Biology subjects

Brumwell, S. L.

Publications and source records attributed to Brumwell, S. L..

12 recordsLinked to original sources

bioRxiv2025

A scalable transposon mutagenesis system for non-model bacteria

Transposon mutagenesis enables genome-wide interrogation of gene function with a single self-contained genetic construct. However, its application to non-model bacteria remains limited because transposition efficiency depends on multiple host factors that are difficult to predict a priori including transposase activity, antibiotic resistance marker performance, and regulatory element compatibility. Here, we present a scalable system to identify functional transposon configurations in non-model bacteria through pooled library screening. We selected 18 promoters across multiple bacterial phyla to independently drive expression of the transposase and antibiotic resistance marker, generating 324 promoter combinatorial variants for each of six antibiotic resistance markers. We developed a high-throughput, automated workflow to deliver all 1,944 mariner-based transposon variants in a single experiment and applied this to 92 non-model bacteria spanning multiple phyla. From this, we identified functional transposons for 43 strains, with high-level mutagenesis (102-104 unique insertions) in 13 species, including seven with no previously described transposon mutagenesis. We then expanded to a dual-transposase system, mariner or Tn5, and devised a single transposon insertion sequencing method for high-throughput screening of 3,888 configurations. To demonstrate the practical utility of our screening approach, we used a top-performing variant to generate a genome-wide transposon mutant library for Comamonas testosteroni KF-1, a bacterium that metabolizes plastic- and lignin-derived polymers. We assayed this C. testosteroni mutant library to identify enzymatic pathways, transporter genes, and regulators essential for the metabolism of plastics-associated monomer terephthalate and lignin-associated monomer 4-hydroxybenzoate. Together, this work establishes a scalable approach to construct and identify genetic perturbation systems in non-model bacteria, expanding our ability to systematically probe gene function across the bacterial tree of life.

synthetic biology↗

bioRxiv2025

A genetic platform for a biocementation bacterium

Sporosarcina pasteurii is the most widely studied bacterium for microbially-induced calcium carbonate precipitation (MICP), a process of intense interest for materials and construction applications. Despite two decades of investigation, S. pasteurii has remained genetically intractable, limiting our mechanistic understanding of biomineralization pathways and constraining efforts to engineer scalable solutions. Here, we present the first genetic toolkit for S. pasteurii, including a stable replicating plasmid, a conjugation-based DNA delivery protocol, engineered inducible promoters, and methods for genome modification. Using homologous recombination, we precisely deleted 5.7 kb of the genome spanning two operons encoding urease activity and demonstrated complete loss of biocementation. We also screened a library of engineered transposon constructs for activity in S. pasteurii and generated a genome-wide mutant library with >15,000 unique insertion sites. Using this library, we identified putative genes affecting ureolytic growth, revealing previously inaccessible aspects of S. pasteurii genetics. This work establishes S. pasteurii as a genetically tractable platform for rational engineering of MICP and constitutes the first genetic modification capability within the Sporosarcina genus.

synthetic biology↗

bioRxiv2025

Active learning guides automated discovery of DNA delivery via electroporation for non-model microbes

