop.

Computational tools addressing various components of design-build-test-learn (DBTL) loops for the construction of synthetic genetic networks exist but do not generally cover the entire DBTL loop. This manuscript introduces an end-to-end sequence of tools that together form a DBTL loop called Design Assemble Round Trip (DART). DART provides rational selection and refinement of genetic parts to construct and test a circuit. Computational support for experimental process, metadata management, standardized data collection and reproducible data analysis is provided via the previously published Round Trip (RT) test-learn loop. The primary focus of this work is on the Design Assemble (DA) part of the tool chain, which improves on previous techniques by screening up to thousands of network topologies for robust performance using a novel robustness score derived from dynamical behavior based on circuit topology only. In addition, novel experimental support software is introduced for the assembly of genetic circuits. A complete design-through-analysis sequence is presented using several OR and NOR circuit designs, with and without structural redundancy, that are implemented in budding yeast. The execution of DART tested the predictions of the design tools, specifically with regard to robust and reproducible performance under different experimental conditions. The data analysis depended on a novel application of machine learning techniques to segment bimodal flow cytometry distributions. Evidence is presented that, in some cases, a more complex build may impart more robustness and reproducibility across experimental conditions. Graphical Abstract.

Experimental Data Connector (XDC): Integrating the Capture of Experimental Data and Metadata Using Standard Formats and Digital Repositories.

Accelerating the development of synthetic biology applications requires reproducible experimental findings. Different standards and repositories exist to exchange experimental data and metadata. However, the associated software tools often do not support a uniform data capture, encoding, and exchange of information. A connection between digital repositories is required to prevent siloing and loss of information. To this end, we developed the Experimental Data Connector (XDC). It captures experimental data and related metadata by encoding it in standard formats and storing the converted data in digital repositories. Experimental data is then uploaded to Flapjack and the metadata to SynBioHub in a consistent manner linking these repositories. This produces complete connected experimental data sets that are exchangeable. The information is captured using a single template Excel Workbook, which can be integrated into existing experimental workflow automation processes and semiautomated capture of results.

Specifications of standards in systems and synthetic biology: status and developments in 2022 and the COMBINE meeting 2022.

This special issue of the Journal of Integrative Bioinformatics contains updated specifications of COMBINE standards in systems and synthetic biology. The 2022 special issue presents three updates to the standards: CellML 2.0.1, SBML Level 3 Package: Spatial Processes, Version 1, Release 1, and Synthetic Biology Open Language (SBOL) Version 3.1.0. This document can also be used to identify the latest specifications for all COMBINE standards. In addition, this editorial provides a brief overview of the COMBINE 2022 meeting in Berlin.

SynBioSuite: A Tool for Improving the Workflow for Genetic Design and Modeling.

Synthetic biology research has led to the development of many software tools for designing, constructing, editing, simulating, and sharing genetic parts and circuits. Among these tools are SBOLCanvas, iBioSim, and SynBioHub, which can be used in conjunction to create a genetic circuit design following the design-build-test-learn process. However, although automation works within these tools, most of these software tools are not integrated, and the process of transferring information between them is a very manual, error-prone process. To address this problem, this work automates some of these processes and presents SynBioSuite, a cloud-based tool that eliminates many of the drawbacks of the current approach by automating the setup and reception of results for simulating a designed genetic circuit via an application programming interface.

Excel-SBOL Converter: Creating SBOL from Excel Templates and Vice Versa.

Standards support synthetic biology research by enabling the exchange of component information. However, using formal representations, such as the Synthetic Biology Open Language (SBOL), typically requires either a thorough understanding of these standards or a suite of tools developed in concurrence with the ontologies. Since these tools may be a barrier for use by many practitioners, the Excel-SBOL Converter was developed to facilitate the use of SBOL and integration into existing workflows. The converter consists of two Python libraries: one that converts Excel templates to SBOL and another that converts SBOL to an Excel workbook. Both libraries can be used either directly or via a SynBioHub plugin.

A Comparison of Weighted Stochastic Simulation Methods for the Analysis of Genetic Circuits.

Rare events are of particular interest in synthetic biology because rare biochemical events may be catastrophic to a biological system by, for example, triggering irreversible events such as off-target drug delivery. To estimate the probability of rare events efficiently, several weighted stochastic simulation methods have been developed. Under optimal parameters and model conditions, these methods can greatly improve simulation efficiency in comparison to traditional stochastic simulation. Unfortunately, the optimal parameters and conditions cannot be deduced a priori. This paper presents a critical survey of weighted stochastic simulation methods. It shows that the methods considered here cannot consistently, efficiently, and exactly accomplish the task of rare event simulation without resorting to a computationally expensive calibration procedure, which undermines their overall efficiency. The results suggest that further development is needed before these methods can be deployed for general use in biological simulations.

