Cell-Free Synthetic Biology PDF
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C e ll- F r e e S y n t h e t ic B io lo g y • S e o k H o o n H o n g Cell-Free Synthetic Biology Edited by Seok Hoon Hong Printed Edition of the Special Issue Published in Methods and Protocols www.mdpi.com/journal/mps Cell-Free Synthetic Biology Cell-Free Synthetic Biology SpecialIssueEditor SeokHoonHong MDPI•Basel•Beijing•Wuhan•Barcelona•Belgrade SpecialIssueEditor SeokHoonHong IllinoisInstituteofTechnology USA EditorialOffice MDPI St.Alban-Anlage66 4052Basel,Switzerland This is a reprint of articles from the Special Issue published online in the open access journal MethodsandProtocols(ISSN2409-9279)in2019(availableat:https://www.mdpi.com/journal/mps/ specialissues/CellfreeSyntheticBiology). Forcitationpurposes,citeeacharticleindependentlyasindicatedonthearticlepageonlineandas indicatedbelow: LastName,A.A.; LastName,B.B.; LastName,C.C.ArticleTitle. JournalNameYear,ArticleNumber, PageRange. ISBN978-3-03928-022-3(Pbk) ISBN978-3-03928-023-0(PDF) CoverimagecourtesyofDr.JavinP.Oza,CaliforniaPolytechnicStateUniversity. (cid:2)c 2020bytheauthors. ArticlesinthisbookareOpenAccessanddistributedundertheCreative Commons Attribution (CC BY) license, which allows users to download, copy and build upon publishedarticles,aslongastheauthorandpublisherareproperlycredited,whichensuresmaximum disseminationandawiderimpactofourpublications. ThebookasawholeisdistributedbyMDPIunderthetermsandconditionsoftheCreativeCommons licenseCCBY-NC-ND. Contents AbouttheSpecialIssueEditor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii SeokHoonHong “Cell-FreeSyntheticBiology”:SyntheticBiologyMeetsCell-FreeProteinSynthesis Reprintedfrom:MethodsandProtocols2019,2,80,doi:10.3390/mps2040080 . . . . . . . . . . . . . 1 NicoleE.Gregorio,MaxZ.LevineandJavinP.Oza AUser’sGuidetoCell-FreeProteinSynthesis Reprintedfrom:MethodsandProtocols2019,2,24,doi:10.3390/mps2010024 . . . . . . . . . . . . . 4 Dohyun Jeong, Melissa Klocke, Siddharth Agarwal, Jeongwon Kim, Seungdo Choi, ElisaFrancoandJongminKim Cell-FreeSyntheticBiologyPlatformforEngineeringSyntheticBiologicalCircuitsandSystems Reprintedfrom:MethodsandProtocols2019,2,39,doi:10.3390/mps2020039 . . . . . . . . . . . . . 38 WeiGao,NingBuandYuanLu EfficientIncorporationofUnnaturalAminoAcidsintoProteinswithaRobustCell-FreeSystem Reprintedfrom:MethodsandProtocols2019,2,16,doi:10.3390/mps2010016 . . . . . . . . . . . . . 63 JeehyeKim,CarolineE.Copeland,SahanaR.PadumaneandYong-ChanKwon ACrudeExtractPreparationandOptimizationfromaGenomicallyEngineeredEscherichiacoli fortheCell-FreeProteinSynthesisSystem:PracticalLaboratoryGuideline Reprintedfrom:MethodsandProtocols2019,2,68,doi:10.3390/mps2030068 . . . . . . . . . . . . . 75 XingJin,WestonKightlingerandSeokHoonHong OptimizingCell-FreeProteinSynthesisforIncreasedYieldandActivityofColicins Reprintedfrom:MethodsandProtocols2019,2,28,doi:10.3390/mps2020028 . . . . . . . . . . . . . 90 Seung-OokYang,GregoryH.Nielsen,KristenM.Wilding,MeridethA.Cooper, DavidW.WoodandBradleyC.Bundy TowardsOn-DemandE.coli-BasedCell-FreeProteinSynthesisofTissuePlasminogenActivator Reprintedfrom:MethodsandProtocols2019,2,52,doi:10.3390/mps2020052 . . . . . . . . . . . . . 102 LenaThoring,AnneZemella,DoreenWu¨stenhagenandStefanKubick Accelerating the Production of Druggable Targets: Eukaryotic Cell-Free Systems Come into Focus Reprintedfrom:MethodsandProtocols2019,2,30,doi:10.3390/mps2020030 . . . . . . . . . . . . . 110 HyeJinLimandDong-MyungKim Cell-FreeMetabolicEngineering:RecentDevelopmentsandFutureProspects Reprintedfrom:MethodsandProtocols2019,2,33,doi:10.3390/mps2020033 . . . . . . . . . . . . . 