Sensitive and Specific Whole-Cell Biosensor for ArsenicDetection

Xiaoqiang Jia,a,b,c Rongrong Bu,a Tingting Zhao,a Kang Wud

aDepartment of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, ChinabFrontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Tianjin University), Ministry of Education, Tianjin, ChinacSynthetic Biology Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, ChinadDepartment of Chemical Engineering, University of New Hampshire, Durham, New Hampshire, USA

ABSTRACT Whole-cell biosensors (WCBs) have been designed to detect As(III), butmost suffer from poor sensitivity and specificity. In this paper, we developed an ar-senic WCB with a positive feedback amplifier in Escherichia coli DH5�. The outputsignal from the reporter mCherry was significantly enhanced by the positive feed-back amplifier. The sensitivity of the WCB with positive feedback is about 1 order ofmagnitude higher than that without positive feedback when evaluated using a half-saturation As(III) concentration. The minimum detection limit for As(III) was reducedby 1 order of magnitude to 0.1 �M, lower than the World Health Organization stan-dard for the arsenic level in drinking water, 0.01 mg/liter or 0.13 �M. Due to the am-plification of the output signal, the WCB was able to give detectable signals within ashorter period, and a fast response is essential for in situ operations. Moreover, theWCB with the positive feedback amplifier showed exceptionally high specificity to-ward As(III) when compared with other metal ions. Collectively, the designed posi-tive feedback amplifier WCB meets the requirements for As(III) detection with highsensitivity and specificity. This work also demonstrates the importance of genetic cir-cuit engineering in designing WCBs, and the use of genetic positive feedback ampli-fiers is a good strategy to improve the performance of WCBs.

IMPORTANCE Arsenic poisoning is a severe public health issue. Rapid and simplemethods for the sensitive and specific monitoring of arsenic concentration in drink-ing water are needed. In this study, we designed an arsenic WCB with a positivefeedback amplifier. It is highly sensitive and able to detect arsenic below the WHOlimit level. In addition, it also significantly improves the specificity of the biosensortoward arsenic, giving a signal that is about 10 to 20 times stronger in response toAs(III) than to other metals. This work not only provides simple but effective arsenicbiosensors but also demonstrates the importance of genetic engineering, particularlythe use of positive feedback amplifiers, in designing WCBs.

KEYWORDS arsenic resistance, positive feedback amplifier, sensitivity, specificity,whole-cell biosensor (WCB)

Arsenic (As)-contaminated groundwater, occurring from mining or agriculture ornatural contamination due to the abundance of arsenic in the Earth’s crust, is a

serious global health issue. Long-term exposure to arsenic can result in various diseasesincluding cancers (1, 2). It is estimated that over 100 million people worldwide may beat risk from consuming water contaminated with arsenic (3). The World Health Orga-nization (WHO) has recommended 0.01 mg/liter (0.13 �M) as a safe permissible level forarsenic in drinking water (4), and the Food and Agriculture Organization (FAO) has seta maximum contamination level (MCL) for arsenic of 0.01 mg/liter in irrigation water (5).

Citation Jia X, Bu R, Zhao T, Wu K. 2019.Sensitive and specific whole-cell biosensor forarsenic detection. Appl Environ Microbiol85:e00694-19. https://doi.org/10.1128/AEM.00694-19.

Editor Robert M. Kelly, North Carolina StateUniversity

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Xiaoqiang Jia,xqjia@tju.edu.cn, or Kang Wu,kang.wu@unh.edu.

Received 23 March 2019Accepted 27 March 2019

Accepted manuscript posted online 5 April2019Published

ENVIRONMENTAL MICROBIOLOGY

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Due to its toxicity and strict arsenic standards for drinking water, cost-effective andsensitive environmental monitoring tools to detect arsenic are needed.

To date, many methods have been reported to detect arsenic at low concentrations,such as chemiluminescent immunoassay (6), inductively coupled plasma optical emis-sion spectrometry (ICP-OES) (7), and atomic absorption spectrometry (AAS) (8). How-ever, these methods often require complicated and expensive instruments and trainedprofessionals to pretreat and analyze samples, making them hard to use in situ (9, 10).To overcome these limitations, biosensors using enzymes, antibodies, and microorgan-ism cells have garnered interest for use in the detection of arsenic in drinking water(11, 12).

