The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for rapid and sensitive protein detection and quantification in simple and robust formats for widespread point-of-care ...applications. Here, we report on nanobody-functionalized organic electrochemical transistors with a modular architecture for the rapid quantification of single-molecule-to-nanomolar levels of specific antigens in complex bodily fluids. The sensors combine a solution-processable conjugated polymer in the transistor channel and high-density and orientation-controlled bioconjugation of nanobody-SpyCatcher fusion proteins on disposable gate electrodes. The devices provide results after 10 min of exposure to 5 μl of unprocessed samples, maintain high specificity and single-molecule sensitivity in human saliva and serum, and can be reprogrammed to detect any protein antigen if a corresponding specific nanobody is available. We used the sensors to detect green fluorescent protein, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) spike proteins, and for the COVID-19 screening of unprocessed clinical nasopharyngeal swab and saliva samples with a wide range of viral loads.
The re-use of previously validated designs is critical to the evolution of synthetic biology from a research discipline to an engineering practice. Here we describe the Synthetic Biology Open ...Language (SBOL), a proposed data standard for exchanging designs within the synthetic biology community. SBOL represents synthetic biology designs in a community-driven, formalized format for exchange between software tools, research groups and commercial service providers. The SBOL Developers Group has implemented SBOL as an XML/RDF serialization and provides software libraries and specification documentation to help developers implement SBOL in their own software. We describe early successes, including a demonstration of the utility of SBOL for information exchange between several different software tools and repositories from both academic and industrial partners. As a community-driven standard, SBOL will be updated as synthetic biology evolves to provide specific capabilities for different aspects of the synthetic biology workflow.
Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic ...platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems.
The Synthetic Biology Open Language (SBOL) is a standard that enables collaborative engineering of biological systems across different institutions and tools. SBOL is developed through careful ...consideration of recent synthetic biology trends, real use cases, and consensus among leading researchers in the field and members of commercial biotechnology enterprises. We demonstrate and discuss how a set of SBOL-enabled software tools can form an integrated, cross-organizational workflow to recapitulate the design of one of the largest published genetic circuits to date, a 4-input AND sensor. This design encompasses the structural components of the system, such as its DNA, RNA, small molecules, and proteins, as well as the interactions between these components that determine the system’s behavior/function. The demonstrated workflow and resulting circuit design illustrate the utility of SBOL 2.0 in automating the exchange of structural and functional specifications for genetic parts, devices, and the biological systems in which they operate.
Protein tyrosine kinases (PTKs) are key signaling molecules and important drug targets. Although the efficient recombinant production of active PTKs is important for both pharmaceutical industry and ...academic research, most PTKs are still obtained from conventional, expensive and time-consuming insect-cell based expression. Host toxicity, kinase inactivity, insolubility and heterogeneity are among the reasons thought to preclude PTK expression in Escherichia coli. Herein we review these presumed roadblocks and their possible solutions for bacterial expression of PTKs, and give an overview on kinase activity assays. Finally, we report our experiences and observations with the kinases Src, Lyn and FAK as examples to illustrate implementation, effects and pitfalls of E. coli expression and in vitro assaying of PTKs.
The COVID-19 pandemic, caused by SARS-CoV-2, has emphasized the necessity for scalable diagnostic workflows using locally produced reagents and basic laboratory equipment with minimal dependence on ...global supply chains. We introduce an open-source automated platform for high-throughput RNA extraction and pathogen diagnosis, which uses reagents almost entirely produced in-house. This platform integrates our methods for self-manufacturing magnetic nanoparticles and qRT-PCR reagents-both of which have received regulatory approval for clinical use-with an in-house, open-source robotic extraction protocol. It also incorporates our "Nanopore Sequencing of Isothermal Rapid Viral Amplification for Near Real-time Analysis" (NIRVANA) technology, designed for tracking SARS-CoV-2 mutations and variants. The platform exhibits high reproducibility and consistency without cross-contamination, and its limit of detection, sensitivity, and specificity are comparable to commercial assays. Automated NIRVANA effectively identifies circulating SARS-CoV-2 variants. Our in-house, cost-effective reagents, automated diagnostic workflows, and portable genomic surveillance strategies provide a scalable and rapid solution for COVID-19 diagnosis and variant tracking, essential for current and future pandemic responses.
