Genetically engineered
The present disclosure relates to the seminal discovery of a generation and use of genetically engineered Vibrio sp. Provided is the use of the genetically engineered bacteria for the construction, maintenance, manipulation, and/or propagation of DNA constructs; protein expression; protein secretion; vectors and other metabolic tools; metabolic engineering; expression of cellular extracts for cell-free biology; shuttle vectors; cloning vectors; and for synthetic biology applications. The disclosure also relates to the use of the replication machinery of Vibrio sp. as a cloning or expression vector for replication of recombinant DNA constructs. The disclosure also relates to methods of use of the above.
1. A method of producing a biomolecule comprising:
a) contacting a Vibrio natriegens organism with a vector and introducing the vector into the Vibrio natriegens organism, wherein the vector comprises a heterologous nucleic acid encoding the biomolecule, wherein the Vibrio natriegens organism comprises a recombinant nucleic acid comprising a nucleotide sequence encoding a T7 RNA polymerase and a recombinant regulatory element 5′ to the nucleotide sequence encoding the T7 RNA polymerase that regulates expression of the T7 RNA polymerase, and wherein the recombinant regulatory element comprises a ribosome binding site having at least 95% sequence identity to SEQ ID NO: 8;
b) expressing the heterologous nucleic acid in a cell-free extract of the Vibrio natriegens organism, thereby producing the biomolecule; and
c) isolating the biomolecule.
2. The method of claim 1 , wherein the vector further comprises an inducible promoter operably linked to the heterologous nucleic acid encoding the biomolecule.
3. The method of claim 1 , wherein the vector is contacted and introduced into the Vibrio natriegens organism by conjugation, chemical competence, natural competence, or electroporation.
4. The method of claim 1 , wherein the expression of the nucleic acid sequence encoding the T7 RNA polymerase is regulated by an inducible promoter, and the inducible promoter and the nucleic acid sequence encoding the T7 RNA polymerase are integrated into a chromosome of the Vibrio natriegens organism.
5. The method of claim 4 , wherein the inducible promoter is inducible with arabinose.
6. The method of claim 4 , wherein the inducible promoter is inducible with IPTG.
7. The method of claim 6 , wherein the Vibrio natriegens organism expresses a lacI repressor for repression of the inducible promoter.
8. The method of claim 1 , wherein the biomolecule is a heterologous protein.
9. The method of claim 1 , wherein the Vibrio natriegens organism further comprises a deletion of at least one extracellular nuclease.
10. The method of claim 9 , wherein the extracellular nuclease is Dns extracellular nuclease.
11. The method of claim 1 , wherein the Vibrio natriegens organism has a doubling time of less than 15 minutes.
12. The method of claim 1 , wherein the sequence of the heterologous nucleic acid is at least 10 kb in size.
13. The method of claim 1 , wherein the biomolecule is a protein or peptide.
14. The method of claim 1 , wherein the vector comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having 90% or greater sequence identity to the nucleotide sequence of SEQ ID NO: 1.
15. The method of claim 14 , wherein the vector is capable of supporting its replication in the Vibrio natriegens organism to produce the biomolecule.