review article recent advances in genetic technique of microbial report...

Review ArticleRecent Advances in Genetic Technique of Microbial ReportCells and Their Applications in Cell Arrays

Do Hyun Kim1 Moon Il Kim1 and Hyun Gyu Park2

1Department of Bionano Technology Gachon University Gyeonggi 461-701 Republic of Korea2Department of Chemical and Biomolecular Engineering (BK21+ Program) Korea Advanced Institute of Science andTechnology (KAIST) Daejeon 305-701 Republic of Korea

Correspondence should be addressed to Moon Il Kim moonilgachonackr and Hyun Gyu Park hgparkkaistackr

Received 23 April 2015 Accepted 26 August 2015

Academic Editor Yun-Peng Chao

Copyright copy 2015 Do Hyun Kim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Microbial cell arrays have attracted consistent attention for their ability to provide unique global data on target analytes at lowcost their capacity for readily detectable and robust cell growth in diverse environments their high degree of convenience andtheir capacity for multiplexing via incorporation of molecularly tailored reporter cells To highlight recent progress in the field ofmicrobial cell arrays this review discusses research on genetic engineering of reporter cells technologies for patterning live cells onsolid surfaces cellular immobilization in different polymers and studies on their application in environmental monitoring diseasediagnostics and other related fields On the basis of these results we discuss current challenges and future prospects for novelmicrobial cell arrays which show promise for use as potent tools for unraveling complex biological processes

1 Introduction

Biosensors which have been widely utilized for the detectionof target molecules are increasingly important owing totheir broad applications in biotechnology and related fieldsincluding disease diagnostics environmental monitoringdrug discovery and food processing [1] Biosensor is oper-ated mainly based on the specificity of biologically activemolecules including nucleic acids enzymes antibodies andantigens receptors and cells and thus these substances arefundamental for the specific recognition of target molecules

Among biosensors cell-based or whole-cell biosensorshave garnered particular interest because they can provideunique data on the global activities of test samples such astheir toxicity genotoxicity or bioavailability from a directassay on live cells [2] The use of live cells also allowsfor reagent-free nondestructive real-time monitoring of thebiological effects as they develop with no need for thepre- or posttreatment steps that are generally required forconventional chemistry-based analyticalmethodsThereforethe use of whole cells as sensing entities can convenientlyprovide a diverse array of data on integrated biological effects

that cannot be achieved using other biosensors although therelative insufficiency of their specificity is inevitable based onthe nature of living systems

Previous cell-based biosensors typically used a singletype cells with a single function to analyze a single samplehowever recent approaches have focused on the developmentof arrays comprised of multiple cells on a mapped solidsurface that are subsequently exposed to mixtures containingmulticomponent analytes which can provide multiplexedoutput signals corresponding to the amount of each targetin the sample mixture [3] Compared to singleplex cell-based biosensors cell arrays enable simultaneous detectionof multiple samples with multiple output signals that can beused to rapidly analyze large numbers of samples Due to theincreased need for multiplex high-throughput analysis capa-bility cell array techniques have recently garnered significantattention [4]

Diverse types of cell arrays have been reported In partic-ular human cells have been extensively employed to developminiaturized cell-based assay platforms which includemicrofluidic and microarray biochips to mimic humanmetabolism [5ndash9]These biochips have been extensively used

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 182107 8 pageshttpdxdoiorg1011552015182107

2 BioMed Research International

Electrochemical

Amperometry

Optical

Digitalsignal

Environmental monitoring

Others

Disease diagnostics

Fluorescent

Bioluminescent

Molecular recognition Transducer Signal processing Application

Colorimetric

Figure 1 A schematic representation of a microbial cell array

in drug discovery to evaluate toxicity and other metabolicactivities during adsorption distribution metabolism andelimination of drug candidates in the human body [5] Othereukaryotic cells including yeast have also been examinedin applications for gene function analysis microphysiometryand therapeutic agent identification based on array platformsprepared using diverse microfabrication strategies such asphotolithography inkjet printing or microcontact printing[10ndash17] Due to the critical need for high-throughput meth-ods for investigating the bioactivity of eukaryotic cells cellarrays based on eukaryotic cells have come into widespreaduse

