IP Library › Granted Patent US 12,344,879
Granted Patent B2
US 12,344,879 · App. 18/324,843 · Granted Jul 1, 2025

Transgenic bacteria with expanded amino acid usage and nucleic acid molecules for use in the same

Inventors: Andrew D. Ellington (Austin, TX); Ross Thyer (Austin, TX)
Assignee: BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM
C12P21/02C12N9/86C12N9/93C12N15/11C12N15/67C12Y305/02006
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Quick Facts
Patent No.
US 12,344,879
App. No.
18/324,843
Granted
Jul 1, 2025
Kind
B2
Abstract

Recombinant bacterial cells are provided that comprise a stable non-canonical amino acid translation pathway. In some aspects, the bacteria comprise nucleic acids encoding a non-canonical amino acid translation pathway (e.g., a tRNA for incorporation of a non-canonical amino acid, such selenocysteine); a marker polypeptide that includes the non-canonical amino acid. Recombinant tRNA and selection marker coding sequences are likewise provided.

Claims (33)

1. A recombinant nucleic acid molecule, wherein the molecule encodes a tRNA and is at least 92% identical to SEQ ID NO: 19 or SEQ ID NO: 20; and wherein the tRNA encoded by the recombinant nucleic acid molecule comprises a U at a position corresponding to position 49.

2. The recombinant nucleic acid molecule of claim 1 , wherein the molecule encodes a tRNA and is at least 95% identical to SEQ ID NO: 19 or SEQ ID NO: 20; and wherein the tRNA encoded by the recombinant nucleic acid molecule comprises a U at a position corresponding to position 49.

3. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (i) a G at a position corresponding to position 7.

4. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (i) a C at a position corresponding to position 7.

5. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (iii) a A at a position corresponding to position 50.

6. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (iii) a C at a position corresponding to position 50.

7. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (iv) a U at a position corresponding to position 64.

8. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (v) a A at a position corresponding to position 65.

9. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (v) a G at a position corresponding to position 65.

10. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (vi) a C at a position corresponding to position 66.

11. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (vi) a G at a position corresponding to position 66.

12. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (vi) a U at a position corresponding to position 66.

13. The recombinant nucleic acid molecule of claim 1 , wherein the molecule comprises two or more of the following features:

(i) a G or C at a position corresponding to position 7;

(ii) a U at a position corresponding to position 49;

(iii) a A or C at a position corresponding to position 50;

(iv) a U at a position corresponding to position 64;

(v) a G or A at a position corresponding to position 65;

(vi) a G, U or C at a position corresponding to position 66.

14. The recombinant nucleic acid molecule of claim 1 , wherein the molecule comprises three or more of the following features:

(i) a G or C at a position corresponding to position 7;

(ii) a U at a position corresponding to position 49;

(iii) a A or C at a position corresponding to position 50;

(iv) a U at a position corresponding to position 64;

(v) a G or A at a position corresponding to position 65;

(vi) a G, U or C at a position corresponding to position 66.

15. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises: (i) a G at a position corresponding to position 7; (ii) a U at a position corresponding to position 49; (iii) a C at a position corresponding to position 50; (iv) a U at a position corresponding to position 64; (v) a G at a position corresponding to position 65; and (vi) a C at a position corresponding to position 66.

16. The recombinant nucleic acid molecule of claim 1 , wherein the recombinant nucleic acid molecule comprises a sequence with at least 95% sequence identity to SEQ ID NO: 19.

17. The recombinant nucleic acid molecule of claim 1 , wherein the recombinant nucleic acid molecule comprises a sequence with at 18 least 95% sequence identity to SEQ ID NO: 20.

18. A transgenic bacterial cell, said cell comprising the recombinant nucleic acid molecule according to claim 1 .

19. A method of producing a recombinant polypeptide comprising a selenocysteine comprising:

(i) providing a bacterial cell comprising (a) the recombinant nucleic acid molecule according to claim 1 and (b) an expression cassette encoding the recombinant polypeptide; and

(ii) incubating the bacterial cell under conditions that allow expression of the polypeptide comprising the selenocysteine.

Continuity (4)
Continuation 16786995 · Feb 10, 2020
Continuation 15380560 · Dec 15, 2016
Provisional Application 62267498 · Dec 15, 2015
Related Publication 20230366003A1 · Nov 16, 2023
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