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US 11,041,217 B2Granted
Auxotrophic strains of staphylococcus bacterium
AssigneeAzitra Inc
InventorsTravis Michael Whitfill, Ming-De Deng, David Richard Dodds, Alexander Tikhonov
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Abstract
The present disclosure provides recombinant Staphylococcus bacterium (e.g. S. epidermidis) that are dependent on D-alanine for growth. In one aspect, the disclosure features a recombinant Staphylococcus bacterium comprising two inactivated alanine racemase genes (Δalr1Δalr2); and an inactivated D-alanine aminotransferase (dat) gene. In another aspect, the disclosure features a method of making the recombinant Staphylococcus bacterium.
Claims12
A recombinant Staphylococcus bacterium comprising:
- two inactivated alanine racemase genes (alr1 and alr2);
- and
- an inactivated D-alanine aminotransferase gene (dat),
- wherein the Staphylococcus bacterium is Staphylococcus epidermidis (S. epidermidis), and subspecies thereof, and
- wherein the Staphylococcus bacterium is dependent on D-alanine for growth.
2 more elements
A method of making a recombinant Staphylococcus bacterium comprising:
- (i) transforming a plasmid comprising D-alanine aminotransferase (dat) knockout into competent cells of a Staphylococcus strain, wherein the Staphylococcus strain comprises inactive alanine racemase genes alr1 and alr2 (SEΔalr1Δalr2);
- (ii) detecting the presence of the knockout plasmid in transformed cells;
- (iii) incubating the transformed cells identified in step (ii);
- (iv) purifying isolated colonies;
- and
- (v) testing the isolated colonies for D-alanine auxotrophy,
- wherein the recombinant Staphylococcus bacterium is Staphylococcus epidermidis (S. epidermis), and subspecies thereof.
4 more elements
Dependencies are inferred from the claim text.