Highly-automated, high-throughput replication of yeast-based logic circuit design assessments.

We describe an experimental campaign that replicated the performance assessment of logic gates engineered into cells of Saccharomyces cerevisiae by Gander et al. Our experimental campaign used a novel high-throughput experimentation framework developed under Defense Advanced Research Projects Agency's Synergistic Discovery and Design program: a remote robotic lab at Strateos executed a parameterized experimental protocol. Using this protocol and robotic execution, we generated two orders of magnitude more flow cytometry data than the original experiments. We discuss our results, which largely, but not completely, agree with the original report and make some remarks about lessons learned. Graphical Abstract.

BioSimulators: a central registry of simulation engines and services for recommending specific tools.

Computational models have great potential to accelerate bioscience, bioengineering, and medicine. However, it remains challenging to reproduce and reuse simulations, in part, because the numerous formats and methods for simulating various subsystems and scales remain siloed by different software tools. For example, each tool must be executed through a distinct interface. To help investigators find and use simulation tools, we developed BioSimulators (https://biosimulators.org), a central registry of the capabilities of simulation tools and consistent Python, command-line and containerized interfaces to each version of each tool. The foundation of BioSimulators is standards, such as CellML, SBML, SED-ML and the COMBINE archive format, and validation tools for simulation projects and simulation tools that ensure these standards are used consistently. To help modelers find tools for particular projects, we have also used the registry to develop recommendation services. We anticipate that BioSimulators will help modelers exchange, reproduce, and combine simulations.

Engineering genetic circuits: advancements in genetic design automation tools and standards for synthetic biology.

Synthetic biology (SynBio) is a field at the intersection of biology and engineering. Inspired by engineering principles, researchers use defined parts to build functionally defined biological circuits. Genetic design automation (GDA) allows scientists to design, model, and analyze their genetic circuits in silico before building them in the lab, saving time, and resources in the process. Establishing SynBio's future is dependent on GDA, since the computational approach opens the field to a broad, interdisciplinary community. However, challenges with part libraries, standards, and software tools are currently stalling progress in the field. This review first covers recent advancements in GDA, followed by an assessment of the challenges ahead, and a proposed automated genetic design workflow for the future.

Genetic circuit design automation with Cello 2.0.

Cells interact with their environment, communicate among themselves, track time and make decisions through functions controlled by natural regulatory genetic circuits consisting of interacting biological components. Synthetic programmable circuits used in therapeutics and other applications can be automatically designed by computer-aided tools. The Cello software designs the DNA sequences for programmable circuits based on a high-level software description and a library of characterized DNA parts representing Boolean logic gates. This process allows for design specification reuse, modular DNA part library curation and formalized circuit transformations based on experimental data. This protocol describes Cello 2.0, a freely available cross-platform software written in Java. Cello 2.0 enables flexible descriptions of the logic gates' structure and their mathematical models representing dynamic behavior, new formal rules for describing the placement of gates in a genome, a new graphical user interface, support for Verilog 2005 syntax and a connection to the SynBioHub parts repository software environment. Collectively, these features expand Cello's capabilities beyond Escherichia coli plasmids to new organisms and broader genetic contexts, including the genome. Designing circuits with Cello 2.0 produces an abstract Boolean network from a Verilog file, assigns biological parts to each node in the Boolean network, constructs a DNA sequence and generates highly structured and annotated sequence representations suitable for downstream processing and fabrication, respectively. The result is a sequence implementing the specified Boolean function in the organism and predictions of circuit performance. Depending on the size of the design space and users' expertise, jobs may take minutes or hours to complete.

Sequence-Based Searching for SynBioHub Using VSEARCH.

The ability to search for a part by its sequence is crucial for a large repository of parts. Prior to this work, however, this was not possible on SynBioHub. Sequence-based search is now integrated into SynBioHub, allowing users to find a part by a sequence provided in plain text or a supported file format. This sequence-based search feature is accessible to users via SynBioHub's web interface, or programmatically through its API. The core implementation of the tool uses VSEARCH, an open source, global alignment search tool, and it is integrated into SBOLExplorer, an open source distributed search engine used by SynBioHub. We present a new approach to scoring part similarity using SBOLExplorer, which takes into account both the popularity and percentage match of parts.

Round Trip: An Automated Pipeline for Experimental Design, Execution, and Analysis.

