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Background and aims: Among other antibiotics macrolides have been used in a large scale for decades which leads into an increase of antibiotics in food, feed and environment and results in antibiotic resistance in bacteria. Therefore, a publicly available and cost-efficient macrolide antibiotic detection method by a whole cell biosensor is developed. The whole cell biosensor in process is constructed in Estericia coli cells containing a self luminescent luciferase plasmid regulated by macrolide 2'-phosphotransferase regulating protein (MphR(E)). The model molecule of MphR(E) is 2'-phosphotransferase I regulating protein MphR(A) which binds to its promoter with the dissociation constant of 574 ± 29 nM. The aim of this study was to require information of the regulatory protein MphR(E) and its affinity towards promoter DNA by making mutations to the DNA binding HTH motif of MphR(E).

Methods: Six different mutations were designed for the DNA binding helix-turn-helix motif of MphR(E) to increase the affinity towards the promoter DNA. The mutations were made by splicing by overlap extension PCR (SOE-PCR), inserted into a Pac400c plasmid and transformed into Escherichia Coli XL-1 Blue. Five of the mutations, MphR(E) and MphR(A) were produced and purified using affinity chromatography. MphR(E) was measured by mass spectrometry. Furthermore, the Dissociation constant (Kd) was calculated for MphR(E) and two mutations both containing histidine tags after fluorescence polarization.

Results: The five mutations were successfully produced in E. Coli and purified along with MphR(E) and MphR(A). In fluorescence polarization the undigested MphR(E) had a Kd of 204 nM, MphR(E)_CG had a Kd of 576 nM and MphR(E)_K had a Kd of 652 nM. Showing that the more MphR(E) becomes like MphR(A) the weaker the affinity towards the promoter DNA becomes. The mass spectrometry showed that MphR(E) was well purified and that the histidine tag was cleaved from the specific cleavage site and from two other sites.

Conclusions: Most of mutations designed were produced and purified. The purification was successful but the cleavage needed further optimization. The aims of this study were met by gaining information of the DNA binding affinity of MphR(E) which was higher than the one of MphR(A) unlike presumed. The mutations made MphR(E) more like MphR(A) and on the contrary did not succeeded in lowering the DNA affinity. All of the proteins can be further analyzed by mass spectrometry and by fluorescence polarization to determine the Kd and to evaluate the cleavage of the histidine tag.