Plant colonization assays using natural microbial barcodes
Inventors
Higgins, Douglas • PRIOR, Andrew Abel
Assignees
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Publication Number
US-12421519-B2
Publication Date
2025-09-23
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 invention provides an engineered bacterial cell comprising a native barcode polynucleotide cassette inserted into a genomic locus of the cell. The cassette includes first, second and third native nucleotide sequences oriented 5′ to 3′, where the first native nucleotide sequence serves as a forward primer binding site, the second serves as a barcode, and the third serves as a reverse primer binding site. The first, second and third native nucleotide sequences comprised within the native barcode polynucleotide cassette are from the same species as the engineered bacterial cell into which they are inserted.
The genomic locus into which the native barcode polynucleotide cassette is inserted is at a heterologous location compared to the location in the genome of the first, second and third native nucleotide sequence. The disclosure also includes optional use of naturally occurring linker sequences between barcode cassette elements, as well as quantitative design ranges for the native barcode polynucleotide cassette, including total cassette length, barcode length, and primer binding site length.
The disclosure further describes related barcoding workflows, including methods for barcoding cells and screening cells in samples by next-generation sequencing-based style analysis. It also describes strategies to mitigate next-generation sequencing coverage bias by maintaining semi-constant insertion distance from the origin of replication, and candidate insertion site selection near transcriptional terminators.
Claims Coverage
The independent claim coverage is directed to an engineered bacterial cell with a native barcode polynucleotide cassette inserted into a heterologous genomic locus. The core inventive features are the native-source cassette composition and orientation, the heterologous insertion location, and the functional organization of the cassette as forward primer binding site, barcode, and reverse primer binding site, with dependent refinements on insertion context and sequence source.
Engineered bacterial cell with native barcode polynucleotide cassette inserted into a heterologous genomic locus
An engineered bacterial cell comprising a native barcode polynucleotide cassette inserted into a genomic locus of the cell, the cassette having first, second and third native nucleotide sequences oriented 5′ to 3′, where the first serves as a forward primer binding site, the second serves as a barcode, and the third serves as a reverse primer binding site; wherein the first, second and third native nucleotide sequences comprised within the native barcode polynucleotide cassette are from the same species as the engineered bacterial cell into which they are being inserted; and wherein the genomic locus into which the native barcode polynucleotide cassette is inserted is at a heterologous location, as compared to the location in the genome of the first, second and third native nucleotide sequence.
Genomic insertion constrained relative to origin of replication
The native barcode polynucleotide cassette is inserted into a genomic locus at a set distance from an origin of replication.
Genomic insertion between coding regions with terminator context
The native barcode polynucleotide cassette is inserted at a genomic locus between two coding regions separated by a terminator region.
Species-native sourcing of cassette sequences
At least one of the first, second, or third native nucleotide sequences is isolated from ribosomal DNA or internal transcribed spacer (ITS) DNA.
Primer binding site selection from specified primer sequences
At least one primer binding site is chosen from a specified list of primer sequences.
Control sequence introduced for nitrogen fixation or assimilation regulatory network
An introduced control sequence is operably connected to at least one gene in a nitrogen fixation or nitrogen assimilation genetic regulatory network.
The claim scope centers on an engineered bacterial cell carrying a native barcode polynucleotide cassette with forward primer binding site, barcode, and reverse primer binding site, all sourced from the same species as the host and inserted at a heterologous genomic locus. Dependent refinements further constrain the genomic insertion context, restrict cassette sequence source, specify primer binding site choices, and optionally introduce nitrogen fixation or assimilation control elements.
Stated Advantages
Detect relative differences in colonization capacity when multiple barcoded strains are co-inoculated.
Rank strains by abundance consistency across plants.
Enable absolute abundance determination using a spiked control ladder barcode that shares universal primer sites.
Compute cells per gram fresh weight for mixed strain populations using endogenous native barcodes and regression against spiked control reads.
Minimize NGS coverage bias.
Avoid altering gene expression by targeting insertion to non-coding regions near terminators.
Documented Applications
Plant root colonization assays in planta and across plant root systems using barcoded microbes to measure relative microbial colonization ability.
Coco-seq plant root colonization assays using native barcode polynucleotide cassettes to analyze colonization capacity and abundance of barcoded strains in plant roots.
Mixed strain population analysis in the context of plant root colonization, including absolute abundance determination and validation against qPCR.
Determining strain abundance and consistency in plant root colonization across multiple barcoded strains co-inoculated in the assay.
Detection and quantification context for identifying colonization and nitrogen-related phenotypes associated with engineered or non-intergeneric remodeled nitrogen-fixing microbes.
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