生物设计研究(英文)最新文献

Probiotics are the treasure of the microbiology fields. They have been widely used in the food industry, clinical treatment, and other fields. The equivocal health-promoting effects and the unknown action mechanism were the largest obstacles for further probiotic's developed applications. In recent years, various genome editing techniques have been developed and applied to explore the mechanisms and functional modifications of probiotics. As important genome editing tools, CRISPR-Cas systems that have opened new improvements in genome editing dedicated to probiotics. The high efficiency, flexibility, and specificity are the advantages of using CRISPR-Cas systems. Here, we summarize the classification and distribution of CRISPR-Cas systems in probiotics, as well as the editing tools developed on the basis of them. Then, we discuss the genome editing of probiotics based on CRISPR-Cas systems and the applications of the engineered probiotics through CRISPR-Cas systems. Finally, we proposed a design route for CRISPR systems that related to the genetically engineered probiotics.

Broad-host-range synthetic biology is an emerging frontier that aims to expand our current engineerable domain of microbial hosts for biodesign applications. As more novel species are brought to "model status," synthetic biologists are discovering that identically engineered genetic circuits can exhibit different performances depending on the organism it operates within, an observation referred to as the "chassis effect." It remains a major challenge to uncover which genome-encoded and biological determinants will underpin chassis effects that govern the performance of engineered genetic devices. In this study, we compared model and novel bacterial hosts to ask whether phylogenomic relatedness or similarity in host physiology is a better predictor of genetic circuit performance. This was accomplished using a comparative framework based on multivariate statistical approaches to systematically demonstrate the chassis effect and characterize the performance dynamics of a genetic inverter circuit operating within 6 Gammaproteobacteria. Our results solidify the notion that genetic devices are strongly impacted by the host context. Furthermore, we formally determined that hosts exhibiting more similar metrics of growth and molecular physiology also exhibit more similar performance of the genetic inverter, indicating that specific bacterial physiology underpins measurable chassis effects. The result of this study contributes to the field of broad-host-range synthetic biology by lending increased predictive power to the implementation of genetic devices in less-established microbial hosts.

High-precision bioengineering and synthetic biology require fine-tuning gene expression at both transcriptional and posttranscriptional levels. Gene transcription is tightly regulated by promoters and terminators. Promoters determine the timing, tissues and cells, and levels of the expression of genes. Terminators mediate transcription termination of genes and affect mRNA levels posttranscriptionally, e.g., the 3'-end processing, stability, translation efficiency, and nuclear to cytoplasmic export of mRNAs. The promoter and terminator combination affects gene expression. In the present article, we review the function and features of plant core promoters, proximal and distal promoters, and terminators, and their effects on and benchmarking strategies for regulating gene expression.

A key functionality of proteins is based on their ability to form interactions with other proteins or peptides. These interactions are neither easily described nor fully understood, which is why the design of specific protein-protein interaction interfaces remains a challenge for protein engineering. We recently developed the software ATLIGATOR to extract common interaction patterns between different types of amino acids and store them in a database. The tool enables the user to better understand frequent interaction patterns and find groups of interactions. Furthermore, frequent motifs can be directly transferred from the database to a user-defined scaffold as a starting point for the engineering of new binding capabilities. Since three-dimensional visualization is a crucial part of ATLIGATOR, we created ATLIGATOR web-a web server offering an intuitive graphical user interface (GUI) available at https://atligator.uni-bayreuth.de. This new interface empowers users to apply ATLIGATOR by providing easy access with having all parts directly connected. Moreover, we extended the web by a design functionality so that, overall, ATLIGATOR web facilitates the use of ATLIGATOR with a more intuitive UI and advanced design options.

MicroRNAs (miRNAs) are a class of endogenous short noncoding RNA. They regulate gene expression and function, essential to biological processes. It is necessary to develop an efficient detection method to determine these valuable biomarkers for the diagnosis of cancers. In this paper, we proposed a general and rapid method for sensitive and quantitative detection of miRNA by combining CRISPR-Cas12a and rolling circle amplification (RCA) with the precircularized probe. Eventually, the detection of miRNA-21 could be completed in 70 min with a limit of detection of 8.1 pM with high specificity. The reaction time was reduced by almost 4 h from more than 5 h to 70 min, which makes detection more efficient. This design improves the efficiency of CRISPR-Cas and RCA-based sensing strategy and shows great potential in lab-based detection and point-of-care test.

