Auxotrophic strains of staphylococcus bacterium

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.

1 . A recombinant  Staphylococcus  bacterium comprising:
 two inactivated alanine racemase genes (alr1 and alr2); and   an inactivated D-alanine aminotransferase gene (dat).   
     
     
         2 . The recombinant  Staphylococcus  bacterium of  claim 1 , wherein the  Staphylococcus  bacterium is dependent on D-alanine for growth. 
     
     
         3 . The recombinant  Staphylococcus  bacterium of  claim 1 , wherein the  Staphylococcus  bacterium is  Staphylococcus epidermidis  ( S. epidermidis ), and subspecies thereof. 
     
     
         4 . The recombinant  Staphylococcus  bacterium of  claim 1 , wherein the  Staphylococcus  bacterium further comprises one or more additional mutations. 
     
     
         5 . The recombinant  Staphylococcus  bacterium of  claim 4  in which the additional mutations comprise inactivated glutamatic acid racemase gene, MurI. 
     
     
         6 . The recombinant  Staphylococcus  bacterium of  claim 1 , wherein the bacterium is transformed with a pUBTR114-based vector. 
     
     
         7 . The recombinant  Staphylococcus  bacterium of  claim 6 , wherein the pUBTR114-based vector is pUBTR119*-Sal-GFP. 
     
     
         8 . 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); and   (iv) purifying isolated colonies.   
     
     
         9 . The method of  claim 8 , further comprising testing the isolated colonies for D-alanine auxotrophy. 
     
     
         10 . The method of  claim 8 , wherein the presence of the knockout plasmid in the transformants is detected using Polymerase Chain Reaction (PCR). 
     
     
         11 . The method of  claim 8 , wherein the recombinant  Staphylococcus  bacterium is  Staphylococcus epidermidis  ( S. epidermis ), and subspecies thereof. 
     
     
         12 . The method of  claim 8 , further comprising transforming the recombinant  Staphylococcus  bacterium with a pUBTR114-based vector. 
     
     
         13 . The method of  claim 12 , wherein the pUBTR114-based vector is pUBTR119*-Sal-GFP. 
     
     
         14 . A recombinant  Staphylococcus  bacterium produced by the method of  claim 8 . 
     
     
         15 . A kit comprising the recombinant  Staphylococcus  bacterium of  claim 1 . 
     
     
         16 . The kit of  claim 15 , further comprising a pUBTR114-based vector.