Construction And Use Of Hg²⁺-biosensor Based On Lux-tagged For The Detection On Bioavailable Mercury In The Soil

The purpose of this study is to construct Hg²⁺-specific biosensor and to test the applicability of these biosensors for analysis of mercury-polluted soils.It included three major parts:1) construction of two lux-tagged Hg²⁺-specific biosensors and their luminesence performance;2) construction of lux-tagged X4(pUC-luxABCDE-merR -Kan-X4) sensor and control strain X4(pUC-luxABCDE -Kan-X4) and determination of toxicity effect of the Hg²⁺ ions on the microorganisms;3) application of X4(pUC-luxABCDE-merR-Kan-X4) sensor in detection mercury biotoxicity in contaminated soils.The results were listed as following:Two Hg²⁺-specific biosensors were constructed using bacterial luciferase as reporter gene and plasmid-free Pseudomonas putida X4,Enterobacter aerogenes NTG-01 as host strains.The performance of X4 biosensor was compared with that of NTG-01 biosensor in the same assay conditions.The maximum bioluminescence for X4(pmerRluxCDABE -Kan) biosensor was found during the mid-exponential phase and that for NTG-01 (pmerRluxCDABE-Kan) was at the late-exponential phase.The shortest induction time of two biosensors was 30 minutes.The maximum light signal output for NTG-01 and X4 sensors was observed at the incubation time of 5 h and 4 h,respectively.The lowest detectable concentration of mercury by the two biosensors were both of 100 pmol/L at 28℃,pH 7 and an initial cell number of 10⁶ CFU/g.Cd²⁺,Zn²⁺,Co²⁺,Cu²⁺ and Pb²⁺ ions at nanomolar level did not interfere with the measurement by the biosensors.The plasmid pX4 of P.putida X4 reconstructed was transformed into plasmid-free P. putida X4 to obtain a sensor which was made by fusing the mercury inducible promoter, P_mer,and its regulatory gene,merR with a promoterless reporter gene IuxABCDE and a control strain which lacked mercury inducible promoter,P_mer,and its regulatory gene, merR.The toxicity effect of mercury was confirmed by using a constitutive lighting strain X4(pUC-luxABCDE-Kan-X4) during the induction measurements.It was found that in the concentration range from 1 pmol/L to 1μmol/L,the luminescence was stable,the luminescence exhibited an apparently slope from 1 to 20μmol/L and at a concentration of 100μmol/L HgCl₂,the luminescence fell to zero due to the toxic effect of mercury ions.The survival of X4(pUC-luxABCDE-merR-Kan-X4) in soil was studied by colonies method.The results showed that X4(pUC-luxABCDE-merR-Kan-X4) successfully survived both in nature and sterilized soil.At the same time,X4(pUC-luxABCDE -merR-Kan-X4) was used to quantify water-extractable mercury concentration in contaminated soil and to compare with chemical analysis.Concentration results with chemical and biosensor analysis were similar in mercury-polluted soil.Therefore,it is concluded that the constructed biosensor can be used to assess bioavailability of mercury in environments.