Biology subjects

Crook, N.

Publications and source records attributed to Crook, N..

6 recordsLinked to original sources

bioRxiv2025

Inducible genome-wide mutagenesis for improvement of pDNA production by E. coli

Plasmid DNA (pDNA) is a cost-driving reagent for the production of gene therapies and DNA vaccines. Improving pDNA production in the most common production host (E. coli) has faced obstacles arising from the complex network of genes responsible for pDNA synthesis, with the specific enzyme(s) limiting pDNA yield remaining unidentified. To address this challenge, we employed an inducible genome-wide mutagenesis strategy, combined with fluorescent screening, to isolate E. coli NEB 5 strains with enhanced pDNA production. Following selection, we successfully isolated an E. coli strain (M3) with elevated plasmid copy numbers (PCNs) across multiple origin types. Specifically, we observed a 5.93-fold increase in PCN for the GFP reporter plasmid, a 1.93-fold increase for the gWiz DNA vaccine plasmid, and an 8.7-fold increase for the pAAV-CAGG-eGFP plasmid, all of which contain pUC origins. In addition, plasmids with p15A and pSC101 origins showed 1.44-fold and 1.68-fold increases in PCN, respectively. Whole-genome sequencing of the adapted strain M3 identified 85 mutations, including one in recG, which encodes an ATP-dependent DNA helicase. Replacement of the mutant recG with its wild-type counterpart in the mutant strain resulted in a 63% reduction in PCN, but the recG mutation alone was insufficient to increase PCN in the wild-type strain. These findings suggest that the recG mutation plays a synergistic role with other genomic mutations to drive PCN increases. Taken together, this study presents the development of a pDNA hyperaccumulating E. coli strain with promising applications in industrial and therapeutic pDNA production, while also offering important insights into key genes involved in pDNA production.

synthetic biology↗

bioRxiv2025

Differential metaproteomics of bacteria grown in vitro and in planta reveals functions used during growth on maize roots

Microbes are ubiquitous in the rhizosphere and play crucial roles in plant health, yet the metabolisms and physiologies of individual species in planta remain poorly understood. In this study, we examined microbial gene expression in response to the maize root environment for seven bacterial species originally isolated from maize roots. We grew each species individually, both in vitro in a minimal medium and in planta, and used differential proteomics to identify functions upregulated specifically when bacteria are grown on maize roots. We identified between 1,500 and 2,100 proteins from each species, with 20-60% of these proteins being differentially abundant between the two conditions. While we found that transporter proteins were upregulated in all species in planta, all other differentially abundant functions varied greatly between species, suggesting niche specialization in root-associated microbes. Indeed, in vitro assays confirmed that Curtobacterium pusillum likely degrades plant hemicellulose, Enterobacter ludwigii may benefit the plant by phosphate solubilization, and Herbaspirillum robiniae colonizes maize roots more effectively when both of its Type VI Secretion Systems are functional. Together, our findings highlight both conserved and species-specific bacterial strategies for growth in the root environment and lay a foundation for future work investigating the mechanisms underlying plant-microbiota interactions. ImportanceBacteria that live on and around plant roots are important for plant growth and health, yet we still know relatively little about how individual bacterial species behave in this environment. In this study, we looked at seven bacterial species originally isolated from maize roots to understand how they change their metabolism and physiology when grown on the plant versus when grown under laboratory conditions. By doing this, we identified key strategies bacteria use to survive and thrive in the root environment, including changes in nutrient uptake, metabolism, and secretion systems. We also substantiated some of these behaviors using lab experiments and bacterial mutants. Understanding these species-specific functions helps us learn how bacteria establish themselves on roots and interact with the plant. This knowledge is critical for future efforts to design effective microbial communities that improve crop performance and resilience.

microbiology↗

bioRxiv2024

Targeted DNA ADP-ribosylation triggers templated repair in bacteria and base mutagenesis in eukaryotes

Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here, we harness the bacterial anti-phage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives efficient homologous recombination in tested bacteria, offering flexible and scar-free genome editing without base replacement nor counterselection. In tested eukaryotes including yeast, plants and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of a chemical moiety to DNA to expand current modalities for precision gene editing.

synthetic biology↗

bioRxiv2023

A cell-free transcription-translation pipeline for recreating methylation patterns boosts DNA transformation in bacteria

The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic-biology chasses. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here we report IMPRINT, a generalized, rapid and scalable approach based on cell-free transcription-translation (TXTL) systems to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from the bacterial hosts restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the host DNAs methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also developed a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to facilitate a library screen for translational rules in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling use of increasingly sophisticated genetic manipulation tools across the bacterial world.

synthetic biology↗

bioRxiv2023

Design of 8-mer Peptides that Block Clostridioides difficile Toxin A in Intestinal Cells

Clostridioides difficile (C. diff.) is a bacterium that causes severe diarrhea and inflammation of the colon. The pathogenicity of C. diff. infection is derived from two major toxins, toxins A (TcdA) and B (TcdB). Peptide inhibitors that can be delivered to the gut to inactivate these toxins are an attractive therapeutic strategy. In this work, we present a new approach that combines a peptide binding design algorithm (PepBD), molecular-level simulations, rapid screening of candidate peptides for toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block the glucosyltransferase activity of TcdA by targeting its glucosyltransferase domain (GTD). Using PepBD and explicit-solvent molecular dynamics simulations, we identified seven candidate peptides, SA1-SA7. These peptides were selected for specific TcdA GTD binding through a custom solid-phase peptide screening system, which eliminated the weaker inhibitors SA5-SA7. The efficacies of SA1-SA4 were then tested using a trans-epithelial electrical resistance (TEER) assay on monolayers of the human gut epithelial culture model. One peptide, SA1, was found to block TcdA toxicity in primary-derived human jejunum (small intestinal) and colon (large intestinal) epithelial cells. SA1 bound TcdA with a KD of 56.1 {+/-} 29.8 nM as measured by surface plasmon resonance (SPR). Significance StatementInfections by Clostridioides difficile, a bacterium that targets the large intestine (colon), impact a significant number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can inhibit the biocatalytic activity of these toxins represent a promising strategy to prevent and treat C. diff. infection. We describe an approach that combines a Peptide Binding Design (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in small intestinal and colon epithelial cells. Importantly, our designed peptide, SA1, bound toxin A with nanomolar affinity and blocked toxicity in colon cells.

bioengineering↗

bioRxiv2018

Adaptive strategies of the candidate probiotic E. coli Nissle in the mammalian gut

Probiotics are living microorganisms that are increasingly used as gastrointestinal therapeutics by virtue of their innate or engineered genetic function. Unlike abiotic therapeutics, probiotics can replicate in their intended site, subjecting their genomes and therapeutic properties to natural selection. By exposing the candidate probiotic E. coli Nissle (EcN) to the mouse gastrointestinal tract over several weeks, we uncovered the consequences of gut transit, inter-species competition, antibiotic pressure, and engineered genetic function on the processes under selective pressure during both within-genome and horizontal evolutionary modes. We then show the utility of EcN as a chassis for engineered function by achieving the highest reported reduction in serum phenylalanine levels in a mouse model of phenylketonuria using an engineered probiotic. Collectively, we demonstrate a generalizable pipeline which can be applied to other probiotic strains to better understand their safety and engineering potential.

evolutionary biology↗