In search of a biosensor for DNT detection Studies of inducer response and specificity of DntR

Abstract: The primary aim of the work presented in this thesis was to change the inducer specificity of the DntR protein in order to improve the response to DNT. The long-term goal is to use this protein in a biosensor for DNT, a signature compound for detection of the explosive TNT. Another aspect of this work was to understand the mechanisms of inducer binding and how the binding of an inducer molecule changes the DntR structure into a state that triggers transcriptional activation.In the papers included in this thesis the inducer specificity of wt DntR has been investigated under different conditions. The functional effects of specific mutations have also been investigated, in some cases in combination with structure determination using X-ray crystallography. In addition, structural data offering insights into the details of inducer binding and conformational changes upon inducer binding are presented and discussed in terms of mechanisms for transcriptional activation by DntR. Furthermore, a directed evolution strategy was employed in order to find variants of DntR with improved response to DNT. A variant with a large improvement in the DNT response was isolated and characterized. In optimized growth conditions, this DntR variant had a nearly 10-fold increase in fluorescence in response to DNT compared to wt DntR. Specific substitutions found in this DntR variant are suggested to be important for changing the inducer response.