Abstract
Efficient DNA assembly is of great value in biological research and biotechnology. Type IIS restriction enzyme-based assembly systems allow assembly of multiple DNA fragments in a one-pot reaction. However, large DNA fragments can only be assembled by alternating use of two or more type IIS restriction enzymes in a multi-step approach. Here, we present MetClo, a DNA assembly method that uses only a single type IIS restriction enzyme for hierarchical DNA assembly. The method is based on in vivo methylation-mediated on/off switching of type IIS restriction enzyme recognition sites that overlap with site-specific methylase recognition sequences. We have developed practical MetClo systems for the type IIS enzymes BsaI, BpiI and LguI, and demonstrated hierarchical assembly of large DNA fragments up to 218 kb. The MetClo approach substantially reduces the need to remove internal restriction sites from components to be assembled. The use of a single type IIS enzyme throughout the different stages of DNA assembly allows novel and powerful design schemes for rapid large-scale hierarchical DNA assembly. The BsaI-based MetClo system is backward-compatible with component libraries of most of the existing type IIS restriction enzyme-based assembly systems, and has potential to become a standard for modular DNA assembly.
Highlights
Advances in biology and biotechnology have led to development of a variety of methods for assembly of large DNA constructs [1,2]
The enzyme-based methods generally leave shorter scar sequences defined by the enzyme recognition sequence or the adhesive end generated by the enzyme, but the DNA fragment to be assembled needs to be free of the recognition sequence of the enzymes used
A standard MetClo modular assembly vector set Because MetClo uses a single type IIS restriction enzyme throughout DNA assembly, DNA parts with compatible adhesive ends from different stages of the assembly can be assembled with suitable assembly vectors
Summary
Advances in biology and biotechnology have led to development of a variety of methods for assembly of large DNA constructs [1,2]. Restriction enzyme- or recombinase-based methods, which use defined enzyme-specific sequences to specify the order of assembly, such as Biobrick and Golden gate assembly [6,7,8]. Among the existing assembly methods, the type IIS restriction enzyme-based DNA assembly approach, such as Golden Gate assembly, is attractive for modular DNA assembly because circular double stranded DNA can be used as an input, it is a simple one-pot reaction, and it leaves a relatively small 3–4 bp scar between assembled fragments [8]. The Golden Gate assembly method relies on the ability of type IIS restriction enzymes to cut DNA at a position outside their recognition sequence. We report the design of a fast and powerful hierarchical assembly method named MetClo which uses only one type IIS restriction enzyme in the assembly