Granted Patent
B2
US 12,209,245 · App. 16/671,036 · Granted Jan 28, 2025
Synthetic gene clusters
Inventors:
Ethan Mirsky (San Francisco, CA); Karsten Temme (San Francisco, CA); Christopher A. Voigt (Belmont, MA); Dehua Zhao (Allston, MA)
C12N15/635C07K14/255C07K14/26C12N9/0095C12N15/52C12N15/63C12N15/67C12N15/74C12Y118/06001G16B25/00G16B30/00G16B30/10G16B30/20
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Abstract
Methods for making synthetic gene clusters are described.
Claims (28)
1. A method of altering regulation of a plurality of native bacterial genes associated with a function in a cell, comprising:
providing a bacterial cell for expressing gene products;
providing a gene cluster having a plurality of native bacterial genes having coding sequences;
modifying the gene cluster by making at least one modification in a coding region or an intergenic region;
operably linking at least one heterologous transcriptional regulatory sequence to at least one coding sequence within the modified gene cluster, wherein the at least one heterologous transcriptional regulatory sequence is from a different species than the plurality of native bacterial genes; and
expressing gene products of the modified gene cluster in the bacterial cell under the control of a polypeptide that binds directly or indirectly to the at least one heterologous transcriptional regulatory sequence,
wherein the bacterial cell is from a nitrogen fixing bacterial species and/or wherein the gene cluster comprises genes collectively associated with nitrogen fixation.
2. The method of claim 1 , wherein the gene cluster comprises nif genes.
3. The method of claim 1 , wherein modifying the gene cluster comprises making at least one modification in a native regulatory sequence.
4. The method of claim 3 , further comprising identifying the native regulatory sequence using a computational algorithm.
5. The method of claim 1 , wherein modifying the gene cluster comprises making at least one modification in a promoter.
6. The method of claim 1 , wherein the polypeptide that binds directly or indirectly to the at least one heterologous transcriptional regulatory sequence is heterologous to the bacterial cell.
7. The method of claim 1 , wherein the polypeptide that binds directly or indirectly to the at least one heterologous transcriptional regulatory sequence is from a different species as the plurality of native bacterial genes.
8. The method of claim 1 , further comprising: detecting the expressed gene products.
9. The method of claim 1 , wherein the modified gene cluster is heterologous to the bacterial cell.
10. The method of claim 1 , further comprising expressing the polypeptide that binds directly or indirectly to the at least one heterologous transcriptional regulatory sequence from an expression cassette.
11. A recombinant bacterial cell comprising a modified gene cluster, wherein the modified gene cluster comprises a plurality of native bacterial genes having coding sequences and comprises at least one modification in a coding region or an intergenic region;
wherein at least one coding sequence within the modified gene cluster is operably linked to at least one heterologous transcriptional regulatory sequence;
wherein the at least one heterologous transcriptional regulatory sequence is from a different species than the plurality of native bacterial genes; and
wherein the expression of gene products of the modified gene cluster in the bacterial cell is under the control of a polypeptide that binds directly or indirectly to the at least one heterologous transcriptional regulatory sequence,
wherein the bacterial cell is from a nitrogen fixing bacterial species and/or wherein the gene cluster comprises genes collectively associated with nitrogen fixation.
12. The recombinant bacterial cell of claim 11 , wherein the modified gene cluster comprises nif genes.
13. The recombinant bacterial cell of claim 11 , comprising at least one modification in a native regulatory sequence.
14. The recombinant bacterial cell of claim 11 , comprising at least one modification in a promoter.
15. The recombinant bacterial cell of claim 11 , wherein the polypeptide that binds directly or indirectly to the at least one heterologous transcriptional regulatory sequence is heterologous to the cell.
16. The recombinant bacterial cell of claim 11 , wherein the polypeptide that binds directly or indirectly to the at least one heterologous transcriptional regulatory sequence is from a different species as the plurality of native bacterial genes.
17. The recombinant bacterial cell of claim 11 , wherein the modified gene cluster is heterologous to the bacterial cell.
18. The recombinant bacterial cell of claim 11 , further comprising an expression cassette comprising a polynucleotide sequence encoding the polypeptide.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded May 22, 2020
From: MIRSKY, ETHAN; TEMME, KARSTEN; VOIGT, CHRISTOPHER A.; ZHAO, DEHUA
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 052733/0277 →
Continuity (5)
Continuation
14126307
Provisional Application
61497781
· Jun 16, 2011
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