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Schemeoflambdaphagestructureandproteincomponents
Head gpA gpB gpC gpD gpE gpF gpW Tail gpG gpH gpI gpJ gpK gpL gpM gpT gpU gpV gpZ

PreparationofLambdaphage TheprocedureforpreparinglambdaphagewasadaptedfromprotocolsinMolecularCloning: ALaboratoryManual[38]. Materialsmediaandbuffer E.colibacteriastrainLE392andlambdaphagestrainwerepurchasedfromPromega(Madison, WI).SterilizedLuriaBertani(LB)medium(10gtrypton,5gyeastextractand10gNaCl/liter, pH=7.5)wasusedforbacterialandphagecultivation,aswellasfortheplaqueassay.Sterilized SMsolution(5.8gofNaCl,2gofMgSO47H2O,50mlof1MTrisHClatpH=7.5,and5mlof2% Gelatin/liter)wasemployedassuspensionbufferforlambdaphage.Agarsolution(7gagarin one liter of LB medium, pH=7.5) was prepared for multiple usage throughout the procedure. Agar solution condensed in Petri dishes is referred to as bottom agar/agar plate, while agar solutionstoredinbottlesforfurtheruseistopagar.

Bacterialgrowth ThebacteriastrainLE392wasstreakedonagarplateandincubatedat37Covernight.Thena single bacterial colony was picked from the streaked plate and incubated with 50 ml of LB mediuminthepresenceof0.2%maltoseovernightforbacterialgrowth.Aftercentrifugationof bacterialculture,thesupernatantwasdiscardedandbacterialcellpelletwasresuspendedwith 10mMMgSO4toafinalconcentrationofOD600=1.0(opticaldensitymeasuredat600nm). Infectingplatingbacteria/Phageplaqueassay Topreparelambdaphagestockandmeasuretheconcentrationofthelambdaphage,plating bacteria were infected with diluted lambda phage to form plaques in agar plate. First, the lambdaphage(initiallypurchasedstockorphageculture)wasserially10xdiluted(101,102,to 108)withSMbuffer.Then100lofeachselecteddilutedlambdaphageserials(e.g.107,108) weregentlymixedseparatelywith200lofOD600=1.0bacteriainsmallculturetubes.After incubating in a 37C water bath for 30 min to allow the phage particles to adsorb to the bacteria,themixtureisaddedto3mlofmoltentopagar(47C)and,immediatelypouredonto the agar plate. After overnight incubation at 37C, transparent concentric plaques could be observed for further counting or selecting: the concentration of lambda phage can be calculatedinplaqueformingunits(pfu)permlaftercountingthenumbersofplaquesonthe plate. The well isolated plaque can be selected and picked to prepare genetically homogeneouslambdaphagestocks. Preparationoflambdaphagestock Onewellisolatedlambdaphageplaquewaspickedfromtheplateandsuspendedin1mlof SM buffer with 50 l of chloroform for at least 2 hours to assist phage particles in diffusing fromtheagar.1/10ofsuspendedindividualplaquesolutionand200lofOD600=1.0bacteria wereusedtoperformtheinfectingplatingbacteria/phageplaqueassaystepon2agarplates.

After overnight incubation at 37C, 5 ml of SM buffer was added to each lysed plate and shakenonashakingplatformat4Cfor4hours.ThecollectedSM(containinglambdaphage and bacterial debris) was added with 0.1 ml of chloroform and centrifuged to remove the bacterialdebris.Supernatantswerecollected,combinedwithchloroformandstoredat4Cas lambdaphagestock.Phageplaqueassaywascarriedouttodeterminetheconcentration((pfu ml1))ofthislambdaphagestock,thuswithproperdilution,acertainnumber(pfu)oflambda phagecanbetakenforthesequentialsteps. Largescalephageculturepreparation 107oflambdaphagefromphagestockweremixedwith2mlofOD600=1bacteria.After30 minincubationat37Cwaterbath,themixturewaspouredintoaflaskcontaining500mlof prewarmedLBmedia.Thenafter9hoursofincubationwithshakingrateof275opm,culture lysiswasobserved.Then10mlofchloroformwasaddedtoflask,andincubationcontinuedfor 20min. Precipitatinglambdaphageparticles The cooled lysates were treated with DNase I to a final concentration of 1 g ml1 and incubatedfor30min.Then29.2gofNaClwasadded.Aftersettlingonicefor2hrs,theculture was centrifuged at 11,000 g for 10 min to remove debris. 50 g solid PEG 8000 was then dissolvedinthesupernatants,whichwerekeptonicefor2hrs.Thetreatedculturewasspun againat11,000gfor10minat4Candthesupernatantswerediscarded,whilephagepellets on the centrifuge bottles were resuspended with 10 ml of SM buffer. Equal volumes of chloroform were mixed with resuspended phage by gentle shaking. After centrifugation at 4000gfor15min,purifiedlambdaphage,whichisthelambdaphageparticlessuspendedin SMbuffer,wasobtainedbyrecoveringtheaqueousphase.

