Table Of ContentMETHODS IN ENZYMOLOGY
Editors-in-Chief
JOHN N. ABELSON MELVIN I. SIMON
AND
Division of Biology
California Institute of Technology
Pasadena, California
Founding Editors
SIDNEY P. COLOWICK NATHAN O. KAPLAN
AND
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C
ONTRIBUTORS
LauraAdam
VirginiaBioinformaticsInstitute,VirginiaTech,Blacksburg,Virginia,USA
RivkaAdar
WeizmannInstituteofScience,BiologicalChemistryDepartment,Rehovot,Israel
AndreuAlibe´s
EMBL/CRG Systems Biology Research Unit, Center for Genomic Regulation
(CRG),UPF,Barcelona,Spain
J.ChristopherAnderson
Department of Bioengineering; SynBERC: Synthetic Biology Engineering
ResearchCenter;QB3:CaliforniaInstituteforQuantitativeBiologicalResearch,
Emeryville, and Physical Biosciences Division, Lawrence Berkeley National
Laboratory,UniversityofCalifornia,Berkeley,California,USA
AseemZ.Ansari
Department of Biochemistry, and Genome Center, University of Wisconsin,
Madison,Wisconsin,USA
SwapnilBhatia
DepartmentofElectricalandComputerEngineering,BostonUniversity,Boston,
Massachusetts,USA
LesiaBilitchenko
Department of Computer Science, California State Polytechnic University,
Pomona,California,USA
JenniferBrophy
Department of Bioengineering, University of California, Berkeley, California,
USA
BenBubenheim
Department of Bioengineering, University of California, Berkeley, California,
USA
GeorgeM.Church
DepartmentofGenetics,HarvardMedicalSchool,Boston,andWyssInstitutefor
BiologicallyInspiredEngineering,HarvardUniversity,Massachusetts,USA
xi
xii Contributors
MaisamDadgar
DepartmentofBiomedicalEngineering,BostonUniversity,Boston,Massachusetts,
USA
SarahE.Davis
Department of Biochemistry, and Genome Center, University of Wisconsin,
Madison,Wisconsin,USA
DouglasDensmore
DepartmentofElectricalandComputerEngineering,andDepartmentofBiomed-
icalEngineering,BostonUniversity,Boston,Massachusetts,USA
AndrewD.Ellington
Applied ResearchLaboratories, and Institute for Cellular and Molecular Biology,
TheUniversityofTexasatAustin,Austin,Texas,USA
DanielG.Gibson1
J.CraigVenterInstituteInc.,SyntheticBiologyGroup,LaJolla,California,USA
JeffGrass
DepartmentofBiochemistry,UniversityofWisconsin,Madison,Wisconsin,USA
IlanGronau
WeizmannInstituteofScience,ComputerScienceandMathematicsDepartment,
Rehovot,Israel
ClaesGustafsson
DNA2.0,Inc.,SuiteA,MenloPark,California,USA
RussellHertzberg
VirginiaBioinformaticsInstitute,VirginiaTech,Blacksburg,Virginia,USA
TonyHo
LifeTechnologiesCorporation,Carlsbad,California,USA
RandallA.Hughes
Applied ResearchLaboratories, and Institute for Cellular and Molecular Biology,
TheUniversityofTexasatAustin,Austin,Texas,USA
MitsuhiroItaya
InstituteforAdvancedBiosciencesofKeioUniversity,Nipponkoku,Tsuruoka-shi,
Yamagata,Japan
ElenitaI.Kanin
Department of Biochemistry, and Genome Center, University of Wisconsin,
Madison,Wisconsin,USA
1 Presentaddress:DanielG.Gibson,ScienceCenterDrive,SanDiego,CA
Contributors xiii
ShaiKaplan
WeizmannInstituteofScience,BiologicalChemistryDepartment,andComputer
ScienceandMathematicsDepartment,Rehovot,Israel
FedericoKatzen
LifeTechnologiesCorporation,Carlsbad,California,USA
YiannisN.Kaznessis
DepartmentofChemicalEngineeringandMaterialsScience,andDigitalTechnol-
ogyCenter,UniversityofMinnesota,Minneapolis,Minnesota,USA
TaeYongKim
MetabolicandBiomolecularEngineeringNationalResearchLaboratory,Department
of Chemical and Biomolecular Engineering (BK21 Program), and Bioinformatics
ResearchCenter,KAIST,Daejeon,RepublicofKorea
ThomasF.Knight2
ComputerScienceandArtificialIntelligenceLaboratory,MassachusettsInstituteof
Technology,Cambridge,Massachusetts,USA
WieslawKudlicki
LifeTechnologiesCorporation,Carlsbad,California,USA
RobertLandick
Department of Biochemistry, and Department of Bacteriology, University of
Wisconsin,Madison,Wisconsin,USA
SangYupLee
MetabolicandBiomolecularEngineeringNationalResearchLaboratory,Depart-
ment of Chemical and Biomolecular Engineering (BK21 Program); BioProcess
Engineering Research Center, Center for Systems and Synthetic Biotechnology,
