Improving the Sensitivity of Bacterial Bioreporters

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132 Bioengineered Bugs Volume 1 Issue 2

Bioengineered Bugs 1:2, 132-138; March/April 2010; 2010 Landes Bioscience

*Correspondence to: Anu Hynninen; Email: [email protected]

Submitted: 09/22/09; Revised: 11/30/09; Accepted: 12/02/09

Previously published online: www.landesbioscience.com/journals/biobugs/article/10902

Introduction

While chemical and physical methodologies or monitoring envi-ronmental pollution can be powerul, accurate and sensitive, theyare also costly and require specialized laboratories. In addition,they ail to provide data on the bioavailability o a pollutant and

its eects on living systems. Thereore, whole-cell-based bio-assays, which are easily applicable and inexpensive, and whichreport not only on the presence o a chemical, but also on itsbioavailability and its biological eects, are being developed.Whole-cel l bioreporters are living microorganisms that producea specic quantiable output in response to target chemicals.On the basis o detection specicity, bioreporters can be broadlydivided into three groups: nonspecic, semi-specic and specic.Nonspecic bioreporters are useul or measuring the toxicity osamples, but do not provide any inormation about the identityo the contaminants.1,2 Recombinant semi-specic bioreportersallow or the detection o compounds that cause certain cellu-lar responses, such as stress3-5 or DNA damage6,7 without ur-

ther characterizing the compound. Specic bioreporters, rstdescribed by King et al.8 respond only to a certain compound orclass o compounds and thereore permit a quantitative analysiso the contaminant concentration.

In general, specic bacterial bioreporters combine a sensingelement, which is responsible or detecting the analyte, and areporter element, which allows or quantication o the signal.9,10

Improving the sensitivity of bacterial bioreportersfor heavy metals

Anu Hynninen,* Karmen Tnismann and Marko Virta

Department o Applied Chemistry and Microbiology; University o Helsinki; Helsinki, Finland

Key words: Zn/Cd/Pb-bioreporter, sensor element, speciicity, limit o detection

Whole-cell bacterial bioreporters represent a convenient testing method or quantiying the bioavailability o

contaminants in environmental samples. Despite the act that several bioreporters have been constructed or measuring

heavy metals, their application to environmental samples has remained minimal. The major drawbacks o the available

bioreporters include a lack o sensitivity and specicity. Here, we report an improvement in the limit o detection o

bacterial bioreporters by interering with the natural metal homeostasis system o the host bacterium. The limit o

detection o a Pseudomonas putida KT2440-based Zn/Cd/Pb-biosensor was improved by a actor o up to 45 by disrupting

our main eux transporters or Zn/Cd/Pb and thereby causing the metals to accumulate in the cell. The specicity o

the bioreporter could be modied by changing the sensor element. A Zn-specic bioreporter was achieved by using the

promoter o the cadA1 gene rom P. putida as a sensor element. The constructed transporter-decient P. putida reporter

strain detected Zn2+ concentrations about 50 times lower than that possible with other available Zn-bioreporters. The

achieved detection limits were signicantly below the permitted limit values or Zn and Pb in water and in soil, allowing

or reliable detection o heavy metals in the environment.

In this manner, bacterial bioreporters have been developed orheavy metals11 and several organic compounds.9,12

In heavy metal bioreporters, the sensor element usually consistso a specic promoter used to a reporter gene, and a gene encod-ing or a transcription actor that in response to certain metal ionsactivates the promoter. Binding o a metal ion to the transcrip-

tion actor induces the expression o the reporter gene, thus con-verting metal sensing into a detectable output signal. The mostimportant characteristics o bacterial bioreportersspecicity(which metals are detected) and sensitivity (what concentrationsare detected; characterized by the limit o detection)dependon the transcription actor used in the sensing element as well ason the microbial metal homeostasis and resistance systems.13 Inbacteria, homeostasis o essential metals and resistance to toxicheavy metals are maintained with the help o infux and efuxtransporters. Importantly, it is the activity o these transportersthat determines the intracellular metal concentration that is ulti-mately available or detection by the bioreporters.

