Composition of matter: engineering of Escherichia coli phage K1E

Patent metadata

Field	Value
Publication number	US-11268072-B2
Application number	US-201916510643-A
Country	US
Kind code	B2
Filing date	Jul 12, 2019
Priority date	Aug 1, 2017
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

The present disclosure provides compositions including recombinant K1E bacteriophages, methods for making the same, and uses thereof. The recombinant K1E bacteriophages disclosed herein are useful for the identification and/or antibiotic susceptibility profiling of specific bacterial strains/species present in a sample.

First claim

The invention claimed is:

1. A method for making a linear recombinant K1E bacteriophage genome of SEQ ID NO: 3 in vitro comprising
(a) contacting a non-recombinant K1E bacteriophage genome of SEQ ID NO: 1 comprising a single PflF1 recognition site with PflF1 in vitro under conditions where PflF1 cleaves the PflF1 recognition site to produce a cleaved linear non-recombinant K1E bacteriophage genome; and
(b) recombining in vitro the cleaved linear non-recombinant K1E bacteriophage genome with a heterologous nucleic acid of SEQ ID NO: 2 in the presence of 5′-3′ exonuclease, a DNA polymerase, and a DNA ligase in an in vitro DNA assembly reaction under conditions to produce an in vitro generated linear recombinant K1E bacteriophage genome of SEQ ID NO: 3, wherein the in vitro DNA assembly reaction is devoid of biological extracts,
wherein the cleaved linear non-recombinant K1E bacteriophage genome comprises a first cleaved linear bacteriophage genomic fragment and a second cleaved linear bacteriophage genomic fragment,
wherein the heterologous nucleic acid comprises a 5′ flanking region that is homologous to the 3′ end of the first cleaved linear bacteriophage genomic fragment, and a 3′ flanking region that is homologous to the 5′ end of the second cleaved linear bacteriophage genomic fragment,
wherein the 5′ flanking region and the 3′ flanking region of the heterologous nucleic acid do not comprise the single PflF1 recognition site,
and
wherein the in vitro generated linear recombinant K1E bacteriophage genome of SEQ ID NO: 3 is capable of producing non-endogenous bioluminescent protein that is functionally active when transformed into a bacterial host cell.

2. The method of claim 1 , further comprising transforming the in vitro generated linear recombinant K1E bacteriophage genome in a bacterial host.

3. The method of claim 2 , wherein the bacterial host is a non-natural bacterial host cell or a natural bacterial host cell for K1E bacteriophage.

Assignees

• Charles Stark Draper Laboratory Inc

Inventors

• Gruszka Sarah
• Holder Jason

Classifications

• C12N2795/10243

  viral genome or elements thereof as genetic vector · CPC title

• C12N7/00 Primary

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N2795/10031

  Uses of virus other than therapeutic or vaccine, e.g. disinfectant · CPC title

• C12N2795/10221

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C12Q1/18

  Testing for antimicrobial activity of a material · CPC title