Abstract
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.
Highlights
A prime target for synthetic biology is the production of natural and commodity chemical products such as drug precursors, fine and specialized chemicals and biofuels using renewable feedstocks.[1]
Golden Gate cloning, is a modular, combinatorial technique used for a wide spectrum of DNA assembly applications, from the construction of simple transcription units (TUs) to diverse biochemical pathway libraries originally developed by Marillonnet and colleagues.[3−5]
We desire to assemble a universal cloning system based on the MoClo standard for prokaryotic host expression to allow an open reading frames (ORF) or DNA part to be portable between multiple systems. To begin building this foundation, we reveal EcoFlex that is designed to provide users a flexible combinatorial library toolbox that can be customized for variety of synthetic biology applications in E. coli
Summary
Focusing on MoClo, this particular assembly scheme allows flexible design of multiple transcription units into modular pathways with each gene under the control of its own promoter, RBS and terminator. Golden Gate and MoClo assembly is renowned to be highly efficient and a restriction digest of DNA is generally considered acceptable for qualitycontrol checking.[33] In contrast, the mutations found in pMB1 and ColE1 variants generally represent deletions (probable recombination events), which form spontaneously during subculturing We speculate that these events are due to repetitive promoter, RBS and terminator sequences[16,34] used during assembly, and strong selection pressures caused by violacein production. Considering all variants of the EcoFlex library were included at approximately the same concentration at the initial Level 1 assembly, it is still unclear why certain terminators become more frequent at the final stage of selection of violacein CFUs. While the EcoFlex kit is primarily designed for E. coli, we are interested in expanding this toolkit toward a universal cloning system between alternative prokaryotic hosts that offer attractive advantages in biotechnology, such as thermophilic growth, utilization of alternative carbon feedstocks, resistance to growth-inhibitory metabolites and high titer natural product synthesis. Table S1−S14 and Figures S1−S13, as well as oligonucleotide and DNA part sequences. (PDF)
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