Search for: All records

Sustainably enhancing crop production is a global necessity to meet the escalating demand for staple crops while sustainably managing their associated carbon/nitrogen inputs. Leveraging plant-associated microbiomes is a promising avenue for addressing this demand. However, studying these communities and engineering them for sustainable enhancement of crop production have remained a challenge due to limited genetic tools and methods. In this work, we detail the development of the Maize Root Microbiome ToolKit (MRMTK), a rapid Modular Cloning (MoClo) toolkit that only takes 2.5 h to generate desired constructs (5400 potential plasmids) that replicate and express heterologous genes in Enterobacter ludwigii strain AA4 (Elu), Pseudomonas putida strain AA7 (Ppu), Herbaspirillum robiniae strain AA6 (Hro), Stenotrophomonas maltophilia strain AA1 (Sma), and Brucella pituitosa strain AA2 (Bpi), which comprise a model maize root synthetic community (SynCom). In addition to these genetic tools, we describe a highly efficient transformation protocol (107–109 transformants/μg of DNA) 1 for each of these strains. Utilizing this highly efficient transformation protocol, we identified endogenous Expression Sequences (ES; promoter and ribosomal binding sites) for each strain via genomic promoter trapping. Overall, MRMTK is a scalable and adaptable platform that expands the genetic engineering toolbox while providing a standardized, high-efficiency transformation method across a diverse group of root commensals. These results unlock the ability to elucidate and engineer plant–microbe interactions promoting plant growth for each of the 5 bacterial strains in this study. 

Abstract Infections by Clostridioides difficile , a bacterium that targets the large intestine (colon), impact a large number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can be delivered to the gut and inhibit the biocatalytic activity of these toxins represent a promising therapeutic strategy to prevent and treat C. diff . infection. We describe an approach that combines a Pep tide B inding D esign (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 colon epithelial cells. One peptide, SA1, is found to block TcdA toxicity in primary-derived human colon (large intestinal) epithelial cells. SA1 binds TcdA with a K D of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR). 

Abstract Poly(ethylene terephthalate) (PET) is a highly recyclable plastic that has been extensively used and manufactured. Like other plastics, PET resists natural degradation, thus accumulating in the environment. Several recycling strategies have been applied to PET, but these tend to result in downcycled products that eventually end up in landfills. This accumulation of landfilled PET waste contributes to the formation of microplastics, which pose a serious threat to marine life and ecosystems, and potentially to human health. To address this issue, our project leveraged synthetic biology to develop a whole‐cell biocatalyst capable of depolymerizing PET in seawater environments by using the fast‐growing, nonpathogenic, moderate halophileVibrio natriegens. By leveraging a two‐enzyme system—comprising a chimera ofIsPETase andIsMHETase fromIdeonella sakaiensis—displayed onV. natriegens, we constructed whole‐cell catalysts that depolymerize PET and convert it into its monomers in salt‐containing media and at a temperature of 30°C.