In contrast arrays using prokaryotic cells have numerousdifferentiated benefits [18] It is easy to grow and to maintainthe viability of prokaryotic cells at low cost large andhomogeneous populations are easily obtainable they arerobust to a variety of physical and chemical environmentsand they show low susceptibility to biological contaminationFurthermore prokaryotic cells are amenable to physicalor chemical manipulation particularly those required forpatterning in an array format Perhaps their most importantcharacteristic based on recent developments in genetic engi-neering technology is that prokaryotic cells can be molec-ularly engineered to respond in a dose-dependent mannerto yield readily quantifiable optical (colorimetric fluorescentor luminescent) or electrochemical signals to predeterminedtargets such as chemicals biomolecules or biological effects[3] This is generally achieved by the fusion of a sensingelement a selective promoter along with its regulatory ele-ments to a suitable molecular reporter system Moreoveradvances in genetic engineering also allow the expression oftwo independent reporter systems in a singlemicroorganismfacilitating multiplex analysis and particular logic operationsof microbial cell arrays [19ndash21] These genetically engineeredsensor cells are patterned on a solid surface incorporated intoa single hardware platform and simultaneously exposed to asample for applications such as environmental monitoringdisease diagnostics and others (Figure 1) Due to the signif-icance and widespread applicability of this state-of-the-arttechnology it has garnered increasing public attention anda reasonable research direction must be set to widely expandits utilization for both laboratory and field use

In this paper we review recent advances in microbial cellarrays Recent research into genetic engineering of reportercells technologies for patterning live cells on solid surfacesand their immobilization in different polymers are exten-sively discussed alongwith studies of related applicationsWepresent the current challenges and future prospects for novelmicrobial cell arrays which can be used as potent tools forunraveling complex biological processes

2 Genetic Engineering ofMicrobial Reporter Cells

Although unmodified bacteria have been used as biosensorsbased on changes in natural bioluminescence as cells growgenetic engineering ofmicrobial cells is extensively employedto rationally produce dose-dependent signals to predeter-mined environmental stimuli [22 23] Typical engineeringmethods include fusion of a reporter gene system to promot-ers from selected stress-response regions resulting in specificcell growth and easily measurable signals that are propor-tional to the quantity of target analytes including chemi-cals nutrients or heavy metals To date fluorescence- andluminescence-based signals have typically been produced inmicrobial cell arrays by the activity of corresponding reportergenes that express fluorescent protein and bacterial luciferase(lux) respectively [24 25] In recent years several attemptshave beenmade to improve the performance of reporter cellssuch as further engineering of regulatory regions splitting ofthe lux operon increasing cellular permeability or shufflingof gene elements [26ndash31]

Recently another class of genetic engineering based onconstruction of mutant bacteria with auxotrophic charac-teristics has garnered attention owing to its capacity tospecifically and sensitively detect diverse types of metabolitespresent in themetabolic pathways of themicrobial cells on thearray [32 33] Several different strategies such as transposonor N-methyl-N1015840-nitro-N-nitrosoguanidine- (NTG-) inducedmutagenesis and chromosomal gene deletion based on linearcassettes have been employed to prepare auxotrophic bacte-ria [34 35] Bioluminescence-producing firefly luciferase orfluorescent protein reporter genes have also been used to pro-duce corresponding optical responses that are proportional to

BioMed Research International 3

the concentrations of target analytes These cell arrays wereproven to enable rapid (lt4 h) and simultaneous analysis ofmultiple targets from complex biological fluids [33 36]

As described above the signals generated by the arrayshave primarily been produced by proteins or by the activity ofan enzyme expressed using a reporter gene system Depend-ing on the type of reporter gene the signals emitted havebeen detected optically or electrochemically Additional sig-naling methods based on commercially available LiveDeadstaining or surface plasmon resonance analysis have alsobeen reported for diversification of cell array detectionmechanisms [37 38]

3 Patterning Microbial Cells on Solid Surfaces

In addition to cell arrays patterned in the wells of premademicrotiter plates target microbial cells can be patternedon a solid surface to maximize the number of cell spotsper unit area while enabling the activity of each spot tobe distinguished from that of its neighbors without cross-contamination Diverse microfabrication strategies such asphotolithography soft lithography and noncontact printinghave been employed to prepare patterned cell arrays onnumerous materials such as silicon glass various polymersand gold [38ndash44]

Photography-based processes have been widely appliedto prepare patterns of immobilized bacterial cells Typicallya water-soluble photoresist polymer is employed for thefabrication of a three-dimensional matrix on the desiredregion by simple exposure toUV light resulting in accommo-dation of both target cells and culture medium in the matrixUsing this strategy silicon chips consisting of microfluidicchannels microchambers valves and additional structureshave successfully been prepared for toxicity monitoringbased on generation ofEscherichia colimicrospots on a planararray [40]

