Sunday, August 10, 2008
P57

Engineering mercury biosensor by synthesis and evolution

Qinhong Wang, Xiangdong Fang, Patrick J Shuler, Yongchun Tang, and William A Goddard III. Division of Chemistry and Chemical Engineering, California Institute of Technology, 738 Arrow Grand Circle, Covina, CA 91722

Mercury has been well known as an environmental pollutant for several decades. Emissions of mercury to the environment could have serious effects on human health. The US Environmental Protection Agency defined the toxic level of Hg2+ ions in drinking water to be 10nM. Hence, for environmental-monitoring Hg2+ ions in drinking water, a detection limit of lower than 10nM is required. At present, there are lost of sensor-based detection methods, but with different advantages and disadvantages. Whole-cell biosensors, constructed by coupling microbes to transducers that convert cellular responses into detectable signals, are easy to reproduce and have a great potential to reduce the cost. In addition, latest advances in protein engineering, microbiology and photo detection technology would significantly improve the effectiveness of whole-cell biosensor. Here, based on the metalloregulator MerR and red fluorescent protein mCherry, we constructed different whole-cell biosensors for mercury detection with different sensitivity. These biosensors have a wide range of mercury detection (2.5nM-5,000nM). They also have high selectivity without response to other metal ions, such as Mg2+, Ca2+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Pb2+, and Mo2+. By introducing the bio-amplification genetic circuit based on the gene cascade expression system of PRM-cI from bacteriophage l and Pm-XylS2 from Pseudomonas putida, we have increased detection sensitivity of the whole-cell biosensors by 1~2 fold in our tested conditions. With directed evolution of MerR and subsequent high-throughput screening based on color assay and microplate screening, we further increase detection sensitivity by 5~10-fold at low concentration of mercury (II) of 1-10nM.

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