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dc.contributor.authorIverson, Sonya Victoriaen_US
dc.date.accessioned2016-02-23T13:46:50Z
dc.date.available2016-02-23T13:46:50Z
dc.date.issued2016
dc.identifier.urihttps://hdl.handle.net/2144/14554
dc.description.abstractDNA assembly and rational design are cornerstones of synthetic biology. While many DNA assembly standards have been published in recent years, only the Modular Cloning standard, or MoClo, has the advantage of publicly available part libraries for use in plant, yeast, and mammalian systems. No multipart modular library has previously been developed for use in prokaryotes. Building upon the existing MoClo assembly framework, we developed a collection of DNA parts and optimized MoClo protocols for use in E. coli. We present this assembly standard and library along with part characterization, design strategies, potential applications, and troubleshooting. Developed as part of the Cross-disciplinary Integration of Design Automation Research (CIDAR) lab collection of tools, the CIDAR MoClo Library is publicly available and contains promoters, ribosomal binding sites, coding sequences, terminators, vectors, and a set of fluorescent control plasmids. Optimized protocols reduce reaction time and cost by >80% from previously published protocols. The CIDAR MoClo Library is the first bacterial DNA part library compatible with a multipart assembly standard. To demonstrate the utility of the CIDAR MoClo system in a traditional biology context, we used the library and previous expression data to create a series of dual expression plasmids. In this manner, we produced a dual expression plasmid capable of expressing equimolar amounts of two variants of rabbit aldolase, a His-tagged wildtype protein and a single-amino-acid substitution mutant deficient in binding actin. This expression plasmid will enable the production of dimer-of-dimer heterotetramers needed for structural determination of the actin-aldolase interaction by electron microscopy. To employ CIDAR MoClo in a synthetic biology context, we produced a bioelectronic pH-mediated genetic logic gate with DNA circuits built using MoClo and integrated with Raspberry Pi computers, Twitter, and 3D printed components. Logic gates are an increasingly common biological tool with applications in cellular memory and biological computation. MoClo facilitates rapid iteration of genetic designs, better enabling the development of cellular logic. The CIDAR MoClo Library and assembly standard enable rapid design-build-test cycles in E. coli making this system advantageous for use in many areas of synthetic biology as well as traditional biological research.en_US
dc.language.isoen_US
dc.rightsAttribution 4.0 Internationalen_US
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectBiomedical engineeringen_US
dc.subjectDNA assemblyen_US
dc.subjectDNA libraryen_US
dc.subjectCloningen_US
dc.subjectGenetic engineeringen_US
dc.subjectModular cloningen_US
dc.subjectSynthetic biologyen_US
dc.titleImproved modular multipart DNA assembly, development of a DNA part toolkit for E. coli, and applications in traditional biology and bioelectronic systemsen_US
dc.typeThesis/Dissertationen_US
dc.date.updated2016-02-13T02:22:02Z
etd.degree.nameDoctor of Philosophyen_US
etd.degree.leveldoctoralen_US
etd.degree.disciplineMolecular Biology, Cell Biology & Biochemistryen_US
etd.degree.grantorBoston Universityen_US


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Attribution 4.0 International
Except where otherwise noted, this item's license is described as Attribution 4.0 International