Transgenic bacteria with expanded amino acid usage and nucleic acid molecules for use in the same
View Patent ↗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.
1. A recombinant tRNA at least 95% identical to SEQ ID NO: 21 and having one of the following sets of features:
(i) a G at position 8, a U at position 67, a U at position 82, a G at position 83, and a C at position 84 of SEQ ID NO: 21;
(ii) a U at position 67, a U at position 82, and a G at position 83 of SEQ ID NO: 21; or
(iii) a U at position 67, an A at position 68, a deletion at position 82, an A at position 83, and a U at position 84 of SEQ ID NO: 21.
2. The recombinant tRNA of claim 1 , wherein the tRNA has a G at position 8, a U at position 67, a C at position 68, a U at position 82, a G at position 83, and a C at position 84 of SEQ ID NO: 21.
3. The recombinant tRNA of claim 2 , wherein the tRNA is encoded by a nucleic acid molecule of SEQ ID NO: 18.
4. The recombinant tRNA of claim 1 , wherein the tRNA has a C at position 8, a U at position 67, a C at position 68, a U at position 82, a G at position 83, and a G at position 84 of SEQ ID NO: 21.
5. The recombinant tRNA of claim 4 , wherein the tRNA is encoded by a nucleic acid molecule of SEQ ID NO: 19.
6. The recombinant tRNA of claim 1 , wherein the tRNA has a C at position 8, a U at position 67, an A at position 68, a deletion at position 82, an A at position 83, and a U at position 84 of SEQ ID NO: 21.
7. The recombinant tRNA of claim 6 , wherein the tRNA is encoded by a nucleic acid molecule of SEQ ID NO: 20.
8. A transgenic bacterial cell comprising a nucleic acid encoding the recombinant tRNA of claim 1 , wherein the transgenic the bacterial cell expresses SelA, wherein the transgenic bacterial cell is a transgenic Escherichia coli cell.
9. The transgenic bacterial cell of claim 8 , further comprising a nucleic acid molecule encoding an enzyme for synthesis of selenocysteine.
10. The transgenic bacterial cell of claim 8 , wherein the cell has been engineered to lack endogenous Amber codons.
11. A population of transgenic bacterial cells in accordance with claim 8 .
12. A method of producing a recombinant polypeptide comprising at least one selenocysteine, the method comprising:
(i) obtaining a bacterial cell of claim 8 and an expression cassette encoding the recombinant polypeptide;
(ii) transforming the bacterial cell with the expression cassette; and
(iii) incubating the bacterial cell under conditions that allow expression of the recombinant polypeptide.
13. The method of claim 12 , further comprising (iv) isolating the expressed recombinant polypeptide.
14. A recombinant DNA molecule encoding the recombinant tRNA of claim 1 .
15. A transgenic bacterial cell comprising a recombinant DNA of claim 14 , wherein the transgenic the bacterial cell expresses SelA, wherein the transgenic bacterial cell is a transgenic Escherichia coli cell.
16. The transgenic bacterial cell of claim 15 , further comprising a nucleic acid molecule encoding an enzyme for synthesis of selenocysteine.
17. The transgenic bacterial cell of claim 15 , wherein the cell has been engineered to lack endogenous Amber codons.
18. A population of transgenic bacterial cells in accordance with claim 15 .
19. A method of producing a recombinant polypeptide comprising at least one selenocysteine, the method comprising:
(i) obtaining a bacterial cell of claim 15 and an expression cassette encoding the recombinant polypeptide;
(ii) transforming the bacterial cell with the expression cassette; and
(iii) incubating the bacterial cell under conditions that allow expression of the recombinant polypeptide.
20. The method of claim 19 , further comprising (iv) isolating the expressed recombinant polypeptide.