Biology subjects

Kuo, R.

Publications and source records attributed to Kuo, R..

4 recordsLinked to original sources

bioRxiv2024

Development of Modular Expression and Genome Editing Across Phylogenetically Distinct Diazotrophs

Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs associate closely with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrate genome editing in Klebsiella michiganensis M5al. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/595406v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@ea73a4org.highwire.dtl.DTLVardef@4e540eorg.highwire.dtl.DTLVardef@706557org.highwire.dtl.DTLVardef@10d4c9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Evidence that SPIO Chain Formation is Essential for High-Resolution MPI

Magnetic Particle Imaging (MPI) is a noninvasive imaging modality that exploits the saturation properties of superparamagnetic iron oxide particles (SPIOs). A major thrust of MPI research aims to sharpen the magnetic resolution of biocompatible SPIOs, which will be crucial for affordable and safe clinical translation. We recently reported on a new class of MPI tracers --called superferromagnetic iron oxide nanoparticles (SFMIOs) -- which offer much sharper magnetic saturation curves. SFMIOs experimentally demonstrate 10-fold improvement in both resolution and sensitivity. However, superferromagnetism is a relatively unexplored branch of physics and the nanoscale physics and dynamics of SFMIOs remain a mystery. Here we show experimentally that chaining of SPIOs can explain SFMIOs boost in SNR and resolution. We show how concentration, viscosity, transmit amplitude, and pre-polarization time can all affect SPIO chain formation and SFMIO behavior. These experiments will inform strategies on SFMIO chemical synthesis as well as SFMIO data acquisition pulse sequences.

bioengineering↗

bioRxiv2022

First superferromagnetic remanence characterization and scan optimization for super-resolution Magnetic Particle Imaging

Magnetic particle imaging (MPI) is a sensitive, high contrast tracer modality that images superparamagnetic iron oxide nanoparticles (SPIOs), enabling radiation-free theranostic imaging. MPI resolution is currently limited by scanner and particle constraints. Recent tracers have experimentally shown 10x resolution and signal improvements, with dramatically sharper M-H curves. Experiments suggest that this results from interparticle interactions, conforming to literature definitions of superferromagnetism. We thus call our tracers superferromagnetic iron oxide nanoparticles (SFMIOs). While SFMIOs provide excellent signal and resolution, they exhibit hysteresis, with non-negligible remanence and coercivity. We provide the first report on MPI scanning with remanence and coercivity, including the first quantitative measurements of SFMIO remanence decay and reformation using a novel multi-echo pulse sequence. We also describe an SNR-optimized pulse sequence for SFMIOs under human electromagnetic safety limitations. The resolution from SFMIOs could enable clinical MPI with 10x reduced scanner selection fields, reducing hardware costs by up to 100x.

bioengineering↗

bioRxiv2021

A high resolution single molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

BackgroundAccurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single molecule long read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation or incomplete cDNA synthesis. ResultsWe present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 160k transcripts - twice that of the best current Arabidopsis transcriptome and including over 1,500 novel genes. 79% of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We developed novel methods to determine splice junctions and transcription start and end sites accurately. Mis- match profiles around splice junctions provided a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identified high confidence transcription start/end sites and removed fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provided higher resolution of transcript expression profiling and identified cold- and light-induced differential transcription start and polyadenylation site usage. ConclusionsAtRTD3 is the most comprehensive Arabidopsis transcriptome currently available. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single molecule sequencing analysis from any species.

plant biology↗