AdditionalImplicationsofthisStudyforPhageTechnologies PhageDisplayTechnology Phage display is a significant technology for which a foreign gene sequence(s) is inserted into thegeneoftheencodingphageproteins,andultimatelydisplayanextensionpeptideorprotein on the viral coat by expressing and amplifying in the host bacterium [48, 49]. Phage display librariesaremixturesofdisplayedphagescarryingadifferentforeigngeneinsert.Byscreening phagedisplaylibrarywithatargetsubstancesuchasmetal,receptororsurface,thosedisplayed peptidesorproteinsthathavespecificaffinitytothetargetsubstancewillbeselectedoutfor their further applications in various areas such as drug discovery, epitope mapping, disease diagnosticsandnewmaterialdevelopment. WhilephagedisplayhasprimarilyfocusedonlysogenicfilamentousphageM13,fdandf1,there aremanystudiesyettobedonewiththeseandevenmoresowithlyticlambdaphage,T4and T7[49,50].Forlysogenicphages,theexpressedpeptides/proteinsneedbeextrudedorsecreted acrossthebacterialmembranes,whichmaycausedifficultiesregardingthesecretionoflarger foreignpeptidesorproteins[49,51,52].Thelyticphagesthatareassembledinthecytoplasm ofhostcellsandreleasedbylysis,donotrequireproteins(includingfusionpeptides/proteins) to be translocated across the host membrane, therefore, represent advantages in ultimately displayingsizeablepeptidesorproteins,andeventhosemaytoxictothehostcells[49].Ithas been shown that gpD and gpV in lambda phage can be employed for phage display and it is worthytonoteagainthattheseproteinsarerichinmetalbindingsulfur.[5355] WiththepossibilityofgpE,gpD,gpVinteractionswiththemetalsdetected(Mn,Fe,Co,Ni,Cu andZn),librariesmightbeselectedordevelopedconsideringtheirpotentialinteractionswith phageproteins,sincegpEisessentialforcapsidformation,whilegpDandgpVhavebeennoted

asphagedisplayproteins[45,54,55].Thegrowthmethodscouldconsiderappropriatemetal concentrationsfordisplayingspecificpeptidesorproteinsongpDorgpV. Aparticularareaofpotentialbenefitisbiomineralization,whichfindssignificantmetalbinding peptidesorproteinsservingastemplatesforbiosynthesizingnanoscalematerialsinvitro.[20, 56]Thedetailedapproachesincludingthepinningprocesshavebeendescribed[50,56,57].In general,bygeneticallyengineeringthephagecoatasapresentationvehicle,numerous(e.g.~ 109peptides)differentviraldisplayedpeptidesorproteinsarescreenedwithtargetinorganic metals to identify peptides/proteins exhibiting preferential affinity to target metals and are potentially useful for metal material biosynthesis [13]. So far, Ag[57], Al[58], Au[18], Co[14], Fe[58],Li[15]andPt[17]nanowirematerialshavebeennucleatedandgrownonthepeptidesor proteinsselectedfromphagedisplaylibraries.Thecurrentphagevectorsystemsengineeredfor preparing these materials are filamentous phages. But charged peptides or cysteinerich peptides that are excellent for metalbinding might have difficulty in fusing on filamentous phages due to the limitations imposed by secretion [49, 59]. The lytic phages (, T4 and T7), which are free of these limitations, can be considered as alternative systems to display peptides/proteinscarryinghighermetalbindingpotential. This study using SECICPMS to screen metals in lambda phage samples, for ultimate metalloprotein identification, indicates phage associated metals and non associated metals generate information to assist selection of preferable target metal pools for existing display libraries.Sinceforeignpeptides/proteinscanbedisplayedongpVandgpD,whilegpEconnects togpDtrimers,ifanyofthelambdaphageassociatedmetalsischosenastarget,theselambda phage proteins will compete with the displayed peptides/proteins for binding with a target metal. Also, in the pinning process, the mutant phages that contain a target metal binding peptide/protein are selected and enriched by affinity selection of a phage display library on

the immobilized target so that binding phages are captured and nonbinding phages are washedaway[50].Somephageswithfusednontargetmetalbindingpeptides/proteinscould betrappedduetotheinteractionbetweenthephageproteinsandimmobilizedtargetmetal, therebycausingfalseselectionofspecificpeptides/proteins.Hence,thoselambdaphagenon associatedmetalsfromthisstudy(Al,K,Ca,Cr,As,Se,Ag,Cd,CsandHg)mayserveasbetter choiceoftargetmetalswhenusinglambdaphagedisplaylibrariestoselectpropertemplates fortargetmetalbiomineralization.ThischromatographyICPMSprocesscanalsobeappliedon other currently known and potential phage display systems to screen and distinguish phage associated and nonassociated metals. Thus, the target metals can be preoptimized and selectedfromphagenonassociatedmetalsaccordingtothephagesystemutilized. Beyond the nonphage associated metals, the findings on the association between lambda phageproteins(gpE,gpDandgpV)andtheirfavoredmetals(Mn,Fe,Co,Ni,CuandZn)isalso beneficialforexploringmetalphagehydrogelthathasbroadpotentialbiomedicalapplications, including tissue engineering, gene/drug delivery and stem cell manipulation. Directassembly andstabilizationofAuphagehydrogelshasbeenachievedviainteractingAunanoparticleswith eithernativeormutantphagebytuningthepH,andtherefore,controllingthephagesurface charge[18].Thus,itispromisingtoconsiderinvolvinglambdaphageanditsassociatedmetals fordevelopingvarioushydrogels. References
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