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dc.contributor.authorOrtiz, Luisen_US
dc.contributor.authorPavan, Marileneen_US
dc.contributor.authorMcCarthy, Lloyden_US
dc.contributor.authorTimmons, Joshuaen_US
dc.contributor.authorDensmore, Douglas M.en_US
dc.coverage.spatialUnited Statesen_US
dc.date.accessioned2018-03-16T21:36:51Z
dc.date.available2018-03-16T21:36:51Z
dc.date.issued2017-12-01
dc.identifierhttps://www.ncbi.nlm.nih.gov/pubmed/29286379
dc.identifier.citationOrtiz, L., Pavan, M., McCarthy, L., Timmons, J., Densmore, D. M. Automated Robotic Liquid Handling Assembly of Modular DNA Devices. J. Vis. Exp. (130), e54703, doi:10.3791/54703 (2017).
dc.identifier.issn1940-087X
dc.identifier.urihttps://hdl.handle.net/2144/27554
dc.description.abstractRecent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available "design space" represented by "devices" created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction. Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols. The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.en_US
dc.languageengen_US
dc.relation.ispartofJ Vis Expen_US
dc.rightsCopyright © 2017 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported.
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/3.0/
dc.subjectScience & technologyen_US
dc.subjectBioengineeringen_US
dc.subjectSynthetic biologyen_US
dc.subjectAutomated liquid handlingen_US
dc.subjectRoboticsen_US
dc.subjectModular cloningen_US
dc.subjectAutomated DNA assemblyen_US
dc.subjectCombinatorial library assemblyen_US
dc.subjectBiofoundryen_US
dc.subjectCancer cellsen_US
dc.titleAutomated robotic liquid handling assembly of modular DNA devicesen_US
dc.typeArticle
dc.identifier.doi10.3791/54703
pubs.elements-sourcepubmeden_US
pubs.notesEmbargo: Not knownen_US
pubs.organisational-groupBoston Universityen_US
pubs.organisational-groupBoston University, College of Engineeringen_US
pubs.organisational-groupBoston University, College of Engineering, Department of Electrical & Computer Engineeringen_US
pubs.publication-statusPublished onlineen_US


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Copyright © 2017 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported.
Except where otherwise noted, this item's license is described as Copyright © 2017 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported.