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Single Day Construction of Multigene Circuits with 3G Assembly

Halleran, Andrew D. and Swaminathan, Anandh and Murray, Richard M. (2018) Single Day Construction of Multigene Circuits with 3G Assembly. ACS Synthetic Biology, 7 (5). pp. 1477-1480. ISSN 2161-5063. https://resolver.caltech.edu/CaltechAUTHORS:20180409-133942183

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Abstract

The ability to rapidly design, build, and test prototypes is of key importance to every engineering discipline. DNA assembly often serves as a rate limiting step of the prototyping cycle for synthetic biology. Recently developed DNA assembly methods such as isothermal assembly and type IIS restriction enzyme systems take different approaches to accelerate DNA construction. We introduce a hybrid method, Golden Gate-Gibson (3G), that takes advantage of modular part libraries introduced by type IIS restriction enzyme systems and isothermal assembly’s ability to build large DNA constructs in single pot reactions. Our method is highly efficient and rapid, facilitating construction of entire multigene circuits in a single day. Additionally, 3G allows generation of variant libraries enabling efficient screening of different possible circuit constructions. We characterize the efficiency and accuracy of 3G assembly for various construct sizes, and demonstrate 3G by characterizing variants of an inducible cell-lysis circuit.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/acssynbio.8b00060DOIArticle
https://pubs.acs.org/doi/suppl/10.1021/acssynbio.8b00060PublisherSupporting Information
https://doi.org/10.1101/260851DOIDiscussion Paper
ORCID:
AuthorORCID
Halleran, Andrew D.0000-0001-8720-1451
Swaminathan, Anandh0000-0001-9935-6530
Murray, Richard M.0000-0002-5785-7481
Alternate Title:Single day construction of multi-gene circuits with 3G assembly
Additional Information:© 2018 American Chemical Society. Received: February 9, 2018; Published: May 1, 2018. pSal, pLas, NahR, and LasR plasmids were generously provided by Adam Meyer.(16) Plasmid vectors were provided by Douglas Densmore at the Cross-disciplinary Integration of Design Automation Research lab (Addgene Kit # 1000000059). The project depicted was sponsored by the Defense Advanced Research Projects Agency (Agreement HR0011-17-2-0008). The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. The authors declare no competing financial interest.
Funders:
Funding AgencyGrant Number
Defense Advanced Research Projects Agency (DARPA)HR0011-17-2-0008
Subject Keywords:assembly methods, genetic circuits, Golden Gate assembly, Gibson assembly
Issue or Number:5
Record Number:CaltechAUTHORS:20180409-133942183
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180409-133942183
Official Citation:Single Day Construction of Multigene Circuits with 3G Assembly. Andrew D. Halleran, Anandh Swaminathan, and Richard M. Murray. ACS Synthetic Biology 2018 7 (5), 1477-1480. DOI: 10.1021/acssynbio.8b00060
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:85692
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:09 Apr 2018 21:14
Last Modified:09 Mar 2020 13:19

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