The bacterial respiratory gene, nuoA, was previously used as a reporter gene in an amperometric, whole cell biosensor for tetracycline (Tet) detection. While the nuoA-based bioassay responded sensitively to Tet, the signal declined at high Tet concentrations, probably partly due to transgene over-expression. Also, at zero concentration of Tet, the assay registered a relatively high background signal when compared to the nuoA knockout Escherichia coli strain without the biosensor transgene construct. This was probably due to incomplete repression of nuoA expression. In order to reduce gene over-expression, the sensor cells were incubated with Tet at a relatively low temperature (15 °C). Also, a low-copy number plasmid pBR322 was used to carry the transgene, instead of the high-copy number plasmid pBluescript in order to reduce over-expression and to reduce background expression. Both assays improved the biosensor response. By using a low-copy number plasmid and tetracycline resistance, the sensor was less inhibited at higher Tet concentrations; but, this did not significantly increase the linear range of the sensor. The low temperature nuoA assay could detect Tet at a range of 0.001-1 μg ml(-1). In contrast, the low-copy number nuoA assay was able to detect Tet at a range of 0.0001-1 μg ml(-1). The detection limit of Tet determined by the low-copy number nuoA assay was 0.00023 μg ml(-1), which is one order of magnitude more sensitive than in the previous nuoA assay.