Microcontact printing one of the most conventionallyused soft lithography methods to prepare patterns witha chemical moiety has been employed to create cellularpatterns on both planar and nonplanar surfaces by deliveringanchor molecules using a polydimethylsiloxane (PDMS)stamp [45] Using this stamp self-assembled monolayerswhich can adsorb to patterned gold surfaces form covalentbonds with a protein that guides a cell to the pattern Usingthis strategy high-resolution printing of massive arrays ofvarious microorganisms such as Lactobacillus plantarumE coli Candida albicans and fungal spores of Aspergillusfumigatus has been reported on porous aluminum oxide[46] Another bacterial array based on a combination ofself-assembled polyelectrolyte multilayers and micromoldedpoly(ethylene glycol)-poly(lactide) diblock copolymers topromote target cell adhesion has also been reported [47]

As an example of noncontact printing piezoelectric inkjetprinters have been used to prepare high-density live cellarrays for screening antimicrobial activity [48] Flickinger etal reported the formulation of reactivemicrobial inks and theuse of piezo tips to spot E coli at designated positions [49] Anoncontact robotic printer was also employed to prepare E

coli arrays with several nanoliter-volume spots on chemicallymodified glass [50]

4 Maintenance of Cell Viability

For practical application of microbial cell arrays cells on thearray should maintain their viability and be able to be storedfor sufficiently long periods of time Thus development ofefficient solid-phase arrays by appropriate immobilizationof cells has garnered consistent attention particularly inindustry Various polymers such as agar agarose alginatecollagen latex polyacrylamide polyethylene glycol diacry-late and carrageenan as well as freezevacuum drying havebeen reported to immobilize cells while retaining sufficientviability [25 33 43 49 51ndash56] In particular further additionof components such as glycerol or trehalose was shown toeffectively provide extracellular or intracellular protectionand thus to enhance the long-term survival rate [50 57]Vacuum drying of As(III) reporter bacteria in the presenceof 34 trehalose and 15 polyvinylpyrrolidone resulted invery effective preservation of initial activity during up to 12weeks of storage at 4∘C [58] An innovative strategy based onthe formation of bacterial spores was also reported for long-term (up to 2 years) preservation of sensing cells at roomtemperature [59]

5 Applications of Microbial Cell Arrays

Based on their abovementioned characteristicsmicrobial cellarrays have been used in diverse applications for monitoringthe global effects of test samples as shown briefly in Table 1In this section we describe recent studies of the applicationof microbial cell arrays categorized by environmental moni-toring disease diagnostics and others

51 Environmental Monitoring Although the envisagedapplications of microbial cell array are numerous they haveprimary been applied in the environmental field Microbialcells have been elaborately modified to produce bothqualitative and quantitative outputs in response to singleor multiple kinds of environmental stimuli and applied toconstruct cell arrays to analyze multiple test samples Dueto their capacity to show the unique responses of live cellsmicrobial cell arrays can serve as a potent analytical route toreplace the conventional yet laborious methods currently inuse

Several microbial cell arrays have been developed forassaying heavy metals which are regarded as major toxicelements Biran et al reported a microbial cell array thatemploys a genetically engineered E coli strain that containsthe lacZ reporter gene which can express 120573-galactosidasefused to a the promoter of a heavy metal-responsive geneA plasmid carrying the gene coding for the enhanced cyanfluorescent protein was subsequently introduced into thissensing strain to produce concomitant optical signals inproportion to the quantity of the target heavymetal mercuryLevels as low as 100 nMHg2+ could be detected after only1 h of incubation [60] Arsenic and cadmium were alsosimultaneously quantified via a multichannel bioluminescent


Table 1 Recent applications of microbial cell arrays

Application field Target Microorganism Detectable output References

Environmentalmonitoring Mercury Recombinant Escherichia coli Fluorescence [60]

Environmentalmonitoring

Cadmium and arsenicIII

Recombinant E coli Bioluminescence [61]


Silver and titaniumoxide nanoparticles

Recombinant E coli Gene expression profile [62]

Environmentalmonitoring Endocrine disruptors Recombinant yeast and E coli Bioluminescence [53]

Environmentalmonitoring Cell-damaging stress Recombinant E coli Bioluminescence [63]