Recently, whole-cell biosensors (WCBs) have been extensively studied for the spe-cific and sensitive detection of toxic heavy metal ions. Often, the regulatory elementsfrom a heavy metal resistance operon, including the transcriptional regulator and itscognate promoter, are coupled to a reporter gene such as fluorescence, luminescence,or enzyme assays so that the signal strength from the reporter is correlated to theconcentration of the heavy metal to be detected. The key to developing a sensitive andspecific WCB is to identify the regulatory elements and then optimize performance byengineering the regulatory elements or the genetic circuit. A relatively well-studiedarsenic resistance operon is the one found in Escherichia coli, which contains arsR(transcriptional regulator), arsB (arsenite permease), and arsC (arsenate reductase) (13,14). When arsenic is absent, the transcription regulator ArsR binds to the ArsR-bindingsite (ABS) within the ars promoter and blocks transcription. Once arsenic is present, itbinds to ArsR and changes the local structure of the promoter to activate the tran-scription of the ars genes and clear arsenic in the cell (14–16). The arsR regulator andthe promoter of this operon have been used to construct arsenic WCBs in variousmicroorganism hosts (11, 17, 18). However, low sensitivity and specificity are majorissues when using them for arsenic detection below the WHO recommended level(19–21).

In this study, we used E. coli as the host to construct arsenic WCBs since it naturallycontains the ars operon. Genetic circuit engineering was done to improve the perfor-mance of the WCB. Positive feedback is common in nature and well known for signalamplification (22). It has been used to improve the sensitivity of WCBs in response tovarious analytes, including antibiotics, amino acids, and heavy metals (23). This workintroduced a positive feedback loop using the LuxR autoregulatory elements to arsenicWCBs for the first time to improve sensitivity and specificity. The comparison of thedesigns with and without the positive feedback amplifier in this work provides usefulinsights for the development of WCBs in the future.

RESULTSDesign and construction of the biosensors. As shown in Fig. 1, two arsenic WCBs

were constructed in E. coli DH5�. The first one simply coupled the arsenic-induciblepromoter (Pars) and its regulatory gene (arsR) with the reporter gene mCherry. Thesignal from mCherry is directly correlated to the concentration of the inducer arsenic.No positive feedback circuit was involved. In the second one (Fig. 1B), the transcrip-tional activator, a variant of luxR, was used to replace mCherry, and it was regulated bythe arsR-Pars circuit, while mCherry together with a second luxR was placed under thepromoter PluxI, which was activated by LuxR. When arsenic is present, it activates theexpression of LuxR in the first plasmid, which turns on the expression of mCherry andLuxR from the second plasmid. The second LuxR activates its own expression as well asthat of mCherry and forms a positive feedback loop to enhance the output signal frommCherry in the second plasmid. These two plasmids work together as the arsenic WCBwith the positive feedback amplifier.

Growth curve of the WCB strains. To understand the toxic effects of arsenic on theengineered strains, the growth curves of DH5�/pCDF-As-mCherry and DH5�/pCDF-As-luxR�pGN68-mCherry at different concentrations of arsenic were measured. Arsenic ata final concentration of 0, 0.1, 1, 10, 100, 200, 300, 400, 500, or 600 �M was added to

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the subculture, and optical density at 600 nm (OD600) was measured every hour for10 h. As shown in Fig. 2, for both strains, no significant effect on the growth wasobserved when the concentration of As(III) was below 10 �M. The bacteria entered thelogarithmic phase after incubation for 2 h and the stationary phase after about 6 h.When the concentration of As(III) was at or above 100 �M, cells grew much slower. Thearsenic toxic effect was more obvious and cells barely grew when the As(III) concen-tration was above 200 �M. Therefore, 0 to 200 �M As(III) was used to obtain the arsenicdose-response curve in the next section.

Arsenic sensitivity and specificity of WCBs. The expression of the reporter mCherryfrom these WCBs accumulates along with time. Not only As(III) concentration but alsothe exposure time affect the output signal of the arsenic WCBs (24–26). Therefore, thetime-response curves were measured before examining the sensitivity and specificity ofthe two arsenic WCBs.

Time-dependent response. The response time of a WCB is an essential factor forpractical application. In addition, a potential issue using a genetic amplifier in WCBs isthat the basal-level expression, either from the sensing module or the amplifyingmodule, may be self-reinforcing and cause a high level of false-positive signal over time.

FIG 1 Schematic of the arsenic WCBs with positive feedback (B) and without (A). (A) The typical arsenicWCB consists of the ArsR-regulated promoter Pars, the regulator arsR, and the reporter gene mCherry. (B)The positive feedback WCB consists of the arsR-Pars regulatory circuit and a positive feedback amplifierwhere LuxR produced in response to arsenite activates the expression of mCherry and LuxR from the PluxI

promoter. The LuxR from the PluxI promoter activates its own expression and forms a positive feedbackloop.

