Bacteriophage-based synthetic biology for the study of infectious diseases

for the study of infectious diseases

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Citation

Citorik, Robert J, Mark Mimee, and Timothy K Lu.

“Bacteriophage-Based Synthetic Biology for the Study of Infectious Diseases.”

Current Opinion in Microbiology 19 (June 2014): 59–69.

As Published

http://dx.doi.org/10.1016/j.mib.2014.05.022

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Elsevier

Version

Final published version

Citable link

http://hdl.handle.net/1721.1/90322

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Article is available under a Creative Commons license; see

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Bacteriophage-based

synthetic

biology

for

the

study

of

infectious

diseases

Robert

J

Citorik

,

Mark

Mimee

and

Timothy

K

Lu

Sincetheirdiscovery,bacteriophageshavecontributed enormouslytoourunderstandingofmolecularbiologyas modelsystems.Furthermore,bacteriophageshaveprovided manytoolsthathaveadvancedthefieldsofgenetic engineeringandsyntheticbiology.Here,wediscuss

bacteriophage-basedtechnologiesandtheirapplicationtothe studyofinfectiousdiseases.Newstrategiesforengineering genomeshavethepotentialtoacceleratethedesignofnovel phagesastherapies,diagnostics,andtools.Thoughalmosta centuryhaselapsedsincetheirdiscovery,bacteriophages continuetohaveamajorimpactonmodernbiological sciences,especiallywiththegrowthofmultidrug-resistant bacteriaandinterestinthemicrobiome.

Addresses

1_{MITMicrobiologyProgram,77MassachusettsAvenue,Cambridge,MA}

02139,USA

2_{MITSyntheticBiologyCenter,500TechnologySquare,Cambridge,MA}

02139,USA

3_{DepartmentofBiologicalEngineering,MassachusettsInstituteof}

Technology,Cambridge,MA,USA

4

DepartmentofElectricalEngineeringandComputerScience, MassachusettsInstituteofTechnology,Cambridge,MA,USA

5_{Theseauthorscontributedequallytothiswork.}

Correspondingauthor:Lu,TimothyK([email protected])

CurrentOpinioninMicrobiology2014,19:59–69

ThisreviewcomesfromathemedissueonNoveltechnologiesin microbiology

EditedbyEmmanuelleCharpentierandLucianoMarraffini

ForacompleteoverviewseetheIssueandtheEditorial

Availableonline3rdJuly2014

http://dx.doi.org/10.1016/j.mib.2014.05.022

1369-5274/#2014TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-SAlicense( http://creative-commons.org/licenses/by-nc-sa/3.0/).

Background

With the discovery of bacteriophages generally being credited to FrederickTwort[1]andFelixd’He´relle[2] in the early 20th century, these virusparticles were so named(Greek,‘bacteriaeaters’)basedontheirobserved abilitytolysebacterialcells.Theuseofnaturally occur-ringphagesastherapeuticsforthetreatmentofbacterial infections wasquicklyrealizedbyd’He´relleandothers, withinterestcontinuingtoflourishuntilthediscoveryand productionofpenicillin[2–4].Antibioticsheraldedanew age of effectivesmall molecule treatments forbacterial infections,withphagetherapyfallingoutoffavorinthe

WesternWorld [5,6].Though phageshaveremainedan importanttoolinthestudyofmolecularbiology,genetics, andbacteria[7],concernsovertheever-dwindlingarsenal of antibiotics for the treatment of multidrug-resistant bacterial pathogens have also resulted in a renaissance inphagestudiesandinphage-basedtherapiesasameans to developalternative therapeutics [8–11]. Correspond-ingly, advances in synthetic biology have refined the ability to design, modify, and synthesize these viruses, whichhasenablednovelstrategiesforcreating bacterio-phage-basedtools for thestudyandtreatment of infec-tiousdiseases. Thegoalof this reviewis toexplore the methodsanddemonstrationsbywhichsuchtoolscanbe employed to engineermodified phage andphage parts. For more information concerning the history, appli-cations, and challenges of phage therapy using natural, unmodifiedviruses,thereaderisreferredtootherreviews [12–15].

