Biology subjects

Rosser, S. J.

Publications and source records attributed to Rosser, S. J..

3 recordsLinked to original sources

bioRxiv2024

Super-resolution imaging of proteins inside live mammalian cells with mLIVE-PAINT

Super-resolution microscopy has revolutionized biological imaging, enabling the visualization of structures at the nanometer length scale. Its application in live cells, however, has remained challenging. To address this, we adapted LIVE-PAINT, an approach we established in yeast, for application in live mammalian cells. Using the 101A/101B coiled-coil peptide pair as a peptide-based targeting system, we successfully demonstrate the super-resolution imaging of two distinct proteins in mammalian cells, one localized in the nucleus, and the second in the cytoplasm. This study highlights the versatility of LIVE-PAINT, suggesting its potential for live-cell super-resolution imaging across a range of protein targets in mammalian cells. We name the mammalian cell version of our original method mLIVE-PAINT.

synthetic biology↗

bioRxiv2022

ACtivE: Assembly and CRISPR-targeted in vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time

Among the numerous genetic tools developed for yeast, CRISPR/Cas system has been a widely used genome editing method thanks to its sophistication. However, CRISPR methods for yeast generally rely on pre-assembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose a convenient, rapid, standardizable CRISPR method, named Assembly and CRISPR-targeted in vivo Editing (ACtivE), which only relies on in vivo assembly of linear DNA fragments for both plasmid and donor DNA construction. Thus, depending on the users need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight ARS-close loci in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing {beta}-carotene pathway. In only a few days, > 80% integration efficiency for single gene integration and > 50% integration efficiency for triplex integration were achieved from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.

synthetic biology↗

bioRxiv2019

Control of ΦC31 integrase-mediated site-specific recombination by protein trans splicing

Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system for rapid regulation of site-specific recombination events in vivo. The{varphi} C31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in E. coli generated a fully functional{varphi} C31 integrase. Protein splicing is necessary for recombination activity; no activity was observed when the{varphi} C31 integrase N-and C-terminal extein domains without the intein sequences were co-expressed, nor when a key intein catalytic residue was mutated. As a proof of principle, we used a bistable switch based on an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system. We used araC and tet inducible promoters to regulate the expression of the two parts of the split recombinase. Inversion of a DNA segment containing a constitutive promoter, catalyzed by trans-spliced integrase, switches between RFP and GFP expression only when both inducible promoters are ON. We used the same split inteins to regulate the reconstitution of a split integrase-RDF fusion that efficiently catalyzed the reverse attR x attL recombination, demonstrating that our split-intein regulated recombination system can function as a reversible AND gate in which the forward reaction is catalyzed by the integrase, and the reverse reaction by the integrase-RDF fusion. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.

synthetic biology↗