Oxygen-responsive bacterial gene switch

Abstract

The invention provided herein relates to sequence determinants that elicit certain levels of gene expression and methods for obtaining engineered ligand-responsive gene switches comprising these sequence determinants. More particularly, the invention provided herein relates to molecular building blocks (i.e., discrete nucleotide sequences), synthetic ligand-responsive gene switches comprising an assembly of these molecular building blocks, and methods of using synthetic ligand-responsive gene switches as customizable and controllable expression systems and sensors.

We claim: 
     
         1 . A vector comprising a polynucleotide and a backbone not natively linked to the polynucleotide, the polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-32. 
     
     
         2 . The vector of  claim 1 , wherein the polynucleotide is operably linked to a promoter. 
     
     
         3 . A bacterial host cell comprising the vector of  claim 2 . 
     
     
         4 . The bacterial host cell of  claim 3 , wherein the cell is an  E. coli  cell. 
     
     
         5 . An expression vector comprising a gene switch operably linked to a non-natively linked target nucleic acid sequence, the gene switch comprising one or more nucleic acid sequences having the sequence of a binding site of a transcriptional activator or transcriptional repressor, the gene switch configured to positively or negatively modify a gene expression level from the target nucleic acid sequence in a host cell comprising the expression vector when the cell is subjected to a change in oxygen concentration. 
     
     
         6 . The expression vector of  claim 5 , wherein the one or more gene switch nucleic acid sequences are selected from the group consisting of SEQ ID NOs:1-32. 
     
     
         7 . The expression vector of  claim 5 , wherein the gene switch comprises 1, 2, 3, 4, or 5 repeats of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-32. 
     
     
         8 . A method for modifying a level of gene expression of a target nucleic acid sequence, the method comprising:
 introducing into a host cell an expression vector of  claim 5 ; and   subjecting the host cell to a change in oxygen concentration until the gene expression level of the target nucleic acid sequence is modified.   
     
     
         9 . The method of  claim 8 , wherein the gene expression level is modified under anaerobic conditions. 
     
     
         10 . The method of  claim 8 , wherein the gene switch comprises one or more nucleic acid sequences selected from the group consisting of SEQ ID NOs:1-32. 
     
     
         11 . The method of  claim 8 , wherein the gene switch comprises 1, 2, 3, 4, or 5 repeats of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-32.