Delivery of recombinant DNA is foundational for understanding and engineering a target organism. Electroporation can be applied to any cell type, yet identification of a working protocol for new organisms remains bespoke and laborious because no systematic framework exists, and appropriate instrumentation is lacking. Here, we describe an automated high-throughput platform which uses active learning to discover electroporation protocols for non-model microbes. We first devised a 24-condition electroporation screen, based on systematic evaluation and selection of key parameters, that can be applied to any microbe. Facilitated by a custom-built fully-programmable electroporator, we successfully identified electroporation protocols for eight non-model bacteria using this screen alone. We then combined this electroporation screen with our pooled POSSUM plasmid library to simultaneously evaluate 408 experimental conditions per organism and identified both a protocol and a replicating plasmid for five non-model Proteobacteria spanning three major classes (alpha-, beta-, and gamma-). We report the first electroporation protocols for Shewanella indica, Shewanella putrefaciens 200, Shewanella putrefaciens 95, Halomonas elongata, Piscinibacter sakaiensis, and Duganella zoogloeoides, as well as multiple alternative protocols for Shewanella amazonensis, Shewanella oneidensis, Azospirillum brasilense, Cupriavidus necator, Pseudomonas alcaliphila, and Escherichia coli. Finally, we developed an active learning pipeline to guide the selection of parameters based on gathered experimental data. Using our robotic platform, we iteratively tested 538 conditions over three iterations to improve electroporation for the emerging industrial chassis C. necator, achieving 8.6-fold higher transformation efficiency than state of the art. This work establishes a discovery platform for DNA delivery to diverse and recalcitrant microbes that can be extended broadly to non-model organisms in our biosphere.

microbiology↗

bioRxiv2025

Functional genomics in a microbe that degrades and metabolizes PET plastic

Piscinibacter sakaiensis (formerly Ideonella sakaiensis) was the first bacterial species known to both completely degrade and assimilate polyethylene terephthalate (PET). However, the absence of efficient genetic tools has limited direct engineering of this organism, forcing most efforts to rely on heterologous expression of PET-degrading enzymes in model hosts. Here, we establish foundational genetic tools to engineer P. sakaiensis. We identify a functional plasmid origin of replication, multiple new selectable markers, and a transposon system for the strain. We use these tools to construct a genome-wide, barcoded transposon mutant library for pooled high-throughput functional screens. We apply this mutant library to growth on PET and identify metabolic and physiological genes that impact PET biodegradation. We also use this library to reveal mutants with improved DNA uptake for genome engineering. Together, these advances provide a platform for functional genomics in P. sakaiensis and positions this naturally evolved plastic-degrading bacterium as an engineerable chassis for synthetic biology and sustainable materials research.

microbiology↗

bioRxiv2025

A reference set of functional plasmids for Vibrio natriegens

New microbial hosts with superior phenotypes, such as fast growth, are attractive for research and biotechnology, but often lack systematic evaluation of functional genetic parts. A reference set of working plasmids would increase reproducibility and encourage use of these hosts. Here, we use the POSSUM toolkit, a collection of 23 origins-of-replication and 6 antibiotic markers, to identify functional genetic parts for strains of Vibrio natriegens. We applied this to the wild-type strain ATCC 14048 and an engineered variant NBx CyClone, evaluating 414 combinations of origins of replication and antibiotic selection conditions. We show that both strains support five replicons (pNG2, pSa, pSC101ts, p15A, and RSF1010) with NBx CyClone supporting an extra replicon RK2. The assay can be performed in under a week and is compatible with multiple DNA delivery methods. This work demonstrates the feasibility of rapidly establishing reference information to accelerate the adoption of new microbial hosts.

synthetic biology↗

bioRxiv2025

Engineering Conjugative Plasmids for Inducible Horizontal DNA Transfer

Rapidly developing microbial resistance to existing antimicrobials poses a growing threat to public health and global food security. Current chemical-based treatments target cells by inhibiting growth or metabolic function, but their effectiveness is diminishing. To address the growing antimicrobial resistance crisis, there is an urgent need for innovative therapies. Conjugative plasmids, a natural mechanism of horizontal gene transfer in bacteria, have been repurposed to deliver toxic genetic cargo to recipient cells, showing promise as next-generation antimicrobial agents. However, the ecological risks posed by unintended gene transfer require robust biocontainment strategies. In this study, we developed inducible conjugative plasmids to solve these challenges. Utilizing an arabinose-inducible promoter, we evaluated 13 plasmids with single essential gene deletions, identifying trbC and trbF as strong candidates for stringent regulation. These plasmids demonstrated inducibility in both cis and trans configurations, with induction resulting in up to a 5-log increase in conjugation efficiency compared to uninduced conditions. Although challenges such as reduced conjugation efficiency and promoter leakiness persist, this work establishes a foundation for the controlled transfer of plasmids, paving the way for safer and more effective antimicrobial technologies.