Despite the importance o whole-cell bioreporters in deter-

mining the biologically relevant raction o metals in the environ-ment, they are rarely used or actual environmental analyses. Thewider application o bioreporters has been partly hindered by lowsensitivity and a lack o specicity. The amount o bioavailablemetal in complex matrices can be less than 1%, even in highlycontaminated samples.14,15 Reliable detection o such low con-centrations requires improved detection limits. In addition, the


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analysis o environmental samples. P. putida KT2440.2431 isa metal transporter-decient mutant o the strain KT2440. InP. putida KT2440.2431, genes or our Zn/Cd/Pb efux trans-

porters (P-type ATPases CadA1 and CadA2 and CBA transport-ers CzcCBA1 and CzcCBA2) have been disrupted, renderingthe cell susceptible to heavy metals. As sensor-reporter elements,we used usions o heavy-metal-regulated cadA1 and czcCBA1promoters rom P. putida KT2440 to bacterial bioluminescenceoperon luxCDABEin a low copy number plasmid. Altogether,our dierent bioreporter strains, KT2440(pDNPczc1lux),KT2440.2431(pDNPczc1lux), KT2440(pDNPcadA1lux) andKT2440.2431(pDNPcadA1lux) (Fig. 1), were tested or theresponse to heavy metals.

Optimization o the assay. To achieve minimal LOD o thebioreporters within a reasonable testing time, dierent assayconditions were tested. First, the inducibility o the bioreporters

at dierent growth stages was tested as metal-induced expres-sion rom PcadA1 and Pczc1 promoters is known to be depen-dent on the growth phase o the bacteria.17 The response o thereporter strains to metals was largely dependent on the growthphase (data not shown). The lowest metal concentrations weredetected with bacteria grown to the early exponential phase.Thereore, bacteria grown to the exponential phase (OD

600

0.0150.017 measured on 96-well plate in 200 l) were usedin urther experiments.

In addition, an eect o incubation time (i.e., contact timeo the reporter bacteria with the metal) on sensor inducibility

ability to specically characterize the contaminants in a samplerequires bioreporters that respond to only one metal.

We hypothesized that, on the removal o specic efux pumps,

bacteria start to accumulate more metal in the cell, and thus,lower extracellular concentrations can be detected. Efux mecha-nisms compete with the detection o metal ions and thus lead toa less sensitive response. I all o the metal ions entering the cellwere immediately removed, no binding to transcription actorswould occur and hence no signal would be generated. However,in the absence o efux transporters, metal ions accumulate inthe cell and the threshold concentration required or the expres-sion o the reporter gene is reached at lower extracellular con-centrations. Our approach was to improve the limit o detection(LOD) o Pseudomonas putida-based Cd/Zn/Pb-bioreportersby altering the efux o respective metals rom the cytoplasm,thereby causing metal ions to accumulate in the bacterial cell. In

P. putida KT2440, genes or the divalent heavy metal efux pro-teins CadA1, CadA2, CzcA1 and CzcA2 were deleted, and themutant P. putida strain was applied as a Cd/Zn/Pb-bioreporterand tested or improved limit o detection.

Results

Constructed bioreporter strains.P. putida strains KT2440,16 andKT2440.2431,17 were used as host bacteria. P. putida KT2440 isa ubiquitous saprophytic bacterium endowed with a remarkableadaptability to diverse environments and is thus suitable or the

Figure 1. Principle oPseudomonas putida KT2440 and KT2440.2431 metal bioreporters. (1) Uptake and eux mechanisms regulate the intracellular

metal level; in the mutant strain KT2440.2431, the eux mechanisms have been removed. (2) Upon binding o the metal ion to the transcription actor,

transcription is initiated rom the PcadA1 or Pczc1 promoter that is used to the luxCDABEoperon. (3) mRNA or the reporter proteins is produced and

subsequently translated into proteins. (4) The bacterial luxoperon encodes or lucierase and the proteins necessary or substrate synthesis; thereore,

luminescence can be measured directly as no additional substrate is required. Over a certain concentration range, the signal output correlates to the

intracellular metal concentration, allowing or quantication o the metal.