InstitutefortheBioCentury;BioinformaticsResearchCenter,andDepartmentof
BioandBrainEngineering,KAIST,Daejeon,RepublicofKorea
MarianaLeguia
DepartmentofBioengineering;QB3:CaliforniaInstituteforQuantitativeBiological
Research, and Synthetic Biology Engineering Research Center, University of
California,Berkeley,California,USA
KeLi
LifeTechnologiesCorporation,Carlsbad,California,USA
XiquanLiang
LifeTechnologiesCorporation,Carlsbad,California,USA
2 Currentaddress:GinkgoBioWorks,Inc.,Boston,Massachusetts,USA
xiv Contributors
GregoryLinshiz
WeizmannInstituteofScience,BiologicalChemistryDepartment,andComputer
ScienceandMathematicsDepartment,Rehovot,Israel
MichaelLiss
LifeTechnologiesInc./GeneArtAG,Regensburg,Germany
AdamLiu
Department of Electrical Engineering and Computer Sciences, University of
California,Berkeley,California,USA
MeaganLizarazo
Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute
ofTechnology,Cambridge,Massachusetts,USA
AleksandrE.Miklos
Applied ResearchLaboratories, and Institute for Cellular and Molecular Biology,
TheUniversityofTexasatAustin,Austin,Texas,USA
JeremyMinshull
DNA2.0,Inc.,SuiteA,MenloPark,California,USA
KentaroMiyazaki
Bioproduction Research Institute, National Institute of Advanced Industrial
ScienceandTechnology(AIST),Central6,1-1-1Higashi,Tsukuba,Ibaraki,Japan
RachelA.Mooney
DepartmentofBiochemistry,UniversityofWisconsin,Madison,Wisconsin,USA
AlejandroD.Nadra
Departamentos de Qu´ımica Biolo´gica y Fisiolog´ıa, Biolog´ıa Molecular y Celular,
FacultaddeCienciasExactasyNaturales,UniversidaddeBuenosAires,Argentina
FrankNotka
LifeTechnologiesInc./GeneArtAG,Regensburg,Germany
JongMyoungPark
MetabolicandBiomolecularEngineeringNationalResearchLaboratory,Depart-
mentofChemicalandBiomolecularEngineering(BK21Program),andBioProcess
Engineering Research Center, Center for Systems and Synthetic Biotechnology,
InstitutefortheBioCentury,KAIST,Daejeon,RepublicofKorea
JeanPeccoud
VirginiaBioinformaticsInstitute,VirginiaTech,Blacksburg,Virginia,USA
LanshaPeng
LifeTechnologiesCorporation,Carlsbad,California,USA
ToddPeterson
LifeTechnologiesCorporation,Carlsbad,California,USA
Contributors xv
JasonPotter
LifeTechnologiesCorporation,Carlsbad,California,USA
SivanRavid
WeizmannInstituteofScience,ComputerScienceandMathematicsDepartment,
Rehovot,Israel
RandyRettberg
Department of Biological Engineering, and Computer Science and Artificial
Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge,
Massachusetts,USA
HowardM.Salis
Department of Chemical Engineering, and Department of Agricultural and
Biological Engineering, Pennsylvania State University, University Park,
Pennsylvania,USA
LuisSerrano
EMBL/CRG Systems Biology ResearchUnit, and ICREA Professor,Centerfor
GenomicRegulation(CRG),UPF,Barcelona,Spain
EhudShapiro
Department of Biological Chemistry, and Department of Computer Science and
AppliedMathematics,WeizmannInstituteofScience,Rehovot,Israel
ReshmaShetty3
Department of Biological Engineering, Massachusetts Institute of Technology,
Cambridge,Massachusetts,USA
JoshShirley
LifeTechnologiesCorporation,Carlsbad,California,USA
TetsuroToyoda
BioinformaticsandSystemsEngineeringdivision,RIKEN,Yokohama,Kanagawa,
Japan
KenjiTsuge
Institute for Advanced Biosciences of Keio University, Nipponkoku, Tsuruoka-
shi,Yamagata,Japan
BillyanaTsvetanova
LifeTechnologiesCorporation,Carlsbad,California,USA
AlanVillalobos
DNA2.0,Inc.,SuiteA,MenloPark,California,USA
3 Currentaddress:GinkgoBioWorks,Inc.,Boston,Massachusetts,USA
xvi Contributors
RalfWagner
Life Technologies Inc./GeneArt AG, and Institute of Medical Microbiology and
Hygiene,MolecularMicrobiologyandGeneTherapy,UniversityofRegensburg,
Regensburg,Germany
HarrisH.Wang
DepartmentofGenetics,HarvardMedicalSchool,Boston,andWyssInstitutefor
BiologicallyInspiredEngineering,HarvardUniversity,Massachusetts,USA
MarkWelch
DNA2.0,Inc.,SuiteA,MenloPark,California,USA
MandyL.Wilson
VirginiaBioinformaticsInstitute,VirginiaTech,Blacksburg,Virginia,USA
BingXia
