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Computer simulations of the experiments are being performed using the Los Alamos National Laboratories’ FLAG magnetohydrodynamics code by graduate student Seth Kreher, and Lawrence Livermore National Laboratory’s Ares by graduate students Robert Masti and Matthew Carrier.
Abstract not provided.
The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications.
Slides to be presented during the NNSA 2021 Stewardship Science Academic Programs (SSAP) Symposium (https://web.cvent.com/event/1893b742-2d98-49d5-87cc-f07b1c3cb66c/summary?environment=P2). The symposium will be held virtually February 16–18, 2021.
I am a staff scientist at Sandia National Laboratories (SNL). I work on multiple fundamental-science projects and lead modeling/data-analysis for the stellar opacity experiments. I also work with theorists to refine plasma material-property calculations to make NNSA simulations more predictable. These are challenging and important problems for national security. In fact, our experiments raised questions about “opacity”, the property of matter that controls energy transport inside stars. This work was published by Nature due to its serious implication over broad applications, and our team received NNSA Defense Program Award of Excellence, both in 2015. Work at SNL is collegial and full of learning through interactions with the world’s finest experimentalists and theorists. When I started my Ph.D. program, working at a national lab was a dream position. Today, I collaborate with top scientists to solve mission-critical problems. The Stewardship Science Academic Programs (SSAP) and National Laser Users’ Facility (NLUF) played a vital role in the efficient learning and seamless transition to SNL.
Presentation for the NNSA's 2022 Stewardship Science Academic Programs (SSAP) Symposium February 15-17, 2022
This document summarizes the activities at the Mykonos Pulsed Power Facility during the calendar year 2025. Section 1 reports on the yearly shot statistics along with some facility highlights. Section 2 discusses the many improvements we were able to complete this year, thanks to the generous Mykonos Academic Access Programs (MAAP) funding from SSAP and Sandia strategic investments. Section 3 focuses on each individual campaign and their respective results.
ABSTRACT Several strains in the Pseudomonas genus are categorized as plant growth-promoting rhizobacteria (PGPR). Although several of these strains are strong candidates for applications as biofertilizers or biopesticides, genome editing approaches are generally limited and require further development. Editing genomes in PGPR could enable more robust agricultural applications, persistence, and biosafety measures. In this study, we investigate the use of five phage-encoded recombinases to develop a recombineering workflow in three PGPR strains: Pseudomonas protegens Pf-5, Pseudomonas protegens CHA0, and Pseudomonas putida KT2440. Using point mutations in the rpoB gene, we reach maximum recombineering efficiencies of 1.5 × 10 −4 , 3 × 10 −4 , and 5 × 10 −5 , respectively, in these strains using λ-Red Beta recombinase from Escherichia coli . We further examine recombineering efficiencies across these strains as a function of selected mutation, editing template concentration, and phosphorothiolate bond protection. This work validates the use of these tools across several environmentally and biotechnologically relevant strains to expand the possibilities of genetic manipulation in the Pseudomonas genus. IMPORTANCE The Pseudomonas genus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol, and synthetic biology. Though several strains have been identified with beneficial properties, chromosomal manipulations to further improve these strains for commercial applications have been limited due to the lack of efficient genetic tools that have been tested across this genus. Here, we test the recombineering efficiencies of five phage-derived recombinases across three biotechnologically relevant Pseudomonas strains: P. putida KT2440, P. protegens Pf-5, and P. protegens CHA0. These results demonstrate a method to generate targeted mutations quickly and efficiently across these strains, ideally introducing a method that can be implemented across the Pseudomonas genus and a strategy that may be applied to develop analogous systems in other nonmodel bacteria.