Deinococcus radiodurans' high resistance to various stressors combined with its ability to utilize sustainable carbon sources makes it an attractive bacterial chassis for synthetic biology and industrial bioproduction. However, to fully harness the capabilities of this microbe, further strain engineering and tool development are required. Methods for creating seamless genome modifications are an essential part of the microbial genetic toolkit to enable strain engineering. Here, we report the development of the SLICER method, which can be used to create seamless gene deletions in D. radiodurans. This process involves (a) integration of a seamless deletion cassette replacing a target gene, (b) introduction of the pSLICER plasmid to mediate cassette excision by I-SceI endonuclease cleavage and homologous recombination, and (c) curing of the helper plasmid. We demonstrate the utility of SLICER for creating multiple gene deletions in D. radiodurans by sequentially targeting 5 putative restriction-modification system genes, recycling the same selective and screening markers for each subsequent deletion. While we observed no significant increase in transformation efficiency for most of the knockout strains, we demonstrated SLICER as a promising method to create a fully restriction-minus strain to expand the synthetic biology applications of D. radiodurans, including its potential as an in vivo DNA assembly platform.

We previously demonstrated that we could hijack the fungal pheromone signaling pathway to provide a living yeast biosensor where peptide biomarkers were recognized by G-protein-coupled receptors and engineered to transcribe a readout. Here, we demonstrated that the protease could be reintroduced to the biosensor to provide a simple mechanism for distinguishing single-amino-acid changes in peptide ligands that, otherwise, would likely be difficult to detect using binding-based assays. We characterized the dose-response curves for five fungal pheromone G-protein-coupled receptors, peptides, and proteases-Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Schizosaccharomyces octosporus, and Schizosaccharomyces japonicus. Alanine scanning was carried out for the most selective of these-S. cerevisiae and C. albicans-with and without the protease. Two peptide variants were discovered, which showed diminished cleavage by the protease (CaPep2A and CaPep2A13A). Those peptides were then distinguished by utilizing the biosensor strains with and without the protease, which selectively cleaved and altered the apparent concentration of peptide required for half-maximal activation for 2 peptides-CaPep and CaPep13A, respectively-by more than one order of magnitude. These results support the hypothesis that the living yeast biosensor with a sequence-specific protease can translate single-amino-acid changes into more than one order of magnitude apparent shift in the concentration of peptide required for half-maximal activation. With further engineering by computational modeling and directed evolution, the biosensor could likely distinguish a wide variety of peptide sequences beyond the alanine scanning carried out here. In the future, we envision incorporating proteases into our living yeast biosensor for use as a point of care diagnostic, a scalable communication language, and other applications.

The growth of biotechnology in recent decades and the dual-use nature of most bioscience research are making their misuse, or accidental misuse or release, more likely and present as threats to biosecurity. A proactive approach is through educating the next generation of scientists to be more security conscious. However, current educational and professional programs in biosecurity are lacking. In this perspective, we recommend that biosecurity educational opportunities should be implemented and expanded for undergraduate and graduate students who will likely use one or more methods in the field of biotechnology. We then propose that biosecurity education is a key factor in a path toward sustainable and safe research. Finally, a set of 17 biosecurity competencies organized into 6 distinct themes is outlined.

Globally, agriculture depends on industrial nitrogen fertilizer to improve crop growth. Fertilizer production consumes fossil fuels and contributes to environmental nitrogen pollution. A potential solution would be to harness nitrogenases-enzymes capable of converting atmospheric nitrogen N₂ to NH₃ in ambient conditions. It is therefore a major goal of synthetic biology to engineer functional nitrogenases into crop plants, or bacteria that form symbiotic relationships with crops, to support growth and reduce dependence on industrially produced fertilizer. This review paper highlights recent work toward understanding the functional requirements for nitrogenase expression and manipulating nitrogenase gene expression in heterologous hosts to improve activity and oxygen tolerance and potentially to engineer synthetic symbiotic relationships with plants.