PDF(Pubmed)
Abstract：
In this study, we designed a novel genetic circuit sensitive to Cd2+, Zn2+ and Pb2+ by mimicking the CadA/CadR operon system mediated heavy metal homeostasis mechanism of Pseudomonas aeruginosa. The regular DNA motifs on natural operon were reconfigured and coupled with the enhanced Green Fluorescent Protein (eGFP) reporter to develop a novel basic NOT type logic gate CadA/CadR-eGFP to respond metal ions mentioned above. A Genetically Engineered Microbial (GEM)-based biosensor (E.coli-BL21:pJET1.2-CadA/CadR-eGFP) was developed by cloning the chemically synthesised CadA/CadR-eGFP gene circuit into pJET1.2-plasmid and transforming into Escherichia coli (E. coli)-BL21 bacterial cells.
The GEM-based biosensor cells indicated the reporter gene expression in the presence of Cd2+, Zn2+ and Pb2+ either singly or in combination. Further, the same biosensor cells calibrated for fluorescent intensity against heavy metal concentration generated linear graphs for Cd2+, Zn2+ and Pb2+ with the R2 values of 0.9809, 0.9761 and 0.9758, respectively as compared to non-specific metals, Fe3+ (0.0373), AsO43- (0.3825) and Ni2+ (0.8498) making our biosensor suitable for the detection of low concentration of the former metal ions in the range of 1-6 ppb. Furthermore, the GEM based biosensor cells were growing naturally within the concentration range of heavy metals, at 37 °C and optimum pH = 7.0 in the medium, resembling the characteristics of wildtype E.coli.
Finally, the novel GEM based biosensor cells developed in this study can be applied for detection of targeted heavy metals in low concentration ranges (1-6 ppb) at normal bacterial physiological conditions.
The GEM-based biosensor cells indicated the reporter gene expression in the presence of Cd2+, Zn2+ and Pb2+ either singly or in combination. Further, the same biosensor cells calibrated for fluorescent intensity against heavy metal concentration generated linear graphs for Cd2+, Zn2+ and Pb2+ with the R2 values of 0.9809, 0.9761 and 0.9758, respectively as compared to non-specific metals, Fe3+ (0.0373), AsO43- (0.3825) and Ni2+ (0.8498) making our biosensor suitable for the detection of low concentration of the former metal ions in the range of 1-6 ppb. Furthermore, the GEM based biosensor cells were growing naturally within the concentration range of heavy metals, at 37 °C and optimum pH = 7.0 in the medium, resembling the characteristics of wildtype E.coli.
Finally, the novel GEM based biosensor cells developed in this study can be applied for detection of targeted heavy metals in low concentration ranges (1-6 ppb) at normal bacterial physiological conditions.
摘要：
背景：在这项研究中，我们设计了一种对Cd2+敏感的新型遗传电路，Zn2+和Pb2+经由过程模仿CadA/CadR操纵子体系介导铜绿假单胞菌的重金属稳态机制。将天然操纵子上的规则DNA基序重新配置，并与增强的绿色荧光蛋白（eGFP）报告基因偶联，以开发新型的基本NOT型逻辑门CadA/CadR-eGFP，以响应上述金属离子。基于基因工程的微生物（GEM）生物传感器（E.coli-BL21：pJET1.2-CadA/CadR-eGFP）是通过将化学合成的CadA/CadR-eGFP基因电路克隆到pJET1.2质粒中并转化为大肠杆菌（E.大肠杆菌)-BL21细菌细胞。
结果：基于GEM的生物传感器细胞表明在Cd2存在下报告基因表达，Zn2+和Pb2+单独或组合。Further,针对重金属浓度的荧光强度校准的相同生物传感器细胞生成Cd2+的线性图，与非特定金属相比，Zn2和Pb2的R2值分别为0.9809，0.9761和0.9758，Fe3+(0.0373),AsO43-（0.3825）和Ni2（0.8498）使我们的生物传感器适用于检测1-6ppb范围内低浓度的前金属离子。此外，基于GEM的生物传感器细胞在重金属浓度范围内自然生长，在37°C和最佳pH=7.0的培养基中，类似于野生型大肠杆菌的特征
结论：最后，在这项研究中开发的新型基于GEM的生物传感器细胞可用于在正常细菌生理条件下检测低浓度范围（1-6ppb）的目标重金属。
结果：基于GEM的生物传感器细胞表明在Cd2存在下报告基因表达，Zn2+和Pb2+单独或组合。Further,针对重金属浓度的荧光强度校准的相同生物传感器细胞生成Cd2+的线性图，与非特定金属相比，Zn2和Pb2的R2值分别为0.9809，0.9761和0.9758，Fe3+(0.0373),AsO43-（0.3825）和Ni2（0.8498）使我们的生物传感器适用于检测1-6ppb范围内低浓度的前金属离子。此外，基于GEM的生物传感器细胞在重金属浓度范围内自然生长，在37°C和最佳pH=7.0的培养基中，类似于野生型大肠杆菌的特征
结论：最后，在这项研究中开发的新型基于GEM的生物传感器细胞可用于在正常细菌生理条件下检测低浓度范围（1-6ppb）的目标重金属。