131 v About the Special Issue Editor SeokHoonHong, Dr., hasbeenanAssistantProfessorattheIllinoisInstituteofTechnology since2015. Hisresearchinterestsaredevelopingcell-freeproteinsynthesisplatformsandapplying syntheticbiologytoolstocontrolmultidrug-resistantbacteriaandbiofilms.BeforejoiningtheIllinois Institute of Technology, he worked on cell-free synthetic biology as a postdoctoral researcher at NorthwesternUniversityforfouryears.Hestudiedtheproductionofnovelprotein-basedmaterials via site-specific incorporation of non-standard amino acids and advanced genome engineering. His cell-free protein synthesis platform significantly improved the yield of modified proteins and enabledtheproductionofsequence-definedbiopolymers. In2011, hereceivedhisPh.D.atTexas A&MUniversity,studyingbacterialbiofilmsandpersistercellformationbyproteinengineeringand synthetic biology. He demonstrated biofilm displacement via a population-driven genetic switch coupled to engineered biofilm dispersal proteins. As of November 2019, his work has led to twenty-eight scientific papers in peer-reviewed journals, one patent, and over sixty presentations atscientificandengineeringresearchconferences. vii Editorial “Cell-Free Synthetic Biology”: Synthetic Biology Meets Cell-Free Protein Synthesis SeokHoonHong DepartmentofChemicalandBiologicalEngineering,IllinoisInstituteofTechnology,Chicago,IL60616,USA; [email protected];Tel.:+1-312-567-8950 Received:30September2019;Accepted:1October2019;Published:8October2019 SinceNirenbergandMatthaeiusedcell-freeproteinsynthesis(CFPS)toelucidatethegeneticcode intheearly1960s[1],thetechnologyhasbeendevelopedoverthecourseofdecadesandappliedto studyingbothfundamentalandappliedbiology[2].Cell-freesyntheticbiologyintegratingCFPSwith syntheticbiologyhasreceivedattentionasapowerfulandrapidapproachtocharacterizeandengineer naturalbiologicalsystems.Theopennatureofcell-free(orinvitro)biologicalplatformscomparedto invivosystemsbringsanunprecedentedlevelofcontrolandfreedomindesign[3]. Thisversatile engineeringtoolkithasbeenusedfordebuggingbiologicalnetworks,constructingartificialcells, screeningproteinlibraries,prototypinggeneticcircuits,developingbiosensors,producingmetabolites, andsynthesizingcomplexproteinsincludingantibodies,toxicproteins,membraneproteins,andnovel proteinscontainingnonstandard(unnatural)aminoacids.TheMethodsandProtocols“Cell-FreeSynthetic Biology”SpecialIssueconsistsofaseriesofreviews,protocols,benchmarks,andresearcharticles describingthecurrentdevelopmentandapplicationsofcell-freesyntheticbiologyindiverseareas. AlthoughinterestinCFPSisgrowing,newusersoftenfacetechnicalandfunctionalissuesin choosingandexecutingtheCFPSplatformthatbestsuitstheirneeds.Anextensivereviewarticleby Gregorioetal.[4]providesaguidetohelpnewusersovercomethebarrierstoimplementingCFPS platformsinresearchlaboratories.CFPSplatformsderivedfromdiversemicroorganismsandcelllines canbedividedintotwocategories,includinghighadoptionandlowadoptionplatforms,byclarifying thesimilaritiesanddifferencesamongcell-freeplatforms.Variousapplicationshavebeenachievedby usingeachoftheseplatforms.Theauthorsalsoreviewmethodologicaldifferencesbetweenplatforms andtheinstrumentalrequirementsfortheirpreparation. Newuserscandeterminewhichtypeof cell-freeplatformcouldbeusedfortheirneeds. AnotherreviewarticlebyJeongetal.[5]summarizestheuseofcell-freeplatformsforengineering syntheticbiologicalcircuitsandsystems. Becausesyntheticbiologicalsystemshavebecomelarger andmorecomplex,decipheringtheintricateinteractionsofsyntheticsystemsandbiologicalentities is a challenging task. Cell-free synthetic biology approaches can facilitate rapid prototyping of syntheticcircuitsandexpeditetheexplorationofsyntheticsystemdesignsbeyondtheconfinesof livingorganisms.Cell-freeplatformscanalsoprovideasuitableplatformforthedevelopmentofDNA nanostructures,riboregulators,andartificialcells,andcanenablevalidationofmathematicalmodels forunderstandingbiologicalregulation. Incorporatingnonstandardaminoacidsintoproteinsisanimportanttechnologytoimprovethe understandingofbiologicalsystemsaswellastocreatenovelproteinswithnewchemicalproperties, structures,andfunctions.ImprovementsinCFPSsystemshavepavedthewaytoaccurateandefficient incorporationofnonstandardaminoacidsintoproteins[6].Gaoetal.[7]describearapidandsimple methodtosynthesizeunnaturalproteinsinaCFPSsystembasedonEscherichiacolicrudeextractby usinganunnaturalorthogonaltranslationalmachinery.Thisprotocolprovidesadetailedprocedure forusingaCFPSsystemtosynthesizeunnaturalproteinsondemand. InCFPSsystems,theactivityofthecrudeextractiscrucialtoensurehigh-yieldproteinsynthesis andtominimizebatch-to-batchvariationsinthecell-freereaction.Kimetal.[8]describeapractical MethodsProtoc.2019,2,80;doi:10.3390/mps2040080 1 www.mdpi.com/journal/mps
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