Abstract：
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.
摘要：
全细胞生物传感器（WCB）是一种方便且经济有效的检测污染物的方法。然而,镉WCBs的实际应用受到性能缺陷的阻碍，如灵敏度低，特异性，响应的力量。在这项研究中,为了提高镉WCB的性能，镉转录因子（CadC）及其DNA结合位点（CadO），生物传感器的关键传感模块，分别进行两步定向进化：对CadC进行6轮随机诱变和对CadO进行2轮饱和诱变。对于实际应用，将GFP报告基因替换为lacZ基因,并通过优化使用洗涤剂的反应系统,建立了用于实际水样的简便快速智能手机检测平台.结果表明，进化镉荧光生物传感器CadO66具有较高的特异性，检出限为0.034μg/L，与野生型镉生物传感器相比，减少了19倍。洗涤剂十二烷基苯磺酸钠有效地增强了WCBB0033-lacZ的可视化。使用荧光WCBCadO66和可视WCBB0033-lacZ分析实际水样的镉含量，结果也与石墨炉原子吸收光谱仪一致。一起来看,这项研究表明，CadC和CadO的两步定向进化可以有效地提高镉WCBs的性能，进一步促进了WCB在实际样品检测中的应用，为样品的快速检测提供了一种有前途且可行的方法。