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was invest igated. Clear induction was already visible ater1 hour o incubation. For the wild-type reporter strains, ur ther

incubation had no eect on the LOD. However, or the mutantreporter strains, up to 10-old improvement could be achievedby extending the incubation to 3 hours. For example, the LODo P. putida KT2440.2431(pDNPczc1lux) or Cd2+ shitedrom 0.1 M to 0.02 M by extending the incubation rom1 to 3 hours. Although even longer incubation times slightlyimproved the sensitivity (as low as 0.008 M LOD or Cd2+could be achieved), a 3-hour incubation time was used in ur-ther experiments to balance the time used or the whole assay.The decrease with time in the LOD o the efux transporter-decient strain could be explained by the continuous accumula-tion o metal ions in the cell and the consequent detection olower extracellular metal concentrations.

Sensitivity and specifcity o the reporter strains. The con-structed bacterial bioreporters were assayed with dierent divalentheavy metals to determine their sensitivity (LOD) and specicity(metals detected) using the optimized assay conditions. The Zn/Cd/Pb efux transporter-decient strain KT2440.2431 was usedin parallel with the wild-type strain KT2440.

As hypothesized, the removal o efux transporters aected theLOD o the bioreporters. P. putida KT2440.2431(pDNPczc1lux)could detect Pb2+ at 45-old, Cd2+ at 12-old, Ni2+ at 10-old andZn2+ at 3-old lower concentrations than its wild-type coun-terpart KT2440(pDNPczc1lux) (Fig. 2 and Table 1). For the

wild-type strain, Zn2+ was the best inducer, whereas or themutant strain, Zn2+, Cd2+ and Pb2+ were detected at the same con-

centrations. For both strains, Ni

2+

was a poor inducer as 100-oldhigher concentrations were needed or induction. No inductionwas observed with Hg2+ or Co2+. Toxic concentrations causing arapid decrease in the luminescence signal were much lower orthe mutant strain (Fig. 2), indicating that metals accumulatedin this strain.

Although substantial improvements in LODs were achievedby using the promoter o the czcCBA1 operon as a sensor ele-ment, the lack o specicity still remained a problem. Thereore,we tested a promoter o the cadA1 gene, which is known to beinduced by Zn2+ and slightly by Cd2+, but not by other met-als,17 as a sensor element. P. putida KT2440(pDNPcadA1lux)was inducible only with Zn2+, and expectedly, the mutant strain

KT2440.2431(pDNPcadA1lux) could detect about eight-oldlower Zn2+ concentrations than the wild-type strain (Fig. 3and Table 1). Although P. putida KT2440.2431(pDNPcadA1lux)could not detect concentrations as low as KT2440.2431(pDNPczc1lux), the great advantage o the reporter strain har-boring the promoter ocadA1 as a sensor element was its specic-ityno metals other than Zn2+ induced this reporter strain.

Perormance o the bioreporters in Zn-contaminated soilsamples. The perormance o the constructed bioreporter strainswas tested on soil samples intentionally contaminated with aknown amount o Zn2+. In the measurements o undiluted soil

Figure 2. Inducibility oP. putida KT2440(pDNPczc1lux) (black squares) and KT2440.2431(pDNPczc1lux) (red circles) with Zn2+ (A), Cd2+ (B), Pb2+ (C) and

Ni2+ (D). Data represent the mean standard deviation o at least three independent experiments.


