CHAPTER 2 Materials and Methods - shodhganga.inflibnet.ac.in

1 CHAPTER2 MaterialsandMethodsChapter2 onLuriaAgar(LA)(Hi-MediaLaboratories,Ind ia). , (LB)containingappropriateantibioticswere usedtoprepareglycerolstockswhichwerestor edat-20 :Recommendeddozesofantibioticsusedinthis study(SambrookandRussell,2001).Antibioti csRichmediumMinimalmediumKanamycin50 g/mlStreptomycin10 g/mlTrimethoprim60 g/ml15 g/mlAmpicillin50 g/mlErythromycin100 g/ml25 g/mlGentamycin20 g/ml5 g/mlTheantibiotic ( ). :Koser sCitratemediumCompositionoftheKoser sCitratemediumincludedmagnesiumsulphate, ;monopotassium phosphate, ;sodiumammoniumphosphate, ;sodiumcitrate, ,15 ,India,andwasusedaccordingtomanufacturer :M9minimalmediumCompositionofM9minimalbr othwasaccordingtoSambrookandRussell(2001 )includingNa2 HPO47H2O,34g/L;KH2PO4,15g/L;NH4Cl,5g/L;N aCl, ;2mMMgSO4; , ; , ; , ;H3BO3.

2 , , usedwereglucose,xylose,arabinose,maltose ,cellobios, ,15 ,micronutrients (preparedat1000 Xstockconcentration)andcarbonsource(2M, )wereautoclaved :TrisrockphosphatebufferedmediumChapter2 :MaterialsandMethods3535 GeneticmanipulationofEnterobacterasburia ePSI3forenhancePhosphorusnutritionThemed iacompositionincluded Tris-Cl(pH= ),100mM; NH4Cl,10mM;KCl,10mM;MgSO4,2mM;CaCl2, ;micronutrientcocktail;Glucose,50-100mMa ndphosphate (P)sources(Sharmaetal.,2005).1mg/mlSeneg alRockphosphate (RP) stockconcentrationandafixedvolume ofeachwasaddedtopre-autoclavedflaskscont ainingsteriledistilledwater( ) :Murashige-Skoog sMediumMurashige-Skoog s(MS) , ; KH2PO4, ;KNO3, ; , ;NH4NO3, (autoclavedseparately), wasobtainedfromHi-MediaLaboratories,Indi a,andwasusedaccordingtomanufacturer s instructionswhilethemacronutrients (SambrookandRussell,2001).

3 Chapter2 :GenomicDNAisolationfromPseudomonasputid aKT2440 FreshPseudomonascultureobtainedbygrowing asinglecolonyinoculatedin3mlLBundershake conditionsat 30 ,pelletedat9, 200xgand washedtwice withsterile ,thecellswereusedforgenomicDNAisolationp erformed usingstandardgenomicDNAextractionprotoco lforbacterialcell(Ausubeletal.,2006).The DNAwasfinallyre-suspendedin40 l (asandwhenapplicable)wascarriedoutaccord ingtoSambrookandRussell(2001). (Cohenetal.,1972) asburiaePSI3wasgrownat37 Atthispoint,thecellswerechilledforabout1 0minutes, (chilled).Aftercentrifugation(5000rpmfor 5minutes), ,incubatedonice-bathfor1hr,centrifuged(5 000rpmfor5minutes)andthenresuspendedin1 (microcentrifugetube)toaddDNAsamples( gisrequired) :MaterialsandMethods3737 GeneticmanipulationofEnterobacterasburia ePSI3forenhancePhosphorusnutritionthensu bjectedtoaheatshockat42 Cfor2mintoenableDNAuptake, Cundershakeconditions.

4 Oftheplatesat37 ,formediatingtheconjugaltransferandthere sultanttransformant ofrecipientandthedonorstrainswereaseptic allymixedin1:1ratio(v/v) ,thebacterialculturemixwascentrifugedat5 000rpm for5minutesandtheresultantpelletwasre-su spended ,thiscellsuspension wereplatedonKoserCitrateagarcontaining theappropriateantibioticsforselection(an tibioticdosewasasdescribedinTable1) :AgarosegelelectrophoresisTheDNAsamplesw eremixedwithappropriatevolumeof6 Xloading buffer( blue,and40%sucroseinwater) (containing1 g/mlethidiumbromide)gelinTris-acetate-ED TAC hapter2:MaterialsandMethods3838 GeneticmanipulationofEnterobacterasburia ePSI3forenhancePhosphorusnutrition(TAE)b ufferat5 (RE)wasusedwiththeappropriate10 Xbufferssuppliedbythemanufacturerinafina lreactionvolumeof10 , ,digestionwithoneenzymeisperformedfollow edbypurificationandsubsequentdigestionwi ththeotherenzyme, (~300ng/ l) lBuffer(10X) lRestrictionEnzyme(10U/ l) :PolymeraseChainReaction(PCR)ThePCRreact ionsetupwasbasedontheguidelinesgiven :MaterialsandMethods3939 GeneticmanipulationofEnterobacterasburia ePSI3forenhancePhosphorusnutritionPCRrea ctionmixture.