Protein-protein association is often accompanied by changes in receptor and ligand structure. This interplay between protein flexibility and protein-protein recognition is currently the largest ...obstacle both to our understanding of and to the reliable prediction of protein complexes. We performed two sets of molecular dynamics simulations for the unbound receptor and ligand structures of 17 protein complexes and applied shape-driven rigid body docking to all combinations of representative snapshots. The crossdocking of structure ensembles increased the likelihood of finding near-native solutions. The free ensembles appeared to contain multiple complementary conformations. These were in general not related to the bound structure. We suggest that protein-protein binding follows a three-step mechanism of diffusion, free conformer selection, and refolding. This model combines previously conflicting ideas and is in better agreement with the current data on interaction forces, time scales, and kinetics.
To better understand the interplay between protein-protein binding and protein dynamics, we analyzed molecular dynamics simulations of 17 protein-protein complexes and their unbound components. ...Complex formation does not restrict the conformational freedom of the partner proteins as a whole, but, rather, it leads to a redistribution of dynamics. We calculate the change in conformational entropy for seven complexes with quasiharmonic analysis. We see significant loss, but also increased or unchanged conformational entropy. Where comparison is possible, the results are consistent with experimental data. However, stringent error estimates based on multiple independent simulations reveal large uncertainties that are usually overlooked. We observe substantial gains of pseudo entropy in individual partner proteins, and we observe that all complexes retain residual stabilizing intermolecular motions. Consequently, protein flexibility has an important influence on the thermodynamics of binding and may disfavor as well as favor association. These results support a recently proposed unified model for flexible protein-protein association.
Biskit is a modular, object-oriented python library that provides intuitive classes for many typical tasks of structural bioinformatics research. It facilitates the manipulation and analysis of ...macromolecular structures, protein complexes and molecular dynamics trajectories. At the same time, Biskit offers a software platform for the rapid integration of external programs and new algorithms into complex structural bioinformatics workflows. Calculations are thus often delegated to established programs like Xplor, Amber, Hex, Prosa, Hmmer and Modeller; interfaces to further software can be easily added. Moreover, Biskit simplifies the parallelization of time consuming calculations via PVM (Parallel Virtual Machine).
Availability: The latest snapshot of Biskit, documentation and examples are freely available under the GNU General Public License at http://biskit.sf.net (alternate url http://biskit.pasteur.fr).
Contact:
johan.leckner@nmr.se, raik.gruenberg@crg.es
Synthetic biology open language (SBOL) version 3.1.0 Buecherl, Lukas; Mitchell, Thomas; Scott-Brown, James ...
Berichte aus der medizinischen Informatik und Bioinformatik/Journal of integrative bioinformatics,
03/2023, Letnik:
20, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Synthetic biology builds upon genetics, molecular biology, and metabolic engineering by applying engineering principles to the design of biological systems. When designing a synthetic system, ...synthetic biologists need to exchange information about multiple types of molecules, the intended behavior of the system, and actual experimental measurements. The
(SBOL) has been developed as a standard to support the specification and exchange of biological design information in synthetic biology, following an open community process involving both bench scientists and scientific modelers and software developers, across academia, industry, and other institutions. This document describes SBOL 3.1.0, which improves on version 3.0.0 by including a number of corrections and clarifications as well as several other updates and enhancements. First, this version includes a complete set of validation rules for checking whether documents are valid SBOL 3. Second, the best practices section has been moved to an online repository that allows for more rapid and interactive of sharing these conventions. Third, it includes updates based upon six community approved enhancement proposals. Two enhancement proposals are related to the representation of an object’s namespace. In particular, the
class has been removed and replaced with a
property on each class. Another enhancement is the generalization of the
class to allow direct use of
and
. Next, the
class now allow
to be
to describe higher-order interactions. Another change is the use of
terms for
. Finally, this version of SBOL has generalized from using Unique Reference Identifiers (URIs) to
(IRIs) to support international character sets.
PDF