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Discussion

Dozens o bioreporters have been constructed, and their suitabil-ity or testing environmental samples has been demonstrated.13Despite this research, bioreporters are not yet widely used in envi-ronmental applications, partly due to their high LODs and lacko specicity. We approached the sensitivity and specicity issuesby disrupting the metal homeostasis o the host bacteria and test-ing dierent sensor elements.

In this study, we constructed new transporter-decient bacte-rial bioreporter strains P. putida KT2440.2431(pDNPczc1lux)and KT2440.2431(pDNPcadA1lux), which, depending on the

metal, could detect 345-old lower metal concentrations ascompared to their wild-type counterparts. The improvementin LODs was achieved due to the disruption o metal efuxtransporters and consequent accumulation o metals in the cell.P. putida KT2440.2431(pDNPczc1lux), whose upper estimateo LOD or zinc was 0.05 M (3.3 g/l), is the most sensitivebacterial sensor or zinc constructed thus ar (Table 1). Althoughthe LOD or lead and cadmium remained on the same level asthose or already existing bioreporters (Table 1), the success ometal homeostasis interruption indicates that improved LOD orthese metals could also be achieved by modiying already existingbioreporter strains. A similar strategy has been shown to work orCu/Ag-bioreporters, but not or As-bioreporters. The removal o

CopA, a Cu/Ag-exporting ATPase, rom E. colirendered a biore-porter employing the copA promoter as a sensor system 15-oldmore sensitive to Cu and eight-old more sensitive to Ag.18 On theother hand, Tauriainen et al.19 compared the perormance o twoE. coliAs-bioreportersone with an intact As detoxication sys-tem and another containing a deletion in the chromosomal arsen-ite-resistance operon. They ound no dierences in perormanceo these bioreporters, showing that the lack o the arsoperon didnot cause a remarkable accumulation o As in the cell.

Almost all bioreporters constructed to date detect only intra-cellular metal. In addition to causing the accumulation o metals

samples wild-type P. putida KT2440(pDNPcad1lux) couldreliably detect zinc rom the soil that contained 65 mg/kg oZn2+; whereas, the more sensitive transporter-decient strainKT2440.2431(pDNPcad1lux) could detect as low concentra-tions as 20 mg/kg o Zn2+ (Fig. 4A).

In highly contaminated samples metal concentrations

become toxic to the cell and measurements o undiluted sam-ples do not give correct estimation o the bioavailable amounto the metal. In such cases additional diluting o the sampleis required in order to reach concentrations which are close tothe LOD o the bioreporter. The transporter-decient reporterstrain KT2440.2431(pDNPcadA1lux) could detect about10-old lower Zn concentrations than the wild-type strainKT2440(pDNPcadA1lux) in the dilution series o soil spikedwith 654 mg/kg o Zn2+ (Fig. 4B). Thus, as was the case withstandard metal solutions, transporter-decient bioreporter per-ormed better also in actual environmental samples.

Table 1. Limits o detection o Zn-, Cd- and Pb-sensing bioluminescent bacterial bioreporters

Bacterial bioreporterLimit of detection, M

ReferenceZn2+ Cd2+ Pb2+

P. fluorescens OS8::KncadRPcadAlux a 4 0.03 0.3 28

E. coliMC1061(pSLzntR/pDNPzntAlux)a 5 0.01 0.7 28

E. coliMC1061(pzntRluc)b 40 0.06 n.t. 29

E. coliMG1655(pZNT-lux)b

1 0.01 0.03 30S. aureus RN4220(pTO024)c 1 0.01 0.03 31

B. subtilis BR151(pTO024)c 1 0.03 n.i. 31

P. putida KT2440(pDNPczc1lux) d 0.16 (0.08) 1.12 (0.49) 0.90 (0.41) This study

P. putida KT2440.2431(pDNPczc1lux)d 0.05 (0.02) 0.09 (0.05) 0.02 (0.02) This study

P. putida KT2440(pDNPcadA1lux)d 0.89 (0.63) n.i. n.i. This study

P. putida KT2440.2431(pDNPcadA1lux)d 0.12 (0.08) n.i. n.i. This study

alimit o detection (LOD) determined as a concentration that caused 2 + 6 SDw/L

w-old induction. bLOD determined as a concentration that caused

two-old induction. cLOD determined as a concentration that c aused Lw

+ 2 SDw

-old induction. dLOD determined as a concentration that caused (1 + 2

CVw)/(1 - 2 CV

w)-old induction; upper estimate o LOD (conidence level 97.7%) and mean LOD (in parenthesis) are presented (see Materials and Meth-

