Plant colonization assays using natural microbial barcodes
Inventors
Higgins, Douglas • PRIOR, Andrew Abel
Assignees
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Publication Number
US-12735713-B2
Publication Date
2026-09-15
Expiration Date
Abstract
The present disclosure is drawn to methods of utilizing nucleic acid barcodes and corresponding amplifying sites in cells in which the barcodes naturally occur. These barcodes and amplifying sites are reconfigured into a single nucleic acid cassette that provides for ease of use in tagging particular species, strains, or variants of cells, each with a different barcode. These barcodes can be used to track the colonization capabilities of the barcoded cells. The present disclosure further provides for assays that utilize natural barcodes to measure relative microbial colonization ability of a plant root system.
Core Innovation
The disclosure describes an engineered barcoded bacterial cell and method of creating the cell for tracking strains and measuring relative plant-root colonization. The native barcode polynucleotide cassette comprises three native nucleotide sequences selected from a donor bacterial cell arranged in a 5′ to 3′ orientation to serve as a forward primer binding site, a barcode, and a reverse primer binding site. The cassette is inserted at a heterologous genomic location in a recipient bacterial cell of the same species as the donor cell.
Barcode abundance in samples from plant-containing growth media is quantified using native-barcode Coco-seq assays with next-generation sequencing or PCR-based methods for comparative colonization assays in mixed microbial populations. Absolute abundance is estimated using known-copy control barcodes and read-count analysis to evaluate strains colonizing plant roots. These approaches support plant colonization, nitrogen fixation, and field-testing platforms.
Claims Coverage
The claims identify one independent claim directed to an engineered barcoded bacterial cell, comprising two inventive features.
Native barcode polynucleotide cassette
The claim covers a cassette comprising three native nucleotide sequences from a donor bacterial cell assembled in a 5′ to 3′ orientation to function as a forward primer binding site, a barcode, and a reverse primer binding site.
Insertion at a heterologous genomic location
The claim covers inserting the native barcode polynucleotide cassette into a genomic locus of a recipient bacterial cell of the same species as the donor cell, wherein the locus is heterologous relative to the native locations of the three sequences.
The independent claim covers constructing a donor-derived native barcode polynucleotide cassette and inserting it at a heterologous genomic location in a same-species recipient bacterial cell.
Stated Advantages
The figures report yield improvements relative to untreated controls.
The assays distinguish bacterial strains across more than two orders of magnitude and discriminate differences in plant-root colonization while supporting strain ranking with fewer plants than absolute-count assays.
The reported insertion-site considerations are described as reducing sequencing bias and unintended effects on gene expression.
Documented Applications
Tracking bacterial strains and measuring relative plant-root colonization, including comparative colonization assays with mixed microbial populations.
Measuring relative and absolute abundance of bacterial strains colonizing plant roots and in pooled samples using next-generation sequencing.
Plant colonization, including corn-root colonization, and field-testing workflows.
Nitrogen-fixing bacterial applications.
Agricultural inputs and crop applications.
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