Environmentalmonitoring Naphthenic acid Recombinant E coli Fluorescence [64]


Paraquat mitomycinC and salicylic acid


Disease diagnostics 16 amino acids Recombinant E coli auxotroph Bioluminescence [33]

Disease diagnostics Homocysteine Recombinant E coli auxotroph Bioluminescence [65]

Disease diagnostics Galactose Recombinant E coli auxotroph Bioluminescence [66]

Disease diagnosticsPhenylalaninemethionine and

leucineRecombinant E coli auxotroph Fluorescence [36]

Carbohydratesdetection

Mono- anddisaccharides

Recombinant E coli O2reduction [55]

Gene expressionanalysis

Growth of E colicolonies


Screening antibiotics Antibiotic activity Staphylococcus aureus Bioluminescence [68]Screeningpharmaceuticals 420 pharmaceuticals Recombinant E coli Bioluminescence [69]

E coli array system although cross-reactivity was observedwhen the two metals were mixed [61]

Other environmental pollutants have also been moni-tored using microbial cell arrays Gou et al utilized a greenfluorescent protein-fused recombinant E coli array to assessthemechanistic toxicity of silver and titaniumoxide nanopar-ticles by measuring real-time gene expression profiles [62]A portable biosensor device based on engineered yeast andbacterial cells fused to a reporter gene expressing luciferasewas reported to be able to detect several endocrine disruptorsincluding androgens and estrogens [53] Ahn et al reportedan E coli array consisting of optically coded functionalmicrobeads containing both bioluminescent reporter bacte-rial cells and fluorescent microspheres for broad-range toxic-ity monitoring [63] A bacterial cell array using recombinantE coli in 384-well plates was also employed in a genome-wide investigation of the toxic mechanisms of naphthenicacids chemicals that pose serious environmental hazards andwhich are present in effluents from petrochemical processing[64] Three different chemicals that cause either superoxidedamage (paraquat) DNA damage (mitomycin C) or pro-teinmembrane damage (salicylic acid) were also successfullydetected within 2 h using a bacterial cell array based onbioluminescent E coli [25]

52 Disease Diagnostics Recently cell-based assays employ-ing fast-growing auxotrophic bacteria supplemented withbioluminescent or fluorescent reporter genes have beenshown to rapidly conveniently and simultaneously detectmultiple target molecules relevant to human diseases In con-trast to conventional diagnosticmethods which often requirenumerous experimental steps or complicated and expen-sive instrumentation bacterial auxotroph-based arrays showrapid specific and sensitive cell growth in direct responseto the concentration of the corresponding molecules Thismethod can also be extended to evaluate or monitor nutri-tional conditions as themetabolic pathways ofmicrobial cellscontain many relevant metabolites

Several cell-based approaches to diagnosis of humandiseases have been reported A multiplexed amino acid arrayfor simultaneously quantifying 16 different amino acids basedon the rapid and specific growth of amino acid-auxotrophicE coli was reported [33] Using this array multiple aminoacids in biological fluids were quantitatively determinedwithin 4 h simply by measuring bioluminescent signals fromimmobilized cells without any pre- or posttreatment Usingthis system two different kinds of metabolic diseases ofnewborn babies phenylketonuria and homocystinuria weresuccessfully diagnosed by measuring luminescence values


from phenylalanine and methionine auxotrophs incubatedwith an eluted mixture from clinical blood paper specimensSimilarly homocysteine an important marker for cardio-vascular disease and other syndromes such as Alzheimerrsquosand Parkinsonrsquos disease neural tube defects pregnancy com-plications and osteoporosis was quantified by employinganother bioluminescent E coli array which showed highspecificity sensitivity and excellent levels of precision andreproducibility [65] Galactosemia a major metabolic disor-der of newborns was also successfully diagnosed by employ-ing galT-knockout E coli [66] Furthermore simultaneousquantification of multiple amino acids in a single biologicalsample was reported and applied in themultiplexed diagnosisof three key metabolic diseases of newborn babies [36] Theassay utilized three E coli auxotrophs that grow only inthe presence of the corresponding target amino acids andcontain three different fluorescent reporter plasmids thatproduce distinguishable fluorescence signals (red green andcyan) in concert with cell growthThe three auxotrophs weremixed and immobilized in the same well of a 96-well plateand consequently yielded three different fluorescence signalsthat corresponded to the reporter plasmids The clinicalutility of this assay system was demonstrated by employingit to identify metabolic diseases of newborns through thequantification of phenylalanine methionine and leucine inclinically derived dried blood specimens