DepartmentofBioengineering,UniversityofCalifornia,Berkeley,California,USA
LieweiXu
LifeTechnologiesCorporation,Carlsbad,California,USA
Jian-PingYang
LifeTechnologiesCorporation,Carlsbad,California,USA
TuvalBenYehezkel
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot,
Israel
P
REFACE
Thisisthesecondvolumeofatwo-partseriesinMethodsinEnzymologyon
tools and techniques used in synthetic biology. Synthetic biology is an
engineering discipline that seeks to construct living systems that do not
existinnature.Thefieldreferstotheprocessbywhichgeneticsystemsare
designed and constructed, as opposed to any particular application. Along
theselines,thesevolumesareorganizedintotwoareas.VolumeIfocuseson
theassaytechniquesanddesignprinciplesunderlyingthecharacterizationof
genetic parts, their combination into devices and programs, and their inte-
gration into various hosts. Volume II focuses on computational tools and
biophysicalmodelstoaidinthedesignandorganizationofgeneticprograms
andmodernmethodstosynthesizeandassembletheassociatedDNA.
The first set of chapters focus on computational tools to predict the
functionofgeneticpartsandtoorganizethemintomorecomplexsystems.
Biophysical methods for modeling common genetic parts are described,
includingpromoters,codonusage,andribosomebindingsites.Computer-
aided design (CAD) will become an increasingly important tool for
synthetic biology, as designs become larger and more complex. Several
academic simulation packages have emerged (e.g., Clotho and SynBioSS)
thatenablethepredictionofhowmultiplegeneticpartswillbehave.Several
innovativegrammars(e.g.,EUGENEandGenoCAD)havebeenproposed
that simplify the rules for the combination of genetic parts in a way that
enables desires to organize large systems and to facilitate the sharing of
designs. Ultimately, these methods will need to be able to be applied to
genome-scale engineering.
The output of the CAD programs is a DNA sequence that needs to be
assembled. The combination of automated DNA synthesis with modern
cloning techniques has enabled a revolution in the ambition of the scale of
geneticconstructs.Severalmethodsforgenesynthesisaredescribed,including
error correction, which becomes critical for large constructs. The “Gibson
Method” and variations thereof provide a convenient one-step method for
theassemblyofmultiplegeneticparts.Thisbuildsonrestrictionenzyme-based
methods, such as BioBricksTM assembly. Our lab has made great use of the
MEGAWHOP method for rapidly inserting large pieces of DNA into a
plasmid(anybodyfamiliarwithQuickchangewillgetthegistofthemethod).
Synthetic biology ultimately needs to work at the scale of whole-
genomeengineering.Methodsaredescribedfortheassembly,engineering,
and analysis of genome-sized fragments of DNA.
xvii
M E
ETHODS IN NZYMOLOGY
VOLUMEI.PreparationandAssayofEnzymes
EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN
VOLUMEII.PreparationandAssayofEnzymes
EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN
VOLUMEIII.PreparationandAssayofSubstrates
EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN
VOLUMEIV.SpecialTechniquesfortheEnzymologist
EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN
VOLUMEV.PreparationandAssayofEnzymes
EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN
VOLUMEVI.PreparationandAssayofEnzymes(Continued)
PreparationandAssayofSubstrates
SpecialTechniques
EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN
VOLUMEVII.CumulativeSubjectIndex
EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN
VOLUMEVIII.ComplexCarbohydrates
EditedbyELIZABETHF.NEUFELDANDVICTORGINSBURG
VOLUMEIX.CarbohydrateMetabolism
EditedbyWILLISA.WOOD
VOLUMEX.OxidationandPhosphorylation
EditedbyRONALDW.ESTABROOKANDMAYNARDE.PULLMAN
VOLUMEXI.EnzymeStructure
EditedbyC.H.W.HIRS
VOLUMEXII.NucleicAcids(PartsAandB)
EditedbyLAWRENCEGROSSMANANDKIVIEMOLDAVE
VOLUMEXIII.CitricAcidCycle
EditedbyJ.M.LOWENSTEIN
VOLUMEXIV.Lipids
EditedbyJ.M.LOWENSTEIN
VOLUMEXV.SteroidsandTerpenoids
EditedbyRAYMONDB.CLAYTON
xix