5 Forwardprimer(10pmol/ml) lReversePrimer(10pmol/ml) ldNTPs( ) lPfupolymerase(3units/ l) lPCRamplificationswereperformed inDNAE nginethermalcycler(BioRad)orVaritithermo cycler (AppliedBiosystem). *Exactprimerannealingtemperatureandprime rextensiontimevariedwithprimers(designed withrespecttodifferenttemplates) , XT-20polymerase, ,buffer, ,India, ,Ocimumbiosolutions,India, ofintermolecularandintramolecularcomplem entaritiestoavoidprimer-primerannealinga ndhairpinstructuresandtheappropriate%G-C wascarriedoutwiththehelpofonlineprimerde signing ,lengthand%G-Ccontentofprimersaresubject tovariationdependingonthepurposeofPCRand willbeChapter2.

6 MaterialsandMethods4040 GeneticmanipulationofEnterobacterasburia ePSI3forenhancePhosphorusnutritiongivena sand whenapplicableinthe CStep-1(Initialdenaturation)Step-2(Denat uration)Step-3(Annealing)940C-1mincycle1 940C-30sec520C-30sec30cycleStep 4(Extension)720C-1min30secStep-5(Finalex tension)720C 5mincycle1 :GelelutionandpurificationTheDNAfragment sofdesiredsizeswererecoveredfromthegelby cuttingtheagarosegelslabaroundtheDNA band. Theagarose piecewasweighedinasterilemicrocentrifuge tube. GelelutionandpurificationwasdonebyusingP ureLinkQuickGelExtractioKit(Invitrogen) :LigationTheligationreactionwasusually donein10 lvolumecontaining thefollowingconstituents:Purifiedvectora ndinsertDNA(volumevarieddependingonthere spectiveconcentrations);10XT4 DNAL igasebuffer,1 l.

7 T4 DNAligase(MBIF ermentas), watertomakeupthevolume 10 C (molarconcentrationscalculatedbytheunder mentionedformula) of1:4wasmaintained, ( g)x1,515pmolesofDNA=-------------------- -------------------SizeoftheDNAfragment( )Chapter2 :SDS-PAGESDS-PAGE slabgelelectrophoresiswascarriedoutusing 10-15%acrylamidegelbyfollowingtheprocedu resdescribedbySambrooketal (2001). :CompositionofSDS-PAGE reagents(SambrookandRussell,2001)(A)Mono mersolution(30%)(Storeat4 Cindark)(B) ( )AdjustpHwithHCl(C) ( ) (D)TankBuffer( )(E)SampleLoadingbuffer(2X)(F) Till2 LAdjustthe pHwithHClSDS4%Glycerol20%Tris-Cl( ) (fresh)TEMED2-3 lWatersaturatedn-butanolSigma proteinmolecularweightmarker SDS6H2-(30,000-200,000).

8 Used 14 gtotalprotein/well(G)SeparatingGel(8%,10 ml)(H)StackingGel ( ,5ml)(I)StainingSolution30% ( ) lTEMED2 l30% ( ) lTEMED3 acid(J)De-stainingsolution(10%methanolan d10%Acetic acid)Chapter2:MaterialsandMethods4242 GeneticmanipulationofEnterobacterasburia ePSI3forenhancePhosphorusnutritionAftere lectrophoresis thegelwasstainedusingthestainingsolution ( )forapproximately1hand thende-stainedwithde-stainingsolution( ) (TRP)agarplateswhichrepresentsamuchmores tringentconditionofscreeningforPSMs(Gyan eshwaretal,1998b). lofitwasaseptically spottedontheabovementionedagarplatesandw asallowedtodrycompletelyfollowedbyincuba tionat37 < :PhysiologicalexperimentsThephysiologica lexperiments , :InoculumpreparationTheinoculum forM9andTrisminimalmediacontainingfreePi waspreparedbygrowing ,washedtwicebynormalsalineandfinallyre-s uspendedin1mlChapter2 (Gyaneshwaretal.)

9 ,1998b).Themediacompositioninthiscasewas sameasmentionedinSection45-100mMglucoseo r50-100mM ,30mlofrelevantminimalbroth withoutfreePiwasinoculatedwithcellsuspen sions ( ).Thebatchculturestudieswereperformedund eraerobicconditionsinOrbitekrotaryshaker maintainedat37 Cwithagitationspeedkeptconstantat200rpm. 1mlsampleswereasepticallyharvestedatregu larintervals(varyingwitheverysetofbatchc ulturedependingonmediaconditions) :AnalyticaltechniquesThecelldensitydeter minations , Cuntilfurtherusedforbiochemicalestimatio ns. Thestoredsampleswerecentrifugedat9,200xg for1minat4 Candtheculturesupernatants ,thecultureChapter2 mnylonmembranes (MDIadvancedmicrodevices,India)andthesec retedmetaboliteswerequantified usingRP-18columnorLunacolumn(Phenomenex) .

10 , mmembranesandweresubjectedtochromatograp hyfordetermining :PreparationofcellsandcellfreeextractsGl ucosegrowncellsunderabovementionedinTRPm ediumconditions werecollectedbycentrifugation9,200xgfor2 minutesat4 forGDHassaywasdonebywashingtheharvestedc ells(mid-latelogphasecultures) ( ) :InvertaseassayThebacterial ,thenwashedwithandresuspended (2ml) , (acetatebuffer, ,phosphatebuffer, ,orTrisClbuffer, ),withanChapter2 Cfor30minandthereducing sugarproducedwasmeasuredbythemethodofMil ler(1959).Oneunitofactivity isdefinedastheamountofenzymethatproduced reducingsugarequivalentto1 :GDHassayGDH(D-glucosephenazinemethosulp hateoxidoreductase, ( )wasdeterminedspectrophotometricallybyfo llowingthecoupledreductionof2,6-dichloro phenolindophenol (DCIP)at600nm(Quayetal.))