Synthetic biology aims to rationallyengineer new func-tionalities in livingsystems byco-opting andmodifying biomoleculescraftedfrommillenniaofevolution[16–18]. Cells operate as highly complex computationalsystems abletodynamicallyinterrogateandrespondtotheir envi-ronment.Forthepastdecade,syntheticbiologistshavelaid thefoundationalrulesofbiologicaldesign[19],constructed a catalog of standardized genetic parts, and assembled simple circuits, such as oscillators [20], switches [21], and Boolean logicgates[22,23]. Rationalengineering has yieldedcellulardevicesableto producepotent anti-malarial compounds [24], to detect and kill pathogenic bacteria [25]andcancercells[26],to reprogramcellfate [27], and to treat metabolic syndrome [28]. Finally, advancements in DNA synthesis and assembly have enabled the rapid development of higher-order genetic circuits [29–31] of medical [32] and industrial [33,34] relevance. This field has been accelerated by phage-derivedtechnologies,whileconcomitantlyenablingnew approachestoengineeringphagesthemselves.

Phage-enabled

technologies

Phagedisplay

Described by Smith in 1985 [35], phage display is a methodology employed extensively in both the study of infectious diseases and the development of novel therapeutics.Librariescomprisingsyntheticrandom pep-tidesornaturalpeptidesderivedfrompathogengenomic orcDNAarefusedwithacoatproteinofabacteriophage, oftenM13,Fd,orl,suchthatthepeptideisdisplayedon thephagesurface.Iterativeselectionstepsareemployed

toenrichforphageparticlesthatbindwithhighaffinityto an immobilized target molecule of interest, which are thenelutedandpropagatedinEscherichiacoli.Sincethe identityofthedisplayedpeptideisgeneticallyencoded inthephagegenome,protein–ligandinteractionscanbe screened inhigh-throughput to identifymolecules with novelbiologicalfunctions.Phagedisplayhasenabledthe discovery and characterization of bacterial adhesins [36,37], which bind to receptors on hostcells or extra-cellularmatrixand are implicatedinestablishing infec-tion,as well as antigens used for vaccine development [38]. Moreover, bioactive peptides that block anthrax toxin binding [39] or inhibit cell wall biosynthesis enzymes in Pseudomonas aeruginosa [40] were isolated fromphagedisplay libraries.Development of antibody-based therapeutics has also greatly benefited from the technology,whichcanbeimplementedtorapidlyscreen random libraries of antigen-binding domains [41]. In a demonstration of direct therapeutic application, phage particles selectedfor high affinity interactionto Staphy-lococcusaureus wereconjugatedto chloramphenicol pro-drugs to deliver localized, lethal payloads [42]. The applicationsof phage display are vastand the readeris referredto other literature [43–48] for amore thorough discussionofadditionalexamples.

Bacteriophage-derivedpartsforsyntheticbiology

Bacteriophageshave formedthebackbone of molecular biology,having championedthedemonstration ofDNA as geneticmaterial[49],theproof of Darwiniannatural selection[50],andthe ubiquitoususe of phage-derived enzymesforcommonlaboratoryprotocols[51].Similarly, bacteriophagecomponentsconstituteacoresetofpartsin

thetoolboxofasyntheticbiologist.TheDNA-dependent RNApolymeraseof T7canspecificallydrive high-level transcriptionfromtheT7promoter(PT7)bothinvitroand

invivo.Thepolymerasehasbeenusedtoreconstitutein vitro genetic circuits [52], such as switches [53] and oscillators[54],whichpermitprecisemathematical mod-elingof biological reactions to inform future predictive design. Moreover, libraries of orthogonal T7 and PT7

variants,whichexhibitlowertoxicity[55]oraresplitinto partstofunction asANDgates [56] (Figure1aandb), havebeenconstructedto permithigher-order construc-tionof artificial geneticcircuits. For example,an AND gatethatonlyoutputsaBooleanTRUEvaluewhenboth inputs are TRUE can be implemented by having an output gene that is only expressed when both parts of asplitT7RNApolymeraseareexpressed.Whencoupled with orthogonal ribosomes that do not translate host mRNAs, a fully insulated transcription-translation net-workwasconstructedinE.coliforproteinexpression[57]. Furthermore, bacteriophage recombinases have been used in the construction of genetic circuits that record memoryofpastreactions[58](Figure1c).Recombinases manipulateDNAbyrecognizingspecificsequences and catalyzingtheexcision,integration,orinversionofDNA segmentsdepending on thelocation and orientation of the recognition sites. Thus, recombinase expression is coupledwithaphysicalchangeingeneticmaterialofthe cell that can be sequenced to assay exposure to past events. The Cre, Bxb1, and PhiC31 recombinases have beenusedintheconstructionofavarietyofsyntheticcircuits including counters [59], a rewritable memory module [60], Boolean logic gates [22,23], and digital-to-analog

Figure1 A (a) (b) (c) A 0 0 0 1 0 0 0 1 0 1 1 1 B B OUT OUT Input A T7-NTerm T7-CTerm Output Output bxb1 phiC31 Input B Input A Input B

Current Opinion in Microbiology

Bacteriophage-derivedpartscanperformmolecularcomputationinmicrobialcells.(a)TruthtableforANDgatefunction.AnANDgatehasaTRUE outputonlywhenbothinputsareTRUE.(b)SplitT7RNApolymerasecanactasatranscriptionalANDgate.InputsAandB,whichcanbeexogenous signalssuchassmall-moleculeinducersorendogenoussignals,controlexpressionoftheN-terminalandC-terminalhalvesofT7RNApolymerase, respectively,suchthatsimultaneousexpressionleadstoareconstitutedpolymerasethatcandriveproductionoftheoutputgene[56_].(c)

Bacteriophage-derivedrecombinasesBxb1andPhiC31implementingANDgatefunctionalitywithintegratedmemory.Recombinaseactivityleadsto inversionoftheinterveningterminatorsequenceflankedbyrecognitionsites.ActivationofBxb1andPhiC31leadstoinversionoftwounidirectional terminators,suchthatRNApolymerasecandriveexpressionoftheoutputgene[22].

converters[22].Sincetheserecombinasesdonotrequire additional host factors, genetic circuits founded on their activity could theoretically function in a wide range of infectioushoststopermitrecordingofgeneexpressionin variousenvironments.

Genomeengineering

Inadditiontotoolsforclassicalmolecularbiology, phage-derived enzymesand technologieshaveledto genome-scale engineering techniques critical to the tailoringof strains for specific applications. Recombineering is a powerfultechniquethatuseshomologousrecombination to introducehighlytargetedmodifications,insertions,or deletions to loci withincells. This techniquehas been enabled through the transformation of DNA products bearing flanking homology to target sequences in con-junctionwiththehighlyactiveRedrecombinationsystem fromphagel.Ithasbeenappliedtowardmodificationsin avarietyofGram-negativebacteria[61],includingE.coli [62], Salmonella enterica [63],Shigella flexneri[64], Vibrio cholerae[65],Yersiniapestis[66],andP.aeruginosa[67]. A system with the potential for high-efficiency modifi-cationsinalargerangeofbacteria,includingboth Gram-positiveandGram-negativeexamples,hasrecentlybeen described by combining broad-host mobile group II introns, or ‘targetrons,’ with the widely used Cre/lox recombination system fromphage P1[68].This tech-nique, known as Genome Editing via Targetrons and Recombinases (GETR), involves first targeting introns containing loxP-derived sites to a specific location in a bacterial genome andsubsequently usingCre recombi-nasetocatalyzerecombinationbetweenloxPsitesonthe chromosomeandonatargetedconstruct.Thistechnique canbeusedtoachieveinsertions,deletions,inversions,or evenrelocationofachromosomallocus,dependingonthe designofthesitesandconstructs.Mutationsinthe wild-type loxPsequenceallowfor controlof directionalityof recombination as well as the generation of orthogonal sitespermittingGETRtobeusedatmultipleloci with-outcrosstalk[68,69].

Acceleratingevolution

An important extension of genome-editing techniques hasbeenthedevelopmentofmultiplexautomated gen-ome engineering (MAGE), a technique for generating geneticdiversitythrough theiterativeprocess of l-Red proteinb-mediatedrecombineeringwithapoolofshort, ssDNA oligonucleotides targeting a single or multiple genomicloci.IntheinitialstudypublishedbyWangetal. [70],MAGEwasusedtogenerateamutantstrainofE.coli withincreasedproductionoflycopeneusing oligonucleo-tidesdesignedto simultaneouslytarget24genetic com-ponents. Automating the growth, transformation, and recovery phases of the procedure has the potential to enablehands-freerapidevolutionofapopulation.Along with specialized genome assembly techniques, MAGE

wasusedtorecodeallUAGstopcodonsfromastrainofE. coli in order to generate a free, customizable codon (UAG),thusdemonstratingthepotentialcapacitytoedit thegeneticcodeinengineeredorganisms[71,72]. MA-GE presentsadditionalopportunitiesas atoolfor infec-tiousdiseaseresearchbyenablingtherapidoptimization ofantimicrobialgenecircuitsorasameansforintroducing diversityintoorganismsandmappingouttheir evolution-ary trajectories. Similarly, MAGE could be applied toward evolving improved or even novel functions in bacteriophagesbydiversifyingkeyphage proteins,such as hostrecognitionelements.

Phage-assisted continuous evolution (PACE) as intro-ducedbyEsveltetal.[73]isanotherstrideinaccelerated evolutionenabledbyphage-basedtechnology.InPACE, thelifecycleofthefilamentousphageM13islinkedwith anactivityofinteresttobeevolved,whichisusedtodrive production of pIII, the minor coat protein required for adsorption of infectious phage particles to the cognate receptoronrecipientcells(Figure2).Thistechniquewas first used to evolve T7 RNA polymerase variants with new properties, such as the ability to recognize novel promotersequences,byreplacingthegeneencodingpIII onthephage genomewiththegenefor T7 RNA poly-meraseandmovingpIIIexpressionunderthecontrolofa targetpromoteronaheterologousplasmid.Inthisway,a mutantT7RNApolymerasewithanimprovedabilityto initiatetranscriptionfrom thetargetpromoter resultsin increasedpIIIproductionandahighertiterofinfectious phages. As with MAGE, the PACE platform enables automatedevolution,thistimebymaintainingproductive phagein continuousculturewithinalagoonwitha con-stantinflowoffreshbacterialcellsandanoutflow ensur-ingremovalofnon-propagativephagevariants.PACEhas beenusedtoexplorehowdifferentparametersaffectthe pathways of genotypic and phenotypic divergence and convergence, contributing toward understanding how evolutionactsonsinglegenesandpotentiallyimproving theoptimizationoffutureengineeringefforts[74,75].

Phage-enabled

therapies

and

diagnostics

Antimicrobialphages

Ratherthanentirelyredesigningorrepurposingisolated phages, some engineering efforts in synthetic biology have been madetoward adding functions or improving existingphages.Forexample,LuandCollins[76] incorp-oratedthegeneencodingDspB,anenzymethatdegrades apolysaccharideadhesinimplicatedinbiofilmformation, into an engineered T7 phage. This modified phage effectivelycleared E. colibiofilmsthroughcycles of in-fection,phage-mediatedlysis,andreleaseofthe recom-binant dispersin enzyme to enzymatically degrade the biofilm material itself and expose protected cells. Additionally, the phage was modified to carry a gene from phage T3 in order to expand its host range and permitinfectionofthebiofilm-formingstrainusedinthe

study. However, despite the promise of phage thera-peutics, bacteria can display resistance toward phages through innate means, such as restriction-modification systems [77,78], as wellas adaptive means, typified by clusteredregularlyinterspacedshortpalindromicrepeats (CRISPR)-CRISPR-associated(Cas)systems[79]. More-over,mechanismsmayemergein abacterialpopulation during the course of selective pressure by phages, in-cluding phenotypic [80] and genotypic [81] causes of decreased phage adsorption, among others [82]. These hurdlesmaybetackledthroughtheuseofphagecocktails [83],high-throughputphageevolution,orperhaps,given

predictableevolutionary pathways,through therational engineeringof phages[10].

Incontrasttotakingadvantageofaphage’snaturalability tolyseatargetcell,somestudieshavefocusedonusing virusparticlesfortheircapacitytodelivernucleicacidsto targetcells. Suchan approach was takenby Westwater et al. [84], in which the group utilized the non-lytic, filamentousphage M13todeliverspecializedphagemid DNAin placeofthephagegenometotargetcells.The engineered phagemids (plasmids carrying signals to enable packaging into phage particles) were designed

Figure2 Infecting Phage pIII pIII T7 RNAP variant T7 RNAP variant Progeny phage (Infectious) Infecting Phage Progeny phage (Non-infectious) Inflow (cells)

gene III gene III

Outflow (cells+phage)

Current Opinion in Microbiology

M13 Accessory

Plasmid

M13 Accessory

Plasmid

Phage-assistedcontinuousevolution.Inphage-assistedcontinuousevolution(PACE),theproteinactivityofinterestislinkedtoexpressionoftheM13 coatproteinpIII,whichisrequiredforbindingandinitiationofthephageinfectioncycle[73].Withininfectedcells(top),theproteinofinterestis expressedfromtheinjectedM13genomeandsuccessfultargetactivitydrivesexpressionofgeneIIIontheaccessoryplasmid,permittingthe assemblyofinfectiousphageprogenythatcanbefurtheramplifiedandselectedthroughsubsequentroundsofinfection(left).Intheexampleshown here,theproteinofinterestisT7RNApolymerase(T7RNAP),whichcansuccessfullydriveproductionofgeneIIIonlyifitrecognizesthepromoterthat controlsgeneIIIexpression,thusenablingtheevolutionofT7RNAPvariantsthatcantargetnewpromotersequences.Ifactivityisinsufficienttodrive expressionofthecoatprotein,progenywillbenon-infectiousandfailtoamplify.Thetargetproteinisevolvedinacontinuousfashioninthe‘lagoon’ (bottom),wheretheencodinginfectiousphagescontinuallyamplifyviatheinputoffreshcells,whilenon-infectiousparticlesfailtoinfectnewcellsand areremovedintheoutflow.

to encodetheaddictiontoxinsGefand ChpBKtoelicit destruction of target cells. Hagens and Bla¨si [85] also applied thistoxicpayloadconceptusingM13to deliver genes encoding therestrictionenzymeBglII or thelS holin tokilltargetE.colibytheintroductionof double-stranded breaks in the chromosome or the creation of cytoplasmic membrane lesions, respectively. Sub-sequently, delivery of BglII was used to rescue mice infected withP.aeruginosa byadaptingthesystem with anengineeredderivativeoftheP.aeruginosafilamentous phagePf3[86].Thesemethodsalsoresultedinamarked decrease in releaseof endotoxin,one of themajor con-cernswithlyticphagetherapy[15],ascomparedtokilling via lysis by a lytic phage [85,86]. More recently, M13-derivedparticleswereusedtoexpressalethalmutantof catabolite activator protein in E. coli O157:H7, a food-borne pathogen that causes outbreaks of hemorrhagic colitis [87]. Biotechnology companies have also begun tomakeuseofrecombinantphagemethods,suchas virus-like particles that deliver genes encoding small, acid-solubleproteins tocausetoxicityto targetcells through non-specific bindingtoDNA[88].