Synthetic biology is a complex discipline that involves creating detailed, purpose-built designs from genetic parts. This process is often phrased as a Design-Build-Test-Learn loop, where iterative design improvements can be made, implemented, measured, and analyzed. Automation can potentially improve both the end-to-end duration of the process and the utility of data produced by the process. One of the most important considerations for the development of effective automation and quality data is a rigorous description of implicit knowledge encoded as a formal knowledge representation. The development of knowledge representation for the process poses a number of challenges, including developing effective human-machine interfaces, protecting against and repairing user error, providing flexibility for terminological mismatches, and supporting extensibility to new experimental types. We address these challenges with the DARPA SD2 Round Trip software architecture. The Round Trip is an open architecture that automates many of the key steps in the Test and Learn phases of a Design-Build-Test-Learn loop for high-throughput laboratory science. The primary contribution of the Round Trip is to assist with and otherwise automate metadata creation, curation, standardization, and linkage with experimental data. The Round Trip's focus on metadata supports fast, automated, and replicable analysis of experiments as well as experimental situational awareness and experimental interpretability. We highlight the major software components and data representations that enable the Round Trip to speed up the design and analysis of experiments by 2 orders of magnitude over prior ad hoc methods. These contributions support a number of experimental protocols and experimental types, demonstrating the Round Trip's breadth and extensibility. We describe both an illustrative use case using the Round Trip for an on-the-loop experimental campaign and overall contributions to reducing experimental analysis time and increasing data product volume in the SD2 program.

Exploring the Gaps in Programming for Men and Women with a Gambling Disorder in the Correctional System in Canada.

The prevalence of problem gambling in the correctional system is about 10 times higher than in the general population, but programming for gambling problems in the correctional system is scarce. Examined barriers to treatment for problem gamblers in the correctional system, sex or gender differences, and options for program design. We conducted interviews with 16 experts who had worked with problem gamblers in the correctional system and analyzed the data in terms of themes. For barriers, the experts discussed a lack of awareness about problem gambling, as well as stigmatization and lockdowns. Experts described the content of current programs as equally applicable to both men and women, but that emotional components were more important for women. Finally, the experts had mixed views on whether programming should be integrated with other programs such as substance abuse. Programming implications for this population are discussed.

Synthetic Biology Curation Tools (SYNBICT).

Much progress has been made in developing tools to generate component-based design representations of biological systems from standard libraries of parts. Most biological designs, however, are still specified at the sequence level. Consequently, there exists a need for a tool that can be used to automatically infer component-based design representations from sequences, particularly in cases when those sequences have minimal levels of annotation. Such a tool would assist computational synthetic biologists in bridging the gap between the outputs of sequence editors and the inputs to more sophisticated design tools, and it would facilitate their development of automated workflows for design curation and quality control. Accordingly, we introduce Synthetic Biology Curation Tools (SYNBICT), a Python tool suite for automation-assisted annotation, curation, and functional inference for genetic designs. We have validated SYNBICT by applying it to genetic designs in the DARPA Synergistic Discovery & Design (SD2) program and the International Genetically Engineered Machines (iGEM) 2018 distribution. Most notably, SYNBICT is more automated and parallelizable than manual design editors, and it can be applied to interpret existing designs instead of only generating new ones.

Stochastic Hazard Analysis of Genetic Circuits in iBioSim and STAMINA.

In synthetic biology, combinational circuits are used to program cells for various new applications like biosensors, drug delivery systems, and biofuels. Similar to asynchronous electronic circuits, some combinational genetic circuits may show unwanted switching variations (glitches) caused by multiple input changes. Depending on the biological circuit, glitches can cause irreversible effects and jeopardize the circuit's functionality. This paper presents a stochastic analysis to predict glitch propensities for three implementations of a genetic circuit with known glitching behavior. The analysis uses STochastic Approximate Model-checker for INfinite-state Analysis (STAMINA), a tool for stochastic verification. The STAMINA results were validated by comparison to stochastic simulation in iBioSim resulting in further improvements of STAMINA. This paper demonstrates that stochastic verification can be utilized by genetic designers to evaluate design choices and input restrictions to achieve a desired reliability of operation.

Synthetic biology open language visual (SBOL visual) version 3.0.

People who engineer biological organisms often find it useful to draw diagrams in order to communicate both the structure of the nucleic acid sequences that they are engineering and the functional relationships between sequence features and other molecular species. Some typical practices and conventions have begun to emerge for such diagrams. SBOL Visual aims to organize and systematize such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 3.0 of SBOL Visual, a new major revision of the standard. The major difference between SBOL Visual 3 and SBOL Visual 2 is that diagrams and glyphs are defined with respect to the SBOL 3 data model rather than the SBOL 2 data model. A byproduct of this change is that the use of dashed undirected lines for subsystem mappings has been removed, pending future determination on how to represent general SBOL 3 constraints; in the interim, this annotation can still be used as an annotation. Finally, deprecated material has been removed from collection of glyphs: the deprecated "insulator" glyph and "macromolecule" alternative glyphs have been removed, as have the deprecated BioPAX alternatives to SBO terms.