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dc.contributor.authorReeves, Analise Z.en_US
dc.contributor.authorSpears, William E.en_US
dc.contributor.authorDu, Juanen_US
dc.contributor.authorTan, Kah Yongen_US
dc.contributor.authorWagers, Amy J.en_US
dc.contributor.authorLesser, Cammie F.en_US
dc.date.accessioned2015-08-03T13:58:57Z
dc.date.issued2015en_US
dc.identifier.citationReeves, Analise Z., William E. Spears, Juan Du, Kah Yong Tan, Amy J. Wagers, and Cammie F. Lesser. 2015. “Engineering Escherichia coli into a Protein Delivery System for Mammalian Cells.” ACS Synthetic Biology 4 (5): 644-654. doi:10.1021/acssynbio.5b00002. http://dx.doi.org/10.1021/acssynbio.5b00002.en
dc.identifier.issn2161-5063en
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:17820642
dc.description.abstractMany Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.en
dc.language.isoen_USen
dc.publisherAmerican Chemical Societyen
dc.relation.isversionofdoi:10.1021/acssynbio.5b00002en
dc.relation.hasversionhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487226/pdf/en
dash.licenseLAAen_US
dc.subjectsynthetic biologyen
dc.subjecttype 3 secretion systemen
dc.subjectprotein deliveryen
dc.subjectbacterial engineeringen
dc.titleEngineering Escherichia coli into a Protein Delivery System for Mammalian Cellsen
dc.typeJournal Articleen_US
dc.description.versionVersion of Recorden
dc.relation.journalACS Synthetic Biologyen
dash.depositing.authorDu, Juanen_US
dc.date.available2015-08-03T13:58:57Z
dc.identifier.doi10.1021/acssynbio.5b00002*
dash.contributor.affiliatedDu, Juan
dash.contributor.affiliatedLesser, Cammie
dash.contributor.affiliatedWagers, Amy


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