Huang--Design and character

DesignandcharacterizationofmoleculartoolsforaSyntheticBiologyapproachtowardsdevelopingcyanobacterialbiotechnologyHsin-HoHuang,DanielCamsund,PeterLindbladandThorstenHeidorn*DepartmentofPhotochemistryandMolecularScience,A˚ngstro¨mLaboratories,UppsalaUniversity,P.O.Box523,SE-75120Uppsala,SwedenReceivedJanuary18,2010;RevisedFebruary28,2010;AcceptedMarch1,2010ABSTRACTCyanobacteriaaresuitableforsustainable,solar-poweredbiotechnologicalapplications.Syntheticbiologyconnectsbiologywithcomputationaldesignandanengineeringperspective,butrequiresefficienttoolsandinformationaboutthefunctionofbiologicalpartsandsystems.ToenablethedevelopmentofcyanobacterialSyntheticBiology,severalmoleculartoolsweredevelopedandcharacterized:(i)abroad-host-rangeBioBrickshuttlevector,pPMQAK1,wasconstructedandconfirmedtoreplicateinEscherichiacoliandthreedifferentcyanobacterialstrains.(ii)ThefluorescentproteinsCerulean,GFPmut3BandEYFPhavebeendemonstratedtoworkasreporterproteinsincyanobacteria,inspiteofthestrongbackgroundofphotosyntheticpigments.(iii)Severalpromoters,likePrnpBandvariantsofPrbcL,andaversionofthepromoterPtrcwithtwooperatorsforenhancedrepression,weredevelopedandcharacterizedinSynechocystissp.strainPCC6803.(iv)Itwasshownthatasystemfortargetedproteindegrad-ation,whichisneededtoenabledynamicexpres-sionstudies,isworkinginSynechocystissp.strainPCC6803.ThepPMQAK1shuttlevectorallowstheuseofthegrowingnumbersofBioBrickpartsinmanyprokaryotes,andtheothertoolshereinimple-mentedfacilitatethedevelopmentofnewpartsandsystemsincyanobacteria.INTRODUCTIONCyanobacteriaaresunlight-drivencellfactoriesthatcanbeharnessedtosustainablyproducenumerousproducts,suchasbiofuels,foods,feedsandotherbiomaterials.Theymayalsoserveasnitrogen-fixingbiofertilisersorbeemployedfortheirbioremediatorypotential(1,2).Cyanobacteriamayusesolarenergyastheirenergysource,CO2fromtheairastheircarbonsource,and,someofthem,atmosphericN2astheirnitrogensource.Withsuchfavorablebasiccharacteristics,welldevelopedmoleculartools(3),andexistingsystemsbiologyanalyses(4),cyanobacteriaareidealcandidatesforasyntheticbiologicalapproach.ForsomeyearsthefieldofSyntheticBiologyhasbeenevolving(5–8),anditisbelievedtobeafuturetechnologywithhighimpact,connectingseveralresearchfieldslikegeneticengineering,systemsbiology,proteindesignandcomputationalmodeling.SyntheticBiologyisthedesignandconstructionofnewbiologicalparts,devicesandsystems,aswellastheredesignofexistingbiologicalsystemsforusefulpurposes().BasicprinciplesofSyntheticBiologyare:.theuseofstandardizedandwellcharacterizedbuildingblocks(BioBricks),.thehierarchicaldesignofnature-inspired,artificialgeneticcircuitsandproteinsinsilico,and.theuseofchemicalDNA-synthesiswhichallowspro-ductionofDNAsequencesthatarenotfoundinnature.TheBioBrickstandard,originallyproposedbyKnightin2003(9),consiststodayofthousandsofBioBrickparts,whichareavailableatthe‘RegistryofStandardBiologicalParts’().Biologicalpartsaree.g.promoters,ribosome-bindingsites,terminators,codingsequencesandreplicationorigins,allinastandardizedformatforeasyassembly.Thepartsareflankedbyastandardsetofrestrictionsitesandtheassemblyofseveralpartscanbeperformedbyestablishedcloningtechniques.Withthesefeaturesarapidconstructionof*Towhomcorrespondenceshouldbeaddressed.Tel:[phone];Fax:[phone];Email:[e-mail]ownthat,intheiropinion,thefirsttwoauthorsshouldberegardedasjointFirstAuthors.Publishedonline17March2010NucleicAcidsResearch,2010,Vol.38,No.82577–2593doi:10.1093/nar/gkq164TheAuthor(s)2010.PublishedbyOxfordUniversityPress.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(),whichpermitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.atEastChinaUniversityofScienceandTechnologyonMay19,2010fferentresearchgroups.WhereassofarmostworkinSyntheticBiologyhasbeendoneinEscherichiacoli,weaimtousetheprinciplesofSyntheticBiologyforthedesignandconstructionofcyanobacterialmoleculartools(e.g.vectors,promoters),andtobuilduparepositoryofwellcharacterizedcyano-bacterialparts.FortheintroductionofBioBrickpartsintocyanobacteria,aBioBrick-compatiblecyanobacterialshuttlevectorwasconstructed.Thisvectorcontainstherepliconofthebroad-host-rangeplasmidRSF1010,designedforreplicationindifferentcyanobacterial,andevennon-cyanobacterial,strains.TheRSF1010repliconoftheIncQgroup(10)iswellknownforallowingplasmidstoreplicateinalargevarietyofgramnegativebacteria,butthereareexamplesofsuccessfulreplicationingrampositivebacteriaaswell(11).Infact,thereplica-tiveandconjugalabilitiesofIncQtypeplasmidsmakethemthemostwide-spreadbacterialrepliconsknown(12).Thisincludes,forexample,bacteriafromthegenusPseudomonas(13),cyanobacterialgenerasuchasSynechococcus(14,15),Prochloro