53 Others Based on their unique advantages the applica-tions for microbial cell arrays are currently being expandedHeld et al reported a bacterial cell array with an automatedflow-injection system for the selective and simultaneousdetermination of various mono- and disaccharides [55] Theselectivity of the array was achieved by combination ofthe metabolic responses of E coli mutants lacking differenttransport systems for individual carbohydrates The arrayenabled simultaneous detection of three major sugars fruc-tose glucose and sucrose in test samples A unique arrayof bacterial colonies has been reported for large-scale geneexpression analysis [67] In this system recombinant E coliclones containing plasmid-encoded copies of several thou-sand individually expressed sequence tags were spotted andincubated for sim6 h to allow bacterial growth and consequentamplification of the cloned tags For use in drug discoveryan array of Staphylococcus aureus fused with lux (luciferase-producing) plasmids was reported for screening antibioticactivity [68] Finally a panel of 15 bioluminescent E colicontaining multiple bacterial reporter genes associated withoxidative stressDNAdamage heat shock and effluxof excessmetals was arrayed to screen a library of 420 pharmaceuticals[69] This work demonstrated that microbial cell arrays canplay a significant role in drug development alongside in vitrotoxicity tests

6 Conclusion and Future Prospects

As described above microbial cell arrays have been widelyinvestigated and have garnered significant attention as apotent analytical paradigm due to their capacity to provideunique global data for live systems The arrays provide

Table 2 Representative advantages and challenges of microbial cellarrays

Advantages Challenges(1) Analysis of globalactivities of target analyte

(1) Limited viability andbiological function

(2) Low cost (2) Insufficient specificity(3) Analysis is moreconvenient than existingtechnologies

(3) Types of target analytes arelimited

(4) Robust to reactionenvironments

(4) Genetic stability ofengineered reporter cell system islow

(5) Simultaneous detectionof multiple analytes

(5) Laws limiting the use ofgenetically modified organisms

(6) Real-time in situmonitoring

(6) Slow diffusion in cellmembranes

the option which was previously unavailable of analyzingbiological reactions via real-timemonitoring of the responsesof an unlimited number of genetically tailored sensor strainswhich provide easily measurable dose-dependent optical orelectrical signals within a short period of time Howeverseveral challenges that significantly hinder the widespreadutilization of microbial cell arrays remain such as their lim-ited viability and biological function after long-term storageinsufficient specificity limited types of target analytes andproblems in genetic engineering of sensor strains (Table 2)However significant progress to overcome these limitations iscontinuously beingmade as shown in someof the approachesreviewed here and microbial cell arrays show great promisefor an increasing number of applications in diverse fields suchas environmental monitoring disease diagnostics and drugdiscovery

For microbial cell arrays to be positioned as a next-generation analytical tool the following technological hur-dles must be overcome before the technology maturesPossibly the most urgent need for practical applications isa dramatic improvement in the maintenance of cell activityand viability over prolonged periods of time Many differ-ent approaches have been suggested such as appropriateimmobilization of cells or addition of particular additives toreduce stress factors however other innovative methods tosignificantly extend the shelf life of arrays are required partic-ularly for commercialization in industry In addition furtherengineering of reporter cells for higher specificity sensitivityand robustness or better methodologies for incorporation ofsuch cells into hardware platforms will also greatly contributeto the widespread utilization of this technology When theymature microbial cell arrays may become an efficient andpractical analytical tool in diverse biotechnological fields

In summary this paper highlights recent progress inthe field of microbial cell arrays Although the relativeinsufficiency of their specificity is inevitable based on thenature of living systems microbial cell arrays can provideunique data on the global activity of test samples with a rangeof advantages that are not achievable using other analyticalmethods Diverse technological advances have provided the


tools materials and elaborately engineered reporter cellsneeded to construct highly integrated arrays Based on theunique advantages and continued progress in this field webelieve that microbial cell arrays will lead a newwave of noveldiagnostic methods in environmental monitoring diseasediagnostics drug screening and other related fields

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) funded by theMinistry of Science ICTamp Future Plan-ning (NRF-2014R1A1A1006016) and by the Gachon Univer-sityGilMedical Center (Grant no 2014-22)This researchwasalso supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) funded bythe Ministry of Education [no 2015R1A2A1A01005393]

