Biology subjects

Rosser, S.

Publications and source records attributed to Rosser, S..

6 recordsLinked to original sources

bioRxiv2023

Biofoundry-scale DNA assembly validation using cost-effective high-throughput long read sequencing

Biofoundries are automated high-throughput facilities specialising in the design, construction and testing of engineered/synthetic DNA constructs (plasmids), often from genetic parts. A critical step of this process is assessing the fidelity of the assembled DNA construct to the desired design. Current methods utilised for this purpose are restriction digest or PCR followed by fragment analysis, and sequencing. The Edinburgh Genome Foundry (EGF) has recently established a single-molecule sequencing quality control step using the Oxford Nanopore sequencing technology, along with a companion Nextflow pipeline and a Python package to perform in-depth analysis and generate a detailed report. Our software enables biofoundry scientists and end-users to rapidly analyse sequencing data, without specialised bioinformatics knowledge. In conclusion, we have created a laboratory and software protocol that validates assembled, cloned or edited plasmids, using Nanopore long reads, which can serve as a useful resource for the genetics, synthetic biology and sequencing communities. Author informationAll authors contributed to the design of the sequencing quality control step and pipeline, and the preparation of the manuscript. P.V. wrote the manuscript, designed and implemented the bioinformatics pipeline and interpreted results. S.D. wrote the manuscript, implemented the laboratory protocol and interpreted results. G.S. designed the bioinformatics pipeline. R.F. wrote the manuscript and contributed to the design of the laboratory protocol and pipeline. Address: Edinburgh Genome Foundry (University of Edinburgh), Michael Swann Building, Max Born Crescent, Edinburgh, EH9 3BF, United Kingdom Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/558498v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@a819e5org.highwire.dtl.DTLVardef@3c34ceorg.highwire.dtl.DTLVardef@aef5d8org.highwire.dtl.DTLVardef@8e5d2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Imaging proteins sensitive to direct fusions using transient peptide-peptide interactions

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of protein subcellular location and function. Some proteins, however, show altered localization and/or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to [~]200 nm, which is two orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs. FOR TABLE OF CONTENTS ONLY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/547312v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@ce1d9borg.highwire.dtl.DTLVardef@1f9b5c8org.highwire.dtl.DTLVardef@10d92ccorg.highwire.dtl.DTLVardef@20ad5d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Live-cell super-resolution imaging of actin using LifeAct-14 with a PAINT-based approach

We present direct-LIVE-PAINT, an easy-to-implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct-LIVE-PAINT with an actin-binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct-LIVE-PAINT can be used to image actin structures below the diffraction-limit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells.

synthetic biology↗

bioRxiv2021

Multiplexed activation in mammalian cells using dFnCas12a-VPR

The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. In particular the recently discovered Cas12a systems offer extended potential, as Cas12a is capable of processing its own crRNA array to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with FnCas12a possessing a shorter PAM sequence than As or Lb variants, enabling denser targeting of genomic loci. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5 of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting.

synthetic biology↗

bioRxiv2020

DNA Features Viewer, a sequence annotation formatting and plotting library for Python

MotivationWhile the Python programming language counts many Bioinformatics and Computational Biology libraries, none offers customizable sequence annotations visualizations with layout optimization. ResultsDNA Features Viewer is a sequence annotations plotting library which optimizes plot readability while letting users tailor other visual aspects (colors, labels, highlights, etc.) to their particular use case. AvailabilityOpen-source code and documentation are available on Github under the MIT licence (https://github.com/Edinburgh-Genome-Foundry/DnaFeaturesViewer). Contactvalentin.zulkower@ed.ac.uk Supplementary informationattached.

bioinformatics↗

bioRxiv2019

DNA Chisel, a versatile sequence optimizer

MotivationAccounting for biological and practical requirements in DNA sequence design often results in challenging optimization problems. Current software solutions are problem-specific and hard to combine. ResultsDNA Chisel is an easy-to-use, easy-to-extend sequence optimization framework allowing to freely define and combine optimization specifications via Python scripts or Genbank annotations. Availabilityas a web application (https://cuba.genomefoundry.org/sculpt_a_sequence) or open-source Python library (code and documentation at https://github.com/Edinburgh-Genome-Foundry/DNAChisel). Contactvalentin.zulkower@ed.ac.uk Supplementary informationattached.

synthetic biology↗