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EcoFlex - a multifunctional MoClo kit for E. coli synthetic biology

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Title: EcoFlex - a multifunctional MoClo kit for E. coli synthetic biology
Authors: Moore, SJ
Lai, H-E
Kelwick, R
Mei, S
Bell, DJ
Polizzi, K
Freemont, PS
Item Type: Journal Article
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.
Issue Date: 21-Oct-2016
Date of Acceptance: 1-Apr-2016
URI: http://hdl.handle.net/10044/1/31290
DOI: https://dx.doi.org/10.1021/acssynbio.6b00031
ISSN: 2161-5063
Publisher: American Chemical Society
Start Page: 1059
End Page: 1069
Journal / Book Title: ACS Synthetic Biology
Volume: 5
Issue: 10
Copyright Statement: © 2016 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY - https://creativecommons.org/licenses/by/4.0/) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
Sponsor/Funder: Engineering & Physical Science Research Council (E
Engineering & Physical Science Research Council (EPSRC)
Engineering & Physical Science Research Council (EPSRC)
Biotechnology and Biological Sciences Research Council (BBSRC)
Imperial College London
Funder's Grant Number: EP/J02175X/1
EP/K038648/1
EP/L011573/1
BB/L027852/1
Imperial College PhD Scholarship
Keywords: Science & Technology
Life Sciences & Biomedicine
Biochemical Research Methods
Biochemistry & Molecular Biology
synthetic biology
Golden Gate
Escherichia coli
recombinant protein production
cell-free protein synthesis
violacein
GENE-EXPRESSION
ONE-POT
CLONING
TRANSCRIPTION
BIOSYNTHESIS
CEREVISIAE
VIOLACEIN
PATHWAYS
STANDARD
SEQUENCE
Gene Library
Genetic Engineering
Genetic Vectors
Indoles
Promoter Regions, Genetic
Recombinant Proteins
Reproducibility of Results
Synthetic Biology
Publication Status: Published
Online Publication Date: 2016-04-20
Appears in Collections:Department of Medicine
Faculty of Medicine
Faculty of Natural Sciences



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