ods or details). Lw, luminescence in water; SD

w, standard deviation o the signal measured in water; CV

w, coeicient o variation o the signal measured

in water; n.t., not tested; n.i., not inducible.

Figure 3. Inducibility oP. putida KT2440(pDNPcadA1lux)

(black squares) and KT2440.2431(pDNPcadA1lux) (red circles) with Zn2+.

Data represent the mean standard deviation o three independent

experiments.


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independent experiments is natural. Thereore, a statistically soundmethod or determining LOD should be used. IUPAC suggeststhat LOD or analytical methods is determined as the concentra-tion which has a response equal to the mean response plus two orthree standard deviations o the blank samples depending on thecondence level desired.26 However, this method eliminates onlythe alse positives and does not take into account the variabilityin the signal measured or the analyte. We used here the methodrecommended or immunoassays.27 This method considers thevariability in the signal or both the blank and also the analyte.The concentration o the analyte where the condence intervaldoes not overlap with the condence interval o the blank is con-sidered as the LOD.27 Thus, the method eliminates both the alse

positives and the alse negatives. In addition to considering thevariation within the experiment we took into account the vari-ability between the independent experiments and reported theupper estimate o LOD in addition to reporting the mean LOD.I only the mean LOD is used or characterizing the bioreporter,50% o the experiments actually have a higher LOD than ini-tially reported. However, the upper estimate o LOD guaranteesthat with a probability o 97.7% the LOD in a single experimentis below the reported level. Thereore, it is important to considerthe variability o the method when reporting the characteristicso the bioreporter.

in the cell by removing efux transporters, permeabilization othe membrane or additional metal uptake mechanisms couldalso improve the LOD o bacterial bioreporters. The infuence ouptake transporters has been tested on a Hg-bioreporter.20 In addi-tion to detoxication proteins, the Hg-resistance operon encodesor a protein that mediates the uptake o Hg2+ ions into the cell.Employing this protein in the bioreporter construct improvedthe sensitivity o the Hg-bioreporter two-old.20 Although spe-cic uptake systems only exist or a limited number o metals,the proteins responsible or the uptake o most metals are known.Overexpression o these systems in combination with the removalo efux transporters would thus be one approach to improving

the LOD o bacterial bioreporters.In addition to improving the LOD o Zn/Cd/Pb-bioreporters,we aimed at more specic reporters by changing the sensor element.P. putida KT2440.2431(pDNPcadA1lux), which carried the cadA1promoter as a sensor element, proved to be Zn2+-specic. AlthoughP. putida KT2440.2431(pDNPcadA1lux) was somewhat less sen-sitive than P. putida KT2440.2431(pDNPczc1lux) (Table 1), itsspecicity is a great advantage over other bioreporters. Restrictedspecicity allows or more accurate quantication o a certaincontaminant regardless o other contaminants in the sample. Theexistence o a Zn-specic sensor element is peculiar, as all o thetranscription actors and resistance mechanisms known or Zn2+thus ar cross-react with Cd2+ and Pb2+ (see Table 1).17,21-24 However,

as only three sensor elements (the promoters ozntA rom E. coli,cadA rom P. putida and cadA rom S. aureus) have been previouslyused or the construction o Zn/Cd/Pb-bioreporters, it is still pos-sible that an open-minded search or new heavy-metal-regulatedpromoters would yield bioreporters with dierent specicities.