Phage-baseddeliveryofantibiotic-sensitizingcassettes

Rather than encoding killing functions directly within phageparticles,Edgaretal.[89]usedphagelasachassis to generateantibiotic-resensitizingparticlesthroughthe delivery of dominantwild-typecopies of rpsL and gyrA (Figure 3a).The transductionofthesegenesintotarget cellsresistanttostreptomycinandfluoroquinolones, con-ferred by mutations in rpsL and gyrA, respectively, resultedintheproductionofwild-typeenzymes suscept-ibletotheformerlyineffectivedrugs.Inanother demon-stration, Lu and Collins [90] engineered M13 to carry genes encoding transcription factors that modify the native regulation of bacterial gene networks (Figure 3b). Constructs encoding the LexA3 repressor orSoxRregulatorwereusedtodisabletheSOSresponse andDNArepairortomodulatetheresponsetooxidative stress in targetcells,respectively, thus potentiating the toxiceffectsofantibiotictreatmentandeven resensitiz-ingaresistantbacterialstrain.Adual-functionphagewas also created and validated by using M13 harboring the globalregulatorcsrA,toinhibitbiofilmformationandthe associatedincreaseinantibioticresistance,andtheporin

Figure3 M13 λ LexA3 Resistant Wild-type Antibiotic Modified Functional SOS Response Antibiotic DNA Damaged

Cell Death Viable Cell

Cell Death Viable Cell

(a) (b)

DNA Repaired

Current Opinion in Microbiology

Bacteriophagecanmodulatetargetcellsbydeliveringgeneticpayloads.Bacteriophagesuchasthetemperatephagel(a)andthefilamentousphage M13(b)havebeenusedtodelivernon-nativeDNAtotargetcells.(a)Deliveryofdominantsensitivegenesencodingwild-typeenzymessuchasgyrA andrpsLresultsintheresensitizationofacelltoantibioticstowhichresistancehadpreviouslybeenconferredbymutationsinthesegenes[89_].(b)

Deliveryoftranscriptionfactorscanmodulateregulatorynetworksinbacteria,suchastheSOSresponsenormallyinducedinordertorespondto cellularstressandrepairdamagedDNA[90].ThedominantLexA3variantcaninhibittheSOSresponseandresensitizecellstosomeantibioticsaswell asreducethenumberofemergingresistantcells.

ompF , to improve drug penetration. Examples such as thesedemonstratethecapacityforbacteriophagestobe engineeredas genedeliverydevicesin orderto perturb geneticnetworksinbacteriaforbothresearchand thera-peuticapplications.With this approach,one canalter a genenetworkataparticularnodeandobservethe qual-itativeandquantitativeeffectsinordertobetter charac-terize native regulatory systems. As models of the interactions in complex regulatory webs of pathogens grow increasingly robust, the ability to know which strandstotugtoelicitdesiredeffectsmayenable ration-ally designed novel therapeutics based on predictable behaviors.

Genomeminingfortherapeutics

The developmentand improvementof next-generation sequencingtechnology hasenabledgenomic and meta-genomic analyses of phage populations [91,92]. For example,sequencingofgutviralmetagenomeshas impli-catedphages as reservoirsof antibiotic-resistance genes [93]and theirrolein influencing theintestinal micro-biota has been of recent interest [94]. Since bacterio-phages must encode mechanisms to control their host cells in order to infect, divert cellularresources to pro-pagate, build progeny phages,and, in many cases, lyse theirhoststoreleasenewparticles,phagegenomes con-stituteavastlibraryofpartsthatcanbeusedto manip-ulatebacteriaforstudyortreatment.Onthebasisofthis concept,Liuandcolleagues[95]developedamethodfor miningsuchtoolstogeneratenoveltherapeuticsagainst S. aureus. Predicted phage open reading frames were cloned with inducible expression into the target strain andscreenedforgrowth-inhibitoryproperties.Identified phageproteinswereusedtopullbacterialtargetsoutof celllysatesandalibraryofsmallmoleculeswasscreened to identifyinhibitorsof the protein–proteininteraction, withthehypothesisthatthesemoleculesmight demon-stratesimilarmodulatoryactiononthehosttarget.Inthis way,theauthorsidentified novelcompoundscapable of inhibiting theinitiationof bacterialDNA replicationin analogywiththephageproteins.Sincecurrentlyavailable drugsthattargetreplication onlyactontopoisomerases, thisworkdemonstratesthatminingphageproteins long evolvedtoinhibitbacterialprocesseshasthepotentialto expandtheantibioticrepertoirebyleadingustodiscover drugsagainstpreviouslyunused targets[95,96].

