The Construction And Application Of Whole-cell Biosensors For Monitoring Mercury

Mercury has been well known as an environmental pollutant forseveral decades. Emissions of mercury to the environment could have seriouseffects on human health. To control mercury pollution and reduce mercurydamage to human health, sensitive determination of mercury is important.There are many types of microbial species in ground water and soilenvironments that can convert Hg into organomercury(CH3Hg). Thoseorganomercury forms are more volatile and lipid soluble, therefore arehundreds-fold more neurotoxic than ionic mercury.There are various conventional techniques reported in the literature formercury detection. Such as Atmotic Absorption Spectrometry (AAS), AtomicFluorescence Spectrometry (AFS), Atomic Emission Spectrometry (AES),Inductively Coupled Plasma–Mass Spectrometry (ICP-MS), but their usagessuffer from the disadvantages of high cost, the need for trained personnel andthe fact that they are mostly laboratory bound. Another major limitation ofthose conventional detection methods is that they are unable to detect thebioavailable concentration of metal contaminations. Whole-cell based biosensors utilize live cells as the recognitionelement to produce biological signals which are then transferred tomeasurement devices. However, the specificity, sensitivity, stability andsimplicity of whole-cell biosensor often need to be further significantlyimproved.Our biosensor design for mercury detection was based on MerR whiceis an activator that controls genes involved in the response to mercurypoisoning.Firstly, we selected mCheery out for fluorescent detection signaldue that the background RFU (Relative Fluorescence Unit) of mCherry inlive cell is smaller than that of other FPs. Then cloned MerR into pUC57toobtain pUC57-MerR, and utilized pUC57-MerR as a template to obtain therecombinant plasmid pUCRR. This plasmid was transformed into E. coliDH5in order to obtain engineered strain E. coli UCRR.This engineeredstrains have highest sensitivity to mercury (II).When the Strain withhigh-copy-number plasmid and high concentration of HgCl2showed wouldfurther increase the RFU of the strain.We established a library of mutated MerR gene by Error-prone PCR inorder to improve MerR protein derived biosensors to detect and monitormercury more efficiently and with higher sensitivity. The mutant MerR genesV109E which have improved functionalities been selected. V109E had highest sensitivity increasing than other mutated mercury biosensors after4-hour incubation time.The whole-cell biosensors based on V109E had highsensitivity and Specificity, the lowest concentration of mercury detectionhad reached to0.5nM. We utilized the the whole-cell biosensors based onV109E to detect45samples surface water of the urban and suburban ofBeijing.The sample which mercury content less than0.001mg/L is27.27%,between0.001-0.005mg/L is43.18%, more than0.005mg/L is29.55%.Theresult of our research illustrated that the water quality of the urban andsuburban of Beijing is in a good condition, and the serious mercurycontamination is not exist.