Genetically engineered Vibrio sp. and uses thereof
Inventors
Weinstock, Matthew T • Wilson, Christopher M. • Hesek, Eric D.
Assignees
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Publication Number
US-11447755-B2
Publication Date
2022-09-20
Expiration Date
Abstract
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.
Core Innovation
The invention relates to a genetically engineered Vibrio natriegens organism comprising recombinant nucleic acid that regulates expression of a T7 RNA polymerase. The recombinant nucleic acid includes a recombinant regulatory element located 5′ to the nucleotide sequence encoding the T7 RNA polymerase, and further encodes a modified ribosome binding site 5′ to the nucleotide sequence encoding the T7 RNA polymerase, where the ribosome binding site comprises the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 8 with 1, 2, or 3 nucleotide substitutions.
The disclosure uses engineered chromosome architectures in Vibrio, including altered or minimized chromosomes, a single-chromosome design, and essential elements from chromosome II moved to chromosome I. It further describes removal of non-essential genes, including nucleases and restriction systems, and the use of replication machinery derived from chromosome II to build and maintain cloning and expression systems, including vector and shuttle vector concepts using Vibrio replication machinery.
The document also describes systems and components for DNA construction and protein expression, including heterologous nucleic acids and vectors. It includes competence cell production and delivery approaches for introducing nucleic acids into Vibrio natriegens, and it further describes inducible promoter-controlled expression including IPTG-, arabinose-, and temperature-induced systems and optional modified or weakened ribosome binding site configurations to reduce basal expression.
Claims Coverage
The partial content provides one independent claim. The independent claim centers on a Vibrio natriegens organism with recombinant nucleic acid encoding T7 RNA polymerase and a modified ribosome binding site based on SEQ ID NO: 8, with translation regulation.
Vibrio natriegens with recombinant T7 RNA polymerase and modified SEQ ID NO: 8 ribosome binding site
A Vibrio natriegens organism comprising a recombinant nucleic acid encoding a T7 RNA polymerase, where a recombinant regulatory element is 5′ to the nucleotide sequence encoding the T7 RNA polymerase to regulate expression, and where the recombinant nucleic acid encodes a modified ribosome binding site 5′ to the nucleotide sequence encoding the T7 RNA polymerase comprising SEQ ID NO: 8 or SEQ ID NO: 8 with 1, 2, or 3 nucleotide substitutions such that the ribosome binding site regulates translation of the T7 RNA polymerase.
Across the provided independent claim coverage, the central inventive concept is a Vibrio natriegens organism engineered with recombinant nucleic acid for regulated expression of T7 RNA polymerase using a recombinant regulatory element and a modified SEQ ID NO: 8 ribosome binding site that regulates translation.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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