FIG 2 Growth curves of the two WCBs at different concentrations of arsenite. (A) WCB without positivefeedback; (B) WCB with positive feedback.

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Therefore, the time-dependent responses of the two WCBs were monitored for 10 hafter adding 0 �M, 0.01 �M, 0.1 �M, or 10 �M As(III). As shown in Fig. 3, the backgroundsignal without As(III) slightly increased when the incubation time was above 6 h forboth WCBs. After incubation for 10 h without arsenic, the fluorescence signal from thepositive feedback biosensor was increased by 5.4 times, while the nonpositive feedbackbiosensor showed a 4.8-times increase. Therefore, the basal level expression wascomparable for the biosensors with and without the positive feedback amplifier. A falsesignal from the amplification of potential leaky expression was not noticed.

The response of the positive feedback amplifier biosensor to As(III) was faster, andthe signal was much higher than that of the one without positive feedback. After As(III)was added at a concentration of 0.1 �M for 6 h, the WCB with the positive feedbackamplifier exhibited fluorescence that was about 3-fold stronger than that without thepositive feedback. In addition, the output signal of the positive feedback amplifierbiosensor exposed to 10 �M As(III) for 4 h was 11 times higher than that of thebiosensor without positive feedback.

Dose-dependent response to arsenite. The amplification effect of the positivefeedback loop with different initial concentrations of arsenic was analyzed. The twoWCBs were compared after exposure to As(III) at 0 to 200 �M for 9 h at 37°C.

Both WCBs displayed a similar dose-dependent pattern with the fluorescenceintensity positively correlated with the concentrations of As(III) (Fig. 4). Also, it is notedthat the sensitivity of the WCB with the positive feedback amplifier was higher byapproximately 1 order of magnitude compared to that of the WCB without positivefeedback. The half-saturation As(III) concentration that gave half of the maximummCherry fluorescence intensity for the WCB with positive feedback was about 0.5 to1 �M, while the half-saturation As(III) concentration for the WCB without positivefeedback is about 10 to 50 �M. Moreover, the WCB with positive feedback significantlyamplified the output signal, about 2.5 to 5.5 times of that from the WCB withoutpositive feedback when As(III) went from 0.1 to 100 �M. The expression of the mCherrygene was noticeable when As(III) was added at a concentration as low as 0.1 �M (P �

0.01) for the WCB with positive feedback and 0.5 �M (P � 0.001) for the one withoutpositive feedback. The detection limit of the WCB with positive feedback is lower thanthe WHO drinking water standards and potentially could be developed as arsenicbiosensors in real application. These results suggested that, compared with the WCBwithout positive feedback, the one with a positive feedback amplifier functions well inenhancing the fluorescence intensity, increasing the detection range, and improvingsensitivity.

Specificity of the WCBs. In addition to sensitivity and strength of output signal,specificity toward arsenic is also an important factor to evaluate the WCB. Both WCBs

FIG 3 Time-dependent response of arsenic biosensors with (A) and without (B) positive feedback.Biosensor cells were grown for 10 h at 0 �M, 0.01 �M, 0.1 �M, and 10 �M As(III). Statistical significancewas shown as follows: *, P � 0.01; **, P � 0.001.

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were exposed to NaAsO2, Pb(NO3)2, ZnCl2, CuCl2, and CdCl2, at a final concentration of0.01 �M, 0.1 �M, 1 �M, or 10 �M, and the fluorescence of mCherry was measured after8 h. As shown in Fig. 5, compared to As(III), the response of the WCB with positivefeedback to other metals was negligible at either 1 �M or 10 �M, which is less than 2%or 10% of that to As(III). At 0.1 �M, the signal from other metals was about 10% to 60%of that from As(III). However, the fluorescence response of the WCB without positivefeedback to other metals was about 37% to 71% at 0.1 �M, 20% to 30% at 1 �M, and15% at 10 �M of that from As(III). By introducing the positive feedback amplifier intothe arsenic WCB, the output signal was enhanced so much that the specificity of theWCB toward arsenic was also significantly increased, which is remarkable as no otherdesigns have been reported to be able to improve the specificity of a WCB throughcircuit engineering.

