EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.
Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimization, 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), characterize 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 minimize pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimization. In summary, EcoFlex provides a standardized and multifunctional kit for a variety of applications in E. coli synthetic biology.
Centre for Synthetic Biology and Innovation, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.|Department of Medicine, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.
Simon J
SJ
Moore
Centre for Synthetic Biology and Innovation, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.|Department of Medicine, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.
Hung-En
HE
Lai
Centre for Synthetic Biology and Innovation, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.|Department of Medicine, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.
Richard J R
RJ
Kelwick
Department of Medicine, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.
Soo Mei
SM
Chee
Department of Medicine, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.
David J
DJ
Bell
Centre for Synthetic Biology and Innovation, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.|Department of Life Sciences, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.
Karen Marie
KM
Polizzi
Centre for Synthetic Biology and Innovation, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.|Department of Medicine, Imperial College London , South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.
Paul S
PS
Freemont
D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D007211:Indoles / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D058615:Synthetic Biology / Q000379:methods*
D007211:Indoles; D011994:Recombinant Proteins; C063155:violacein
Escherichia coli; Golden Gate; cell-free protein synthesis; recombinant protein production; synthetic biology; violacein