synthetic biology↗

bioRxiv2024

Design and construction towards a pan-microbial toolkit

Establishing genetic tractability in non-model microbes requires identifying genetic parts that function in a target host. However, the paucity and purported narrow host range of available parts means that successful identification is governed by serendipity. Instead, a more comprehensive and scalable process would be desirable. Here, we describe the design principles for a pan-microbial genetic toolkit in which phylogenetically-diverse parts can be assembled and tested for function in microbes using high-throughput readouts. The architecture is based on Golden Gate Assembly, which simplifies the addition of parts and the construction of combinatorial libraries. We used this framework to develop two modules: first, the POSSUM (Plasmid Origins and Selection MarkerS for Undomesticated Microbes) module for identification of replicating plasmids in non-model microbes which includes 29 plasmid origin of replication sequences, 23 selection markers, and 30 unique DNA sequences for tracking by sequencing; second, the MACKEREL (Modular, NGS-trACKable ExpRession ELement) module, for identification of functional gene expression cassettes which includes 426 bacterial promoter-RBS sequences driving fluorescent reporter expression, trackable by flow cytometry. We demonstrate the use of these libraries to screen for functional promoter-RBS variants in 6 non-model microbes. Continued efforts to expand this pan-microbial toolbox will accelerate efforts to improve genetic tractability and guide research across the tree of life.

synthetic biology↗

bioRxiv2023

A scalable framework for high-throughput identification of functional origins of replication in non-model bacteria

Microbial genetic manipulation requires access to engineerable plasmids that can be programmed to perturb genes, pathways and genomes. The extensive repertoire of plasmids available for model microbes, such as Escherichia coli, has facilitated fundamental biology studies and synthetic biology applications. However, the scarcity of plasmids for non-model microbes hinders efforts to broaden our biological knowledge and constrains the development of biotechnological solutions. In this study, we introduce a molecular toolkit and multiplexed screen to evaluate functional plasmids in non-model microbes. We constructed a collection of genetic parts consisting of 22 origins of replication (ORIs), 20 antibiotic selectable markers, and 30 molecular barcodes, which can be assembled combinatorially to create a library of plasmids trackable by next-generation DNA sequencing. We demonstrate our approach by delivering a pooled library of 22 ORIs to 12 bacterial species including extremophiles, electroactive bacteria and bioproduction strains. We report, for the first time, DNA delivery by conjugation and functional ORIs for Halomonas alkaliphila, Halomonas neptunia, and Shewanella electrodiphila. Furthermore, we expand the list of functional ORIs for Duganella zoogloeoides, Pseudomonas alcaliphila, Shewanella oneidensis and Shewanella putrefaciens. This screen provides a scalable high-throughput system to rapidly build and identify functional plasmids to establish genetic tractability in non-model microbes.

synthetic biology↗

bioRxiv2022

SLICER: Seamless Loss of Integrated Cassettes Using Endonuclease Cleavage and Recombination in Deinococcus radiodurans

Methods for creating seamless genome modifications are an essential part of the microbial genetic toolkit that allows for strain engineering through the recycling of selectable markers. Here, we report the development of a method, termed SLICER, which can be used to create seamless genome modifications in D. radiodurans. We used SLICER to sequentially target four putative restriction-modification (R-M) system genes, recycling the same selective and screening markers for each subsequent deletion. A fifth R-M gene was replaced by a selectable marker to create a final D. radiodurans strain with 5 of the 6 putative R-M systems deleted. While we observed no significant increase in transformation efficiency, SLICER is a promising method to obtain a fully restriction-minus strain and expand the synthetic biology applications of D. radiodurans including as an in vivo DNA assembly platform. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/512367v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@13a4f39org.highwire.dtl.DTLVardef@127655corg.highwire.dtl.DTLVardef@16faddforg.highwire.dtl.DTLVardef@10023cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Conjugation-based genome engineering in Deinococcus radiodurans