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[53] A Roda L Cevenini E Michelini and B R Branchini ldquoAportable bioluminescence engineered cell-based biosensor foron-site applicationsrdquo Biosensors amp Bioelectronics vol 26 no 8pp 3647ndash3653 2011

[54] D O Fesenko T V Nasedkina D V Prokopenko and A DMirzabekov ldquoBiosensing and monitoring of cell populationsusing the hydrogel bacterial microchiprdquo Biosensors amp Bioelec-tronics vol 20 no 9 pp 1860ndash1865 2005

[55] MHeldW SchuhmannK Jahreis andH-L Schmidt ldquoMicro-bial biosensor array with transport mutants of Escherichiacoli K12 for the simultaneous determination of mono-anddisaccharidesrdquo Biosensors and Bioelectronics vol 17 no 11-12pp 1089ndash1094 2002

[56] R Pedahzur R Rosen and S Belkin ldquoStabilization of recom-binant bioluminescent bacteria for biosensor applicationsrdquo CellPreservation Technology vol 2 no 4 pp 260ndash269 2004

[57] B Kempf and E Bremer ldquoUptake and synthesis of compatiblesolutes as microbial stress responses to high-osmolality envi-ronmentsrdquo Archives of Microbiology vol 170 no 5 pp 319ndash3301998

[58] A Kuppardt A Chatzinotas U Breuer J R van der Meer andH Harms ldquoOptimization of preservation conditions of As (III)bioreporter bacteriardquo Applied Microbiology and Biotechnologyvol 82 no 4 pp 785ndash792 2009

[59] A Date P Pasini and S Daunert ldquoFluorescent and biolumi-nescent cell-based sensors strategies for their preservationrdquo


in Whole Cell Sensing Systems I vol 117 of Advances in Bio-chemical EngineeringBiotechnology pp 57ndash75 Springer BerlinGermany 2010

[60] I Biran D M Rissin E Z Ron and D R Walt ldquoOptical imag-ing fiber-based live bacterial cell array biosensorrdquo AnalyticalBiochemistry vol 315 no 1 pp 106ndash113 2003

[61] T Charrier C Chapeau L Bendria P Picart P Daniel and GThouand ldquoA multi-channel bioluminescent bacterial biosensorfor the on-line detection ofmetals and toxicity Part II technicaldevelopment and proof of concept of the biosensorrdquo Analyticaland Bioanalytical Chemistry vol 400 no 4 pp 1061ndash1070 2011

[62] N Gou A Onnis-Hayden and A Z Gu ldquoMechanistic toxicityassessment of nanomaterials by whole-cell-array stress genesexpression analysisrdquo Environmental Science amp Technology vol44 no 15 pp 5964ndash5970 2010

[63] J-M Ahn J H Kim J H Kim and M B Gu ldquoRandomlydistributed arrays of optically coded functional microbeads fortoxicity screening and monitoringrdquo Lab on a Chip vol 10 no20 pp 2695ndash2701 2010

[64] X Zhang S Wiseman H Yu H Liu J P Giesy andM HeckerldquoAssessing the toxicity of naphthenic acids using a microbialgenome wide live cell reporter array systemrdquo EnvironmentalScience and Technology vol 45 no 5 pp 1984ndash1991 2011

[65] M-A Woo M I Kim B J Yu et al ldquoCell-based quantifica-tion of homocysteine utilizing bioluminescent Escherichia coliauxotrophsrdquoAnalytical Chemistry vol 83 no 8 pp 3089ndash30952011

[66] M-A Woo M I Kim D Cho and H G Park ldquoCell-basedgalactosemia diagnosis system based on a galactose assay usinga bioluminescent Escherichia coli arrayrdquo Analytical Chemistryvol 85 no 22 pp 11083ndash11089 2013

[67] C Barsalobres-Cavallari V De Rosa F Nogueira et al ldquoAnovel system for large-scale gene expression analysis bacterialcolonies arrayrdquo Applied Microbiology and Biotechnology vol 71no 6 pp 963ndash969 2006

[68] L RMesak S Qi I Villanueva VMiao and J Davies ldquoStaphy-lococcus aureus promoter-lux reporters for drug discoveryrdquoJournal of Antibiotics vol 63 no 8 pp 492ndash498 2010

