Abstract
Assembly of DNA ‘parts’ to create larger constructs is an essential enabling technique for bioengineering and synthetic biology. Here we describe a simple method, PaperClip, which allows flexible assembly of multiple DNA parts from currently existing libraries cloned in any vector. No restriction enzymes, mutagenesis of internal restriction sites, or reamplification to add end homology are required. Order of assembly is directed by double stranded oligonucleotides—‘Clips’. Clips are formed by ligation of pairs of oligonucleotides corresponding to the ends of each part. PaperClip assembly can be performed by polymerase chain reaction or by cell extract-mediated recombination. Once multi-use Clips have been prepared, assembly of at least six DNA parts in any order can be accomplished with high efficiency within several hours.
Highlights
Of DNA sequences together is a critical enabling technology for synthetic biology
Linker based methods such as GenBrick (BBF RFC98) and MODAL [14] overcome some of these issues through a pre-assembly step in which oligonucleotide linkers are ligated to the ends generated by Type IIS restriction enzymes, allowing rapid flexible assembly of multiple parts, but still require libraries to be cloned in specific donor plasmids and to be free of internal sites for the relevant enzymes
The order of the DNA parts in PaperClip assembly is determined by multi-use bridging double-stranded oligonucleotides, designated ‘Clips’
Summary
Of DNA sequences (parts) together is a critical enabling technology for synthetic biology. The homology regions must usually be added by polymerase chain reaction (PCR) amplification prior to assembly, requiring new primers and sequence confirmation of products for each assembly Methods such as MoClo [6], the original Golden Gate [7] and GoldenBraid [8,9], based on Type IIS restriction enzymes generating user-defined overhangs, are rapid but may require mutagenesis of internal restriction sites. BioBrickTM-like methods (BBF RFC10) [13] are completely flexible in terms of part order, but require relatively slow pairwise assembly, and all parts must be free of internal sites for the relevant restriction enzymes Linker based methods such as GenBrick (BBF RFC98) and MODAL [14] overcome some of these issues through a pre-assembly step in which oligonucleotide linkers are ligated to the ends generated by Type IIS restriction enzymes, allowing rapid flexible assembly of multiple parts, but still require libraries to be cloned in specific donor plasmids and to be free of internal sites for the relevant enzymes. We demonstrate assembly by PCR and by Escherichia coli cell extract expressing recombination proteins
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