In addition to random-discovery screens, phage lysins have been specifically investigated in recent years as potentialantimicrobials.Theseenzymesare employed bybacteriophagestodegradethebacterialcellwalland permit the release of progeny phages[97]. In another functionalmetagenomicstudy,phageDNAwasisolated fromamixtureoffecesfromnineanimalspecies,cloned intoashotgunlibraryforinducibleexpressioninE.coli, and used in primary and secondary screens to detect lysins from the phage DNA pool [98]. As a discovery

tool, a specific lysin from a phage of Bacillusanthracis wasusedtodevelopanovelantimicrobialbyidentifying anenzymeinvolvedintheproductionofthelysintarget anddesigningacognatechemicalinhibitor[99].Though lysins are considered useful antimicrobials for Gram-positive pathogens,Gram-negative bacteria possess an outer membrane that prevents access of these extra-cellularenzymestothecellwall[100].Toovercomethis barrier,achimericproteincomposedofthetranslocation domain of the Yersinia pestis bacteriocin, pesticin, and the enzymatic domain of lysozyme from the E. coli phage T4 was engineered. The hybrid bacteriocin was shown to be active against E. coli and Y. pestis strains, including those expressing the cognate immu-nityproteinconferringresistancetounmodifiedpesticin [101,102].

Detectionofpathogens

Bacteriophages havealsobeen usedto implement real-worldapplicationsofbiosensing[103–107].Inareasfrom healthcareand hospitalsurfacestofoodpreparation and otherindustrial processes, methodsfor therapid detec-tionofpathogenicorganismsareparamountinpreventing disease and avoiding the public relations and financial burdensofrecallingcontaminatedproducts.Theamount of time necessary for many conventional detection methods is long due to the requirement for bacterial enrichmentbeforedetectionof thefewbacteriapresent in complex samplesin order to achieve sufficient assay sensitivity and specificity [108]. Engineered bacterio-phage-baseddetectorshavetheadvantageofrapid read-outs,highsensitivityandspecificity,anddetectionoflive cells[109].Acommondesign strategyisthecreationof reporter-based constructs packaged within phage or phage-likeparticlesthatinfecttargetcellsandultimately result in the production of fluorescent, colorimetric, or luminescent signals. Furthermore, sensor designs can includegeneticallyengineeredphagethatexpressa pro-ductcausingicenucleation[110]orthatincorporatetags forlinkingtodetectableelementssuchasquantumdots [111]. Though most of these examples of specifically modified phages have been enabled by advancements inengineeringandsyntheticbiologytoachievereal-world applicability,theconceptof usingnaturalphageas sen-sing tools is not a new one. Phage typing and other techniqueshave made use of the narrow host range of phagetoidentifyspeciesorstrainsofbacteriabasedona targetbacteria’sabilitytobind,propagate,orbelysedby non-engineeredviruses[109].

New

phage

engineering

strategies

Historically, modifications to bacteriophage relied on randommutagenesisorhomologousrecombination,both ofwhichareinefficientandnecessitateintensive screen-ingto identify mutants of interest.The relatively large size ofmostbacteriophage genomes and their inherent toxicity to bacterial hosts has confounded the use of

conventionalmolecularbiologytechniquesfor engineer-ing. However, recent synthetic biologytools have revi-talized the ability to make rational additions or modifications to phagegenomes. Amongsuch improve-ments,thephagedefensefunctionencodedby CRISPR-Cassystems,previouslyadaptedforgenomeediting[112– 117] and reviewed in [118], has been described for improving recombineering in bacteriophages by coun-ter-selecting unmodified phages with wild-type target sequences [119]. In vitro assembly of large constructs has also been made possible with techniques such as Gibson assembly [120], which enzymatically stitches together DNA fragments with overlapping homology, thus allowing for insertions of heterologous DNA and site-directed mutagenesis using PCR. Moreover, trans-formationofoverlappingfragmentsintoyeastin conjunc-tionwithacompatibleyeastartificialchromosomeleads to in vivo recombination-based assembly of large con-structs[121,122].Genomescanbeassembledwith modi-fications or be modified post-assembly in yeast, where they are non-toxic to the host, and then purified and rebooted in bacteria to produce engineered phage pro-geny.CurrentDNAsynthesistechnology,inconcertwith in vivoand in vitro recombination, alsopermits de novo chemicalsynthesisofbacteriophagegenomes.Smithetal. [123] utilized this approach to synthesize, clone, and produce infectious particles of the 5386bp phage FX174, and a similar scaled-up approach has created the first bacterial cell with a synthetic genome of 1.1Mb [124]. By rendering bacteriophages genetically accessible, synthetic biology can permit more precise studies of their underlying biologyand inspire creation of noveltherapeuticagents.