Although the LOD is an important characteristic o bacte-rial bioreporters, no common method or calculating the LODhas been established in the literature (see Table 1). Several othe methods applied or calculating the LOD lack any statisti-cal basis. However, as bacterial bioreporters are living systems,some variability in the signal within one experiment and between

Figure 4. Perormance oP. putida KT2440(pDNPcadA1lux) (black squares) and KT244 0.2431(pDNPcadA1lux) (red circles) in Zn-contaminated soil

samples. (A) Induction o the bioreporters as a unction o Zn2+ concentration in the soil samples. The x-axis shows the spiked Zn concentration in the

soil. Each measurement point corresponds to a soil sample spiked with diferent concentration o Zn. (B) Induction o the bioreporters in the dilution

series o the soil sample spiked with 654 mg/kg o Zn 2+. The x-axis shows the actual Zn concentration in the diluted samples. Data represent the mean

standard deviation o three independent experiments.

Table 2. Limit values o zinc, cadmium and lead in the European Union,compared to the upper estimates o the limit o detection o the bacte-

rial bioreporter P. putida KT2440.2431(pDNPczc1lux)

Metal

Limit valueUpper estimate of the

limit of detection, mg/lSurface water,

mg/laSoil, mg/kgb

Zn 3 150300 0.003

Cd 0.005 13 0.010

Pb 0.05 50300 0.004

aCouncil Directive 75/440/EEC; bCouncil Directive 86/278/EEC.


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The optimal incubation time was determined by measuring induc-tion plates at 1-h intervals.

Determination o the limit o detection. The limit o detec-tion or bacterial bioreporters was determined as the metal con-centration which induced the reporter to above the thresholdlevel LOD

IC, calculated according to the equation LOD

IC=

(1 + 2 CVw

)/(1 - 2 CVw

). Here CVw

is coecient o variation

o the signal determined in water (background or blank). Thesignal in water was always measured in at least 12 parallels, butas it was not reasonable to measure samples in as many parallelsit was assumed that CV or samples is the same as or water.This equation assures that, within the condence interval o97.7% the lowest signal rom the sample, L

M- 2 SD

M, is higher

than the highest va lue or the blank within the same condenceinterval, L

W+ 2 SD

W, thus eliminating both alse positives

and alse negatives. The lowest detectable concentration wasdetermined separately or each measurement (according to theLOD

ICo each independent measurement). An upper estimate

o LOD was determined as the mean plus two standard devia-tions o the mean, calculated rom the independent measure-

ments. This corresponds to a 97.7% condence level, i.e., theprobability that the LOD determined rom a single experimentis below this upper limit is 97.7%. In order to acilitate thecomparison to previously published bioreporters also the meanLOD was presented.

Analyses o the soil samples. Articial Zn-contaminated soilsamples were made by spiking industrially produced garden soil(Rainbow, Finland) with dierent Zn2+ concentrations. Onegram o soil (dry weight) was mixed with 9 ml o ZnCl

2solution

(nal concentrations ranging rom 6.54 to 654 mg o Zn 2+ per 1 kgo soil) and then shaken on vortex or 1 h. Non-spiked soil sample(MilliQ water added instead o ZnCl

2solution) was used as a back-

ground control to take into account potential changes in the lumi-nescence not caused by zinc. This includes potential intererenceo the sample matrix (stimulation or quenching o the lumines-cence) due to the presence o nutrients, toxicants or turbidity o thesample, and changes in the luminescence due to the changes in thephysiological state o the cell. Samples were analyzed immediatelyand stored or urther measurements at -20C.

