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dc.contributor.authorZou, Ruiyang
dc.contributor.authorZhou, Kang
dc.contributor.authorStephanopoulos, Gregory
dc.contributor.authorToo, Heng Phon
dc.date.accessioned2014-01-10T18:54:30Z
dc.date.available2014-01-10T18:54:30Z
dc.date.issued2013-11
dc.date.submitted2013-07
dc.identifier.issn1932-6203
dc.identifier.urihttp://hdl.handle.net/1721.1/83864
dc.description.abstractThe ability to assemble multiple fragments of DNA into a plasmid in a single step is invaluable to studies in metabolic engineering and synthetic biology. Using phosphorothioate chemistry for high efficiency and site specific cleavage of sequences, a novel ligase independent cloning method (cross-lapping in vitro assembly, CLIVA) was systematically and rationally optimized in E. coli. A series of 16 constructs combinatorially expressing genes encoding enzymes in the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway were assembled using multiple DNA modules. A plasmid (21.6 kb) containing 16 pathway genes, was successfully assembled from 7 modules with high efficiency (2.0 x 10[superscript 3] cfu/ µg input DNA) within 2 days. Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene. Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC), an intermediate metabolite in the DXP pathway. The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene. This study demonstrated the utility of CLIVA in the assembly of multiple fragments of DNA into a plasmid which enabled the rapid exploration of biological pathways.en_US
dc.description.sponsorshipSingapore-MIT Allianceen_US
dc.language.isoen_US
dc.publisherPublic Library of Scienceen_US
dc.relation.isversionofhttp://dx.doi.org/10.1371/journal.pone.0079557en_US
dc.rights.urihttp://creativecommons.org/licenses/by/2.5/en_US
dc.sourcePLoSen_US
dc.titleCombinatorial Engineering of 1-Deoxy-D-Xylulose 5-Phosphate Pathway Using Cross-Lapping In Vitro Assembly (CLIVA) Methoden_US
dc.typeArticleen_US
dc.identifier.citationZou, Ruiyang, Kang Zhou, Gregory Stephanopoulos, and Heng Phon Too. “Combinatorial Engineering of 1-Deoxy-D-Xylulose 5-Phosphate Pathway Using Cross-Lapping In Vitro Assembly (CLIVA) Method.” Edited by Jorg D. Hoheisel. PLoS ONE 8, no. 11 (November 5, 2013): e79557.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemical Engineeringen_US
dc.contributor.departmentSingapore-MIT Alliance in Research and Technology (SMART)en_US
dc.contributor.mitauthorZhou, Kangen_US
dc.contributor.mitauthorStephanopoulos, Gregoryen_US
dc.relation.journalPLoS ONEen_US
dc.eprint.versionFinal published versionen_US
dc.type.urihttp://purl.org/eprint/type/JournalArticleen_US
eprint.statushttp://purl.org/eprint/status/PeerRevieweden_US
dspace.orderedauthorsZou, Ruiyang; Zhou, Kang; Stephanopoulos, Gregory; Too, Heng Phonen_US
dc.identifier.orcidhttps://orcid.org/0000-0001-6909-4568
mit.licensePUBLISHER_CCen_US
mit.metadata.statusComplete


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