FIG 4 Dose-response curves of arsenic biosensors with (red solid squares) and without (black solidsquares) the positive feedback amplifier. Biosensors were grown for 9 h at different arsenite concentra-tions from 0 �M to 200 �M. The right y axis indicates that the maximum FIR value is set to 1. Statisticalsignificance was shown as follows: *, P � 0.01; **, P � 0.001.

FIG 5 Arsenic specificity of the biosensors with and without the positive feedback amplifier. Fluorescence intensityof the biosensors was measured after exposure to various metals at concentrations of 0.01 �M, 0.1 �M, 1 �M, and10 �M for 8 h.

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DISCUSSION

WCBs have been extensively explored in order to detect toxic heavy metals andmetalloids in environments, including cadmium, lead, mercury, and arsenic (27–30).Although many WCBs have been constructed and studied for the detection of arsenic(31–33), most of these WCBs have not been used for environmental monitoringbecause of the high requirements of sensitivity and specificity (34–37). K. de Mora andcolleagues have reported a sensitive biosensor for arsenic in which pH is an inputsignal, and a color change is the output signal (34). It can detect less than 10 �g/liter(10 ppb) of As(III) with static overnight incubation. Nevertheless, the incubation time istoo long for practical applications. L. A. Pola-Lopez et al. developed a new vector wherethe RNA polymerase of bacteriophage T7 was used as an amplifier with green fluores-cent protein (GFP) as the reporter (31). The detection range of this biosensor wasbetween 5 and 140 �g/liter. As(III) concentrations below WHO standards can bedetected. However, in contrast to our designs, the amplifier did not translate into animprovement of biosensor performance, and the amplification effect was not evaluatedusing the unamplified biosensor as a control.

Genetic amplifiers have been used to construct WCBs to intensify the output signaland increase sensitivity to analytes (22, 23), but the effect of a positive feedback loopon increasing the sensitivity of the circuit is different from case to case, depending onthe genetic context and the regulatory element it is coupled with. The LuxR positivefeedback loop has been used for designing various WCBs, either increasing the outputsignal or improving the sensitivity, but has not been reported for detecting arsenic. Ourwork showed that when coupled with the arsR regulatory circuit, the LuxR positivefeedback circuit not only increased its sensitivity but also improved its selectivitytoward As(III).

One concern about incorporating a positive feedback circuit in WCBs in general isthe high noise level and the false-positive signal from amplification of the basal-levelexpression from leaky promoters. K. Bansal et al. applied the positive feedback amplifierto modulate the expression kinetics of membrane proteins (38). This showed a statis-tically significant increase in the rate of production of the bd oxidase membraneprotein. In addition, the positive feedback plays a role in implementing bistability,which is conventionally named high/low or ON/OFF in steady-state levels of geneexpression. In other words, the gene expression levels of the biosensor are determinedby the initial concentration of the inducer, and the level of the initial input inducer cancause changes in gene expression between the two steady-state levels. Single-cellmeasurements showed that whether positive feedback amplifiers increased cell noisecompared with nonpositive feedback controls depends on the activity of LuxR proteins.Similar noise levels were observed for both the positive feedback and nonpositivefeedback systems. In contrast, the increased noise at higher inducer concentrationsmay be a result of increased system burden by the reduced growth rates of the cells.In our work, the variation of fluorescence from the WCB with positive feedback wasactually slightly lower than that from the WCB without positive feedback. The time-response curve in the absence of As(III) showed that the output signal accumulated toroughly the same extent for the two WCBs. Amplification of leaky signals was notobserved.

The arsenic biosensor with the positive feedback amplifier that we designed has abroader detection range and a lower detection limit than the one without positivefeedback because the positive feedback loop can amplify the output signal, which alsoincreases sensitivity, as the accumulation of fluorescent proteins occurs at a lowconcentration of inducer arsenic. The positive feedback biosensor constructed in thisstudy can detect arsenic as low as 0.1 �M and is more sensitive than the biosensorsconstructed thus far (39, 40). The half-saturation As(III) concentration of the biosensorwith positive feedback is about 1 order of magnitude lower than that without positivefeedback. An amplifier has been applied to detect cadmium and lead. It used T7 RNApolymerases to modulate multiple circuits by decoupling the transcription from the

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host and enhancing the expression (41, 42). It can reduce the detection limit forcadmium ions but has little effect on the minimum detection limit of lead ions. Inaddition to improving the sensitivity of biosensors, the positive feedback amplificationsystem demonstrated improved specificity toward arsenite, as it significantly amplifiedthe signal in response to arsenite but only marginally increased the signal in responseto other metals. So overall, it showed a dramatic difference in the fluorescence inresponse to arsenite and other metals. Many arsenite WCBs have specificity issue (39,43). Generally, the specificity may be improved by protein engineering to modify theinteraction between the regulator and the inducer metal ions. It is surprising that thepositive feedback loop introduced to the arsenite WCB in this study differentiallyenhanced the output signal in response to arsenite and other metals. The arsenite-induced signal was 10- to 40-fold higher than that of other metals, which is sufficientto detect As(III) in the presence of other metals.

