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EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology

Moore, Simon J., Lai, Hung-En, Kelwick, Richard J. R., Chee, Soo Mei, Bell, David J., Polizzi, Karen Marie, Freemont, Paul S. (2016) EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology. ACS Synthetic Biology, 5 (10). pp. 1059-1069. ISSN 2161-5063. (doi:10.1021/acssynbio.6b00031) (KAR id:69453)

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https://doi.org/10.1021/acssynbio.6b00031

Abstract

Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimisation, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli, has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterise in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimise pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimisation. In summary, EcoFlex provides a standardised and multifunctional kit for a variety of applications in E. coli synthetic biology.

Item Type: Article
DOI/Identification number: 10.1021/acssynbio.6b00031
Uncontrolled keywords: Synthetic biology, Golden Gate, Escherichia coli, recombinant protein production, cell-free protein synthesis, violacei
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Simon Moore
Date Deposited: 09 Oct 2018 10:36 UTC
Last Modified: 08 Jul 2019 10:15 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/69453 (The current URI for this page, for reference purposes)
Moore, Simon J.: https://orcid.org/0000-0002-1968-206X
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