For the induction assay 50 l o the soil suspension was platedinto a 96-well microplate (Thermo Labsystems, Finland) andmixed with an equal volume o a bacterial suspension at an earlyexponential growth phase (OD

600 0.0150.020). Plates were

then incubated or 3 h at 30C, and luminescence was measuredwith a Victor Wallac 1420 multilabel counter. All measurements

were obtained using two replicates in three independent experi-ments. Sample specic IC was calculated by dividing lumines-cence measured in spiked sample with luminescence measuredin non-spiked sample at respective dilution. The soil sample wasconsidered to induce the bioreporter i higher IC than LOD

IC

(see above) was achieved.Note that the measurement protocol causes 20-old dilution

o the initial sample (10-old in preparation o suspension andadditional two-old in actual measurement). The soil suspensionswhich induced the bioreporters were urther diluted up to 10,000-old with MilliQ water in order to achieve IC close to LOD

IC.

The new bioreporters constructed in this work,P. putida KT2440.2431(pDNPczc1lux) and KT2440.2431-(pDNPcadA1lux), are suitable or measuring zinc, cadmium orlead rom environmental samples, as their upper estimate o LODremained well below the limit values o water and soil contami-nants set by the European Union (Table 2). Our results show thatthe limit o detection o bacterial heavy metal bioreporters could

be improved by removing heavy metal efux pumps, therebyarticially increasing the intracellular concentration o the metal.This study indicates that the disruption o metal homeostasis inbacteria is a easible approach or the development o bacterialbioreporters with increased sensitivity. The use o hyper-sensitivebioreporters in environmental risk assessment opens the possibil-ity or the detection o low-level pollution.

Materials and Methods

Bacterial strains and growth conditions. As bioreporters,P. putida strains KT2440,16 and KT2440.2431,17 were used.Both strains were transormed with the sensor plasmids pDN-

PcadA1lux and pDNPczc1lux.17 These plasmids contain a luxCD-ABEoperon rom Photorhabdus luminescensunder the control othe cadA1 and czcCBA1 promoters rom P. putida, respectively.

Bacteria were grown in a heavy metal MOPS (HMM)medium25 supplemented with 0.05% caseine hydrolysate, 0.4%glucose and 12 g/ml tetracycline. For maintaining the strains,HMM plates containing 1.5% agar were used.

Induction assay. Inducibility o the constructed strainswas tested with dierent heavy metals. Solutions o CdCl

2

(stock solution 1 M), ZnCl2

(0.1 M), HgCl2

(0.1 M), NiSO4

(1 M), CoCl2

(1 M) and Pb (CH3COO)

2(0.05 M) at dier-

ent concentrations were prepared in MilliQ water (Millipore,

France). First, metal dilutions were prepared with a BiomekNXP (Beckman Coulter, Fullerton) robot using a dilution ac-tor o 1:3. Then, 20 l o the metal dilution was dispensed toa white 384-well plate (Thermo Labsystems, Finland), and anequal amount o bacterial suspension was added using a BiomekNXP pipetting robot. The plates were incubated or 3 h at 30C,and the luminescence was measured with a Victor Wallac 1420multilabel counter (PerkinElmer, Finland). The samples inevery assay were analyzed in our parallels, i.e., each metal con-centration was plated in our wells. At least three independentassays were perormed or every strain. Induction o the biore-porters was reported as an induction coecient (IC), calculatedas ollows: IC = L

M/L

W, where L

Mis the luminescence in the

metal solution and LW is the background luminescence mea-sured in MilliQ water.

Testing conditions. The growth and test conditions were opti-mized to achieve a satisactory limit o detection within a minimaltime. The cells were grown overnight in HMM medium, diluted100-old in resh medium and then grown at 30C with shaking.Aliquots o bacteria were removed at dierent time points and usedin the induction assays. Growth curves were determined by mea-suring the OD

600values at 1-h intervals, and growth phases were

assigned according to dierent stages o the growth curve. OD600

was measured with Victor Wallac 1420 on 96-well plate in 200l.


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Acknowledgements

This study was nancially supported by the EnSTe GraduateSchool and Academy o Finland. We thank Dr. Teemu Hynninenor ruitul discussions and Leena Pitknen or a critical readingo the manuscript.

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Improving the Sensitivity of Bacterial Bioreporters

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