The minimum detection limit of heavy metal biosensors often refers to the finalconcentration of the metal in the medium under optimal culture conditions (44). Inthis work, the same definition was used to describe the detection limit of thebiosensors so that it can be compared with the work of other groups. A concern forthe practical application is the difference between the real detection limit and thefinal concentration since the analyte is diluted when adding the sample water tothe culture. It may not be detectable with high dilution ratios even if the originalconcentration is within the reported detection limit. One potential solution is tomake Luria-Bertani (LB) broth directly from the sample water and inoculate it withthe WCBs, but the cells may grow slower with an initial high concentration of heavymetals. Another method is to use high sample water culture ratios to minimize thedifference. We tested the growth of the two biosensors in media with dilutednutrients. WCBs were grown in a medium with a nutrient concentration two timeshigher than that in LB broth and the same NaCl concentration to maintain elec-trolyte balance. Sodium chloride solution (10 g/liter) was mixed with the culture ina ratio of 5:1 or 10:1. In both cases, the growth of the two WCBs was similar to thatcultured in regular LB broth. So for practical applications, the concentration de-tected largely represents the concentration in the original sample if using a verylow dilution ratio. Due to the variation of live systems, WCBs are mainly forqualitative or semiquantitative analysis. With a low dilution ratio, the biosensors wedesigned can be used for an initial test of whether the sample is polluted.

Another major concern about WCBs is whether they are functional in fieldapplications since the samples may contain highly toxic components or contami-nating species, which interfere with cell growth or the accuracy of sensing. We usedbacteria as the host in this work, as many other WCBs do (34–36), because bacteriado not require strict conditions for rapid growth and reproduction. They can surviveat room temperature and have relatively low requirements for pH and humidity.Although relatively more robust, bacterial WCBs are still subject to environmentalfluctuations since the expression of the reporter protein relies on cell growth (45,46). To solve this issue, the biosensing components have to be decoupled from cellgrowth. One potential strategy is to use a cell-free expression system, and it isbecoming feasible with decreasing costs. Another method is based on the differ-ential interaction between the transcription factor and its cognate promoter inresponse to the analyte to develop a quick detection assay in vitro. This work hasdemonstrated the sensitivity of the regulatory elements in As(III) detection and theeffect of positive feedback on improving the sensitivity of the biosensor. The sameregulatory elements could be applied for the design of in vitro As(III) biosensors toavoid the issue of using live cells.

Overall, our results indicate that the positive feedback WCB is superior to the onelacking positive feedback in terms of response time, sensitivity, and specificity. Thisamplifier biosensor is able to detect arsenic below the WHO and FAO standards andcould be potentially used as a test tool in situ to routinely monitor arsenic levels indrinking water. Our work shows the importance of genetic circuit engineering in

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improving the performance of WCBs and provides insights into the design of otherbiosensors. The positive feedback loop, though maybe having different effects on thegene circuit depending on the genetic context, is a useful strategy for designing andoptimizing WCBs to detect other heavy metals or pollutants.

MATERIALS AND METHODSBacterial strains, reagents, and growth conditions. Construction and characterization of the

designed WCBs were performed in E. coli DH5�. Cells were grown in LB broth (10 g/liter peptone,5 g/liter NaCl, 5 g/liter yeast extract). Solid plates were made using the same medium added with1.5% (wt/vol) agar. All experiments were performed at 37°C unless otherwise noted. Antibiotics wereused at the following concentrations: streptomycin (Sm) at 50 �g/ml and chloramphenicol (Cm) at30 �g/ml.

PCR reagents, restriction endonucleases, and T4 DNA ligase were purchased from TransGen Biotech.NaAsO2, Pb(NO3)2, ZnCl2, CuCl2, and CdCl2 were purchased from Shandong Western Chemical IndustryCo. Ltd., China. Oligo primer synthesis and sequencing were performed by Genewiz (China).