D. radiodurans has become an attractive microbial platform for the study of extremophile biology and industrial bioproduction. To improve the genomic manipulation and tractability of this species, the development of tools for whole genome engineering and design is necessary. Here, we report the development of a simple and robust conjugation-based transformation system from E. coli to D. radiodurans allowing for the introduction of stable, replicating plasmids expressing antibiotic resistance markers. Using this method with nonreplicating plasmids, we developed a protocol for creating sequential gene deletions in D. radiodurans by targeting re-striction-modification system genes. Importantly, we demonstrated a conjugation-based method for cloning the large (178 kb), high G+C content MP1 megaplasmid from D. radiodurans in E. coli. The conjugation-based tools described here will facilitate the development of D. radiodurans strains with synthetic genomes for biological studies and industrial applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/464295v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@128437borg.highwire.dtl.DTLVardef@6669ddorg.highwire.dtl.DTLVardef@1b041eaorg.highwire.dtl.DTLVardef@b77f07_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

Rapid method for generating designer algal mitochondrial genomes

With synthetic biology, we can turn algae into bio-factories that produce high-value molecules (e.g. medicines or biofuels) or tackle global challenges (e.g. malnutrition and climate change). This realization has provoked rapid progress towards the creation of genetic tools for multiple algal species, notably Phaeodactylum tricornutum. The power of synthetic biology to generate more useful or productive organisms is contingent on the ability to produce diverse DNA molecules and rapidly screen them for beneficial variants. However, it is still relatively expensive to synthesize DNA, and delivering large DNA (>50 kbp) to eukaryotic cellular compartments remains challenging. In this study, we establish a robust system for building algal mitochondrial genomes as a practical alternative to DNA synthesis. Our approach permits an inexpensive and rapid generation of mitochondrial derivatives designed for testing targeted DNA delivery. First, we cloned the mitochondrial genome of P. tricornutum into the eukaryotic host organism S. cerevisiae using two different techniques: transformation-associated recombination; and PCR-based cloning. Next, we analyzed the cloned genomes by multiplex PCR, and correct genomes were transferred to prokaryotic host E. coli. Then these genomes were again analyzed by multiplex PCR, followed by diagnostic digest and complete-plasmid sequencing to evaluate the fidelity of each cloning method. Finally, we assessed the burden on eukaryotic and prokaryotic hosts to propagate the cloned genomes. We conclude that our system can reliably generate variants for genome-level engineering of algal mitochondria. HIGHLIGHTSO_LITAR-cloned mitochondrial genome of P. tricornutum in yeast C_LIO_LIDeveloped PCR-based cloning method to create designer algal mitochondrial genomes C_LIO_LIStably propagated algal mitochondrial genomes in S. cerevisiae and E. coli hosts C_LI

synthetic biology↗

bioRxiv2018

Designer Sinorhizobium meliloti strains and multi-functional vectors for direct inter-kingdom transfer of high G+C content DNA

Storage and manipulation of large DNA fragments is crucial for synthetic biology applications, yet DNA with high G+C content can be unstable in many host organisms. Here, we report the development of Sinorhizobium meliloti as a new universal host that can store DNA, including high G+C content, and mobilize DNA to Escherichia coli, Saccharomyces cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. We deleted the S. meliloti hsdR restriction-system to enable DNA transformation with up to 1.4 x 105 efficiency. Multi-host and multi-functional shuttle vectors (MHS) were constructed and shown to stably replicate in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum, with a copy-number inducible E. coli origin for isolating plasmid DNA. Crucially, we demonstrated that S. meliloti can act as a universal conjugative donor for MHS plasmids with a cargo of at least 62 kb of G+C rich DNA derived from Deinococcus radiodurans.

synthetic biology↗