Cell-Free Synthetic Biology

154 Pages·2020·3.241 MB·English

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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.:[phone]
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