Imperial College London

ProfessorPaulFreemont

Faculty of MedicineDepartment of Medicine

Chair in Protein Crystallography
 
 
 
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Contact

 

+44 (0)20 7594 5327p.freemont

 
 
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Location

 

259Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Citation

BibTex format

@article{Moore:2016:10.1021/acssynbio.6b00031,
author = {Moore, SJ and lai, H-E and Kelwick, R and Mei, S and Bell, DJ and Polizzi, K and Freemont, PS},
doi = {10.1021/acssynbio.6b00031},
journal = {ACS Synthetic Biology},
pages = {1059--1069},
title = {EcoFlex - a multifunctional MoClo kit for E. coli synthetic biology},
url = {http://dx.doi.org/10.1021/acssynbio.6b00031},
volume = {5},
year = {2016}
}

RIS format (EndNote, RefMan)

TY  - JOUR
AB - 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.
AU - Moore,SJ
AU - lai,H-E
AU - Kelwick,R
AU - Mei,S
AU - Bell,DJ
AU - Polizzi,K
AU - Freemont,PS
DO - 10.1021/acssynbio.6b00031
EP - 1069
PY - 2016///
SN - 2161-5063
SP - 1059
TI - EcoFlex - a multifunctional MoClo kit for E. coli synthetic biology
T2 - ACS Synthetic Biology
UR - http://dx.doi.org/10.1021/acssynbio.6b00031
UR - http://hdl.handle.net/10044/1/31290
VL - 5
ER -