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 vector operable in a Vibrio sp., comprising:
a Vibrio chromosome II sequence comprising at least 90% sequence identity to SEQ ID NO: 1;
at least one selectable marker; and
a heterologous DNA sequence of greater than 10 kb.
2. The vector of claim 1 further comprising an origin of transfer.
3. The vector of claim 2 wherein the origin of transfer is oriT for RP4-mediated conjugal transfer.
4. The vector of claim 1 further comprising an origin of replication operable in a non- Vibrio and non- E. coli organism.
5. The vector of claim 1 further comprising a selectable marker operable in a yeast.
6. The vector of claim 4 further comprising an autonomously replicating sequence (ARS) and a yeast centromere sequence (CEN).
7. The vector of claim 1 where said vector replicates at a copy number of less than 10.
8. The vector of claim 7 having a copy number of less than 5.
9. The vector of claim 1 wherein the heterologous DNA sequence is greater than 50 kb.
10. A Vibrio organism comprising the vector of claim 1 .
11. A method of producing a biomolecule comprising:
a) contacting Vibrio sp. bacteria with a vector comprising a heterologous nucleic acid encoding the biomolecule and further comprising SEQ ID NO: 1 or a variant thereof, such that the heterologous nucleic acid is introduced into the bacteria;
b) growing the bacteria in a growth-conducive medium wherein the heterologous nucleic acid is expressed, thereby producing the biomolecule; and
c) isolating the biomolecule.
12. The method of claim 11 , wherein the heterologous nucleic acid is at least 10 k in size.
13. The method of claim 12 , wherein the vector further comprises an inducible promoter operably linked to the nucleic acid encoding the biomolecule.
14. The method of claim 11 , wherein the nucleic acid is introduced by conjugation, chemical competence, nature competence, or electroporation.
15. The method of claim 13 , wherein the biomolecule is a protein or peptide.
16. The method of claim 13 wherein the biomolecule is a nucleic acid.
17. The vector of claim 1 wherein the Vibrio sp. is Vibrio natriegens.
18. The vector of claim 1 comprising at least 95% sequence identity to SEQ ID NO: 1.
19. The vector of claim 17 comprising at least 95% sequence identity to SEQ ID NO: 1.