A whole-cell biosensor (WCB) is a convenient and cost-effective method for detecting contaminants. However, the practical application of the cadmium WCBs has been hampered by performance deficiencies, such as low sensitivity, specificity, and responsive strength. In this study, to improve the performance of cadmium WCBs, the cadmium transcription factor (CadC) and its DNA binding site (CadO), the key sensing module of the biosensor, were successively and separately subjected to a two-step directed evolution: 6-round random mutagenesis for CadC and 2-round saturation mutagenesis for CadO. For practical application, the GFP reporter gene was replaced with the lacZ gene and a facile and rapid smartphone detection platform for actual water samples was established by optimizing the reaction systems with detergents. The results showed that the evolved cadmium fluorescent biosensor CadO66 exhibited a higher specificity and a detection limit of 0.034 μg/L, representing a 19-fold reduction compared to the wild-type cadmium biosensor. The detergent sodium dodecylbenzenesulfonate effectively enhanced the visualization of WCB B0033-lacZ. Using the fluorescent WCB CadO66 and the visual WCB B0033-lacZ to analyze the cadmium contents of the actual water samples, the results were also consistent with a graphite furnace atomic absorption spectrometer. Taken together, this study indicates that the two-step directed evolution of CadC and CadO can efficiently improve the performance of cadmium WCBs, further promoting the utilization of WCB in actual sample detection and presenting a promising and feasible method for rapid sample detection.
Keywords: DNA binding site; cadmium; detergent; directed evolution; smartphone detection; whole-cell biosensor (WCB).