Refactoringandgenomestability

Despite advances in rational engineering of bacterio-phages,tamperingwithsystemsfinelytunedby evolu-tion can lead to fitness defects [125]. For example, roughly 30% of the genome of the bacteriophage T7 was ‘refactored,’ a process whereby genes and their respectiveregulatoryelementswereseparatedinto dis-tinctmodulestopermitsystematicanalysisandcontrol [126].Therefactoredgenomeproducedviable bacterio-phage,albeitwithsignificantlyreducedfitness.Multiple roundsofinvitroevolutionrestoredwild-typeviability attheexpenseofsomeofthedesignelements,implying that rational design can be coupled with evolution to ensure the creation of robustbiological systems [127]. Similarly, the evolutionary stability of a T7 phage engineeredtoinfect encapsulatedE.coliby producing acapsule-degradingendosialidaseasanexoenzymewas investigatedinvitro[128].Whiletheengineeredphage permitted replication in the encapsulated strain, the benefit conferred by endosialidase production was shared by wild-type, non-producing ‘cheater’ phages, whichcouldquicklyoutcompetetheengineeredviruses duetotheirhigherfitness.Althoughthesestudiespoint

to the fragility of current synthetic biology efforts, bacteriophage-based systemscan serveasan excellent platform to understand the constraints placed on syn-thetic genetic circuits by evolution and inform future designs.

Conclusions

Bacteriophages and functional components derived fromtheirgenomeshavelongbeenpowerfultoolsthat haveallowedustounderstandbasicbiologicalprocesses andthatsparkedthefieldofmolecularbiology. Mount-ing concerns over the spread of multidrug-resistant bacterial pathogens, as well as the development of enabling technologies from synthetic biology, have resulted in the resurgence of studies involving these highly evolved and specialized viruses. Recent efforts havemadestridesinengineeringphageswithmodified properties,endowingentirelynewfunctions,and deriv-ingrepurposedpartsforthestudy,detection,and treat-mentofinfectiousdiseases (Figure4).Asourabilityto engineer phages throughgenome synthesis and modi-ficationcontinuestoimprove,wewillbeabletofurther leverage thesefinely tuned productsof evolution that constitutethemostnumerousbiologicalentitiesknown to man.

Acknowledgements

TKLacknowledgessupportfromtheNIHNewInnovatorAward(DP2 OD008435),anNIHNationalCentersforSystemsBiologygrant(P50 GM098792),theDefenseThreatReductionAgency(022744-001)andthe U.S.ArmyResearchLaboratoryandtheU.S.ArmyResearchOfficethrough theInstituteforSoldierNanotechnologies(W911NF13D0001).RJCis supportedbyfundingfromtheNIH/NIGMSInterdepartmental BiotechnologyTrainingProgram(5T32GM008334).MMisaHoward HughesMedicalInstituteInternationalStudentResearchfellowanda recipientofaFondsderecherchesante´ Que´becMaster’sTrainingAward. Figure4

Bacteriophage Synthetic Biology

Genome Engineering and Evolution Phage Display Parts for complex

Genetic Circuits Improved Phage Therapeutics Genome mining for therapeutics Genetic payload Delivery Diagnostic tools

Current Opinion in Microbiology

Synergybetweensyntheticbiologyandbacteriophage.Newtoolsand techniquesfromsyntheticbiologyhaveenabledphageresearchandthe developmentofnoveltherapies.Likewise,bacteriophagecomponents havefueledinnovationinsyntheticbiology.

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