Construction of the sensor plasmids. The plasmid of the arsenic resistance biosensor was madefirst by PCR amplification of the arsenite-sensing element (Pars and arsR) (GenBank accession no.NC_000913.3) (21) using E. coli DH5� genomic DNA as the template. The primers were designed asfollows with the restriction enzyme cutting sites underlined: 1F (5=-CGGGATCCCTCCTTTCAAATGAATAGCC-3=) and 1R (5=-GGAATTCTTAACTGCAAATGTTC-3=). The amplified DNA fragment was purified anddigested with BamHI and EcoRI. The BamHI EcoRI DNA fragment was subsequently inserted into thepCDFDuet-1 plasmid, yielding pCDF-As. The mCherry gene was amplified from the plasmid pmCherryusing primers 2F (5=-GGAATTCCGTATTTAAATCAGGAGTGGAAATGGTGAAGCGGGCGAGG-3=) and 2R (5=-ATAAGAATGCGGCCGCCTACTTGTACAGCTCGTCCATGC-3=). The resulting fragment was then cloned intothe EcoRI and NotI restriction sites of the pCDF-As, yielding the plasmid pCDF-As-mCherry.

The luxR expression plasmid was constructed first by amplifying the luxR gene from the plasmidpGN68 (23) using primers 3F (5=-GGAATTCAACTAAAGATTAAC-3=) and 3R (5=-ATAAGAATGCGGCCGCTTATTAATTTTTAAAG-3=). The 304-bp DNA fragment was digested with EcoRI and NotI and then inserted intoplasmid pCDF-As to give pCDF-As-luxR.

The reporter gene mCherry was amplified using primers 4F (5=-GGAATTCATGGTGAGCAAGGGCGAGGAG-3=) and 4R (5=-CGGGATCCCTACTTGTACAGCTCGTCCATGC-3=). The resulting PCR product was di-gested with EcoRI and BamHI and then subcloned into the respective sites of pGN68, yielding plasmidpGN68-mCherry. All constructs were confirmed by PCR/gel electrophoresis and Sanger sequencing.

Growth curve of the WCB strains. A single colony of E. coli harboring the sensor plasmid(s) wasgrown overnight in LB medium containing appropriate antibiotics at 37°C. The OD600 of the overnightcultures was adjusted to 2.0 with fresh LB medium, and then they were used as the seed to inoculate thesubculture by adding 0.5 ml of the seed culture to 50 ml fresh liquid medium containing As(III) atdifferent final concentrations (0, 0.1, 1, 10, 100, 200, 300, 400, 500, 600 �M). The subcultures wereincubated at 37°C in an orbital shaker at 220 rpm. The optical density at 600 nm was first measured byspectrophotometry (UV-2000; Unico, USA) after 2 h of incubation and then measured every hour.

Fluorescence measurement. An overnight culture with an OD600 adjusted to 2.0 was used as theseed culture to inoculate the 5-ml subculture with a dilution rate of 1:100. The heavy metal inducer,NaAsO2, Pb(NO3)2, ZnCl2, CuCl2, or CdCl2, was added to a specific concentration (24). The culture wasincubated at 37°C in an orbital shaker. Every hour, 200 �l of each subculture was transferred to a 96-wellmicroplate, and the optical density at 600 nm and the fluorescence intensity were measured by thefluorescence microplate reader (M2; SpectraMax, USA) with an excitation/emission of 580/610 nm. Allexperiments were performed in triplicate, and the E. coli DH5� strain containing the plasmid pCDFDuet-1was used as the negative control.

The fluorescence induction ratios (FIRs) were calculated using the formula FIR � AFU/BFU, where thearbitrary units of fluorescence (AFU) are defined as the relative fluorescence unit (RFU) divided by theoptical density at 600 nm at specific arsenic concentrations and time points. The background fluores-cence unit (BFU) was defined by dividing the RFU of the E. coli DH5� culture containing no metal(negative control) by its optical density at 600 nm. The fluorescence induction ratios (FIRs) of all othersamples were normalized to BFU.

ACKNOWLEDGMENTSWe wish to acknowledge the financial support provided by the National Basic

Research Program of China (“973” Program: 2014CB745100), the National NaturalScience Foundation of China (no. 21576197), and Tianjin Research Program of Appli-cation Foundation and Advanced Technology (no. 18JCYBJC23500).

Moreover, we thank Accdon for providing linguistic assistance during the prepara-tion of this manuscript.

We declare that there is no conflict of interest regarding the publication of thisarticle.

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