Abstract
Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.
Highlights
Recombinant DNA technologies have been critical for driving biotechnological advances and facilitating studies aimed at understanding basic biological principles
E. coli is able to recombine DNA fragments that share more than ~20-base end homology and the efficiency increases with increasing homology length regardless of the recombination mechanism [2,16,22,23,25,28]
Bubeck et al previously showed that E. coli DH5α has the ability to recombine linear DNA fragments sharing short homologous ends, making it possible to carry out simple in vivo fragment assembly [25]
Summary
Recombinant DNA technologies have been critical for driving biotechnological advances and facilitating studies aimed at understanding basic biological principles. Techniques for the seamless assembly of DNA have been rapidly expanding, enabling more precise genetic manipulation in synthetic biology and metabolic engineering. Many of these methods rely on the annealing of strands from neighboring DNA fragments, allowing for assembly of fragments with overlapping ends. Both in vitro [1,2,3,4,5,6,7,8,9,10] and in vivo [11,12,13,14,15,16,17,18,19,20,21] techniques have been developed for this purpose.
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