[69] T Elad H B Seo S Belkin and M B Gu ldquoHigh-throughputprescreening of pharmaceuticals using a genome-wide bacterialbioreporter arrayrdquo Biosensors amp Bioelectronics vol 68 pp 699ndash704 2015

Submit your manuscripts athttpwwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


Electrochemical

Amperometry

Optical

Digitalsignal

Environmental monitoring

Others

Disease diagnostics

Fluorescent

Bioluminescent

Molecular recognition Transducer Signal processing Application

Colorimetric

Figure 1 A schematic representation of a microbial cell array

in drug discovery to evaluate toxicity and other metabolicactivities during adsorption distribution metabolism andelimination of drug candidates in the human body [5] Othereukaryotic cells including yeast have also been examinedin applications for gene function analysis microphysiometryand therapeutic agent identification based on array platformsprepared using diverse microfabrication strategies such asphotolithography inkjet printing or microcontact printing[10ndash17] Due to the critical need for high-throughput meth-ods for investigating the bioactivity of eukaryotic cells cellarrays based on eukaryotic cells have come into widespreaduse

In contrast arrays using prokaryotic cells have numerousdifferentiated benefits [18] It is easy to grow and to maintainthe viability of prokaryotic cells at low cost large andhomogeneous populations are easily obtainable they arerobust to a variety of physical and chemical environmentsand they show low susceptibility to biological contaminationFurthermore prokaryotic cells are amenable to physicalor chemical manipulation particularly those required forpatterning in an array format Perhaps their most importantcharacteristic based on recent developments in genetic engi-neering technology is that prokaryotic cells can be molec-ularly engineered to respond in a dose-dependent mannerto yield readily quantifiable optical (colorimetric fluorescentor luminescent) or electrochemical signals to predeterminedtargets such as chemicals biomolecules or biological effects[3] This is generally achieved by the fusion of a sensingelement a selective promoter along with its regulatory ele-ments to a suitable molecular reporter system Moreoveradvances in genetic engineering also allow the expression oftwo independent reporter systems in a singlemicroorganismfacilitating multiplex analysis and particular logic operationsof microbial cell arrays [19ndash21] These genetically engineeredsensor cells are patterned on a solid surface incorporated intoa single hardware platform and simultaneously exposed to asample for applications such as environmental monitoringdisease diagnostics and others (Figure 1) Due to the signif-icance and widespread applicability of this state-of-the-arttechnology it has garnered increasing public attention anda reasonable research direction must be set to widely expandits utilization for both laboratory and field use

In this paper we review recent advances in microbial cellarrays Recent research into genetic engineering of reportercells technologies for patterning live cells on solid surfacesand their immobilization in different polymers are exten-sively discussed alongwith studies of related applicationsWepresent the current challenges and future prospects for novelmicrobial cell arrays which can be used as potent tools forunraveling complex biological processes

2 Genetic Engineering ofMicrobial Reporter Cells

Although unmodified bacteria have been used as biosensorsbased on changes in natural bioluminescence as cells growgenetic engineering ofmicrobial cells is extensively employedto rationally produce dose-dependent signals to predeter-mined environmental stimuli [22 23] Typical engineeringmethods include fusion of a reporter gene system to promot-ers from selected stress-response regions resulting in specificcell growth and easily measurable signals that are propor-tional to the quantity of target analytes including chemi-cals nutrients or heavy metals To date fluorescence- andluminescence-based signals have typically been produced inmicrobial cell arrays by the activity of corresponding reportergenes that express fluorescent protein and bacterial luciferase(lux) respectively [24 25] In recent years several attemptshave beenmade to improve the performance of reporter cellssuch as further engineering of regulatory regions splitting ofthe lux operon increasing cellular permeability or shufflingof gene elements [26ndash31]

Recently another class of genetic engineering based onconstruction of mutant bacteria with auxotrophic charac-teristics has garnered attention owing to its capacity tospecifically and sensitively detect diverse types of metabolitespresent in themetabolic pathways of themicrobial cells on thearray [32 33] Several different strategies such as transposonor N-methyl-N1015840-nitro-N-nitrosoguanidine- (NTG-) inducedmutagenesis and chromosomal gene deletion based on linearcassettes have been employed to prepare auxotrophic bacte-ria [34 35] Bioluminescence-producing firefly luciferase orfluorescent protein reporter genes have also been used to pro-duce corresponding optical responses that are proportional to







































































Acknowledgments


References













































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







































































Acknowledgments


References













































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

review article recent advances in genetic technique of microbial report...

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