Granted Patent B2

US 12,735,713 · App. 19/282,553 · Granted Sep 15, 2026

Inventors: Douglas Higgins (Berkeley, CA); Andrew Abel Prior (Oakland, CA)

Assignee: Pivot Bio, Inc.

C12N15/74

Abstract

Claims (28)

1 . A method of creating an engineered barcoded bacterial cell, comprising:

(a) obtaining a donor bacterial cell;

(b) selecting and isolating sequences from the donor bacterial cell to construct a native barcode polynucleotide cassette, comprising:

(i) selecting and isolating from the donor bacterial cell a first native nucleotide sequence to function as a forward primer binding site;

(ii) selecting and isolating from the donor bacterial cell a second native nucleotide sequence to function as a barcode; and

(iii) selecting and isolating from the donor bacterial cell a third native nucleotide sequence to function as a reverse primer binding site,

(c) assembling a native barcode polynucleotide cassette comprising the first, second, and third native nucleotide sequences in a 5′ to 3′ orientation; and

(d) inserting the native barcode polynucleotide cassette into a genomic locus of a recipient bacterial cell from the same species as the donor bacterial cell to thereby create an engineered barcoded bacterial cell,

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.

2 . The method of claim 1 , wherein the barcode is unique to different strains of the engineered barcoded bacterial cell.

3 . The method of claim 1 , wherein the barcode is unique to different species of the engineered barcoded bacterial cell.

4 . The method of claim 1 , wherein the nucleotide sequence of at least one of the first, the second, or the third native nucleotide sequence is isolated from ribosomal DNA or internal transcribed spacer (ITS) DNA.

5 . The method of claim 1 , wherein the nucleotide sequence of at least one of the first, the second, or the third native nucleotide sequence is 16S rDNA or 18S rDNA.

6 . The method of claim 1 , wherein at least one of the primer binding sites is selected from 8F, 27F, CCF, 357F, 515F, 533F, 16S.1100.F16, 804F, 1237F, 338R, 519R, CDR, 806R, 907R, 1100R, 1391R, 1392R, 1492R(l), and 1492R(s).

7 . The method of claim 1 , wherein the barcode comprises a constant barcode region and a variable barcode region.

8 . The method of claim 1 , wherein the engineered barcoded bacterial cell is a non-intergeneric remodeled bacterial cell.

9 . The method of claim 1 , wherein the engineered barcoded bacterial cell is capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.

10 . The method of claim 1 , wherein the engineered barcoded bacterial cell comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network.

11 . The method of claim 1 , wherein the engineered barcoded bacterial cell comprises an introduced control sequence operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.

12 . The method of claim 1 , wherein the engineered barcoded bacterial cell comprises a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.

13 . The method of claim 1 , wherein the engineered barcoded bacterial cell comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a gene associated with biosynthesis of a nitrogenase enzyme, and combinations thereof.

14 . The method of claim 1 , wherein the engineered barcoded bacterial cell comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; and decreased uridylyl-removing activity of GlnD.

15 . The method of claim 1 , wherein the engineered barcoded bacterial cell comprises at least one of: a mutated nifL gene comprising a heterologous promoter in said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; and combinations thereof.

16 . The method of claim 1 , wherein the engineered barcoded bacterial cell is selected from Rahnella aquatilis, Klebsiella varicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum , and Kosakonia sacchari.

17 . The method of claim 1 , wherein the engineered barcoded bacterial cell is endophytic, epiphytic, or rhizospheric.

18 . The method of claim 1 , wherein the genomic locus into which the native barcode polynucleotide cassette is inserted is between two coding regions separated by a terminator region.

19 . The method of claim 1 , wherein the genomic locus into which the native barcode polynucleotide cassette is inserted is at a set distance from an origin of replication.

20 . An engineered barcoded bacterial cell made by the method of claim 1 .

Assignments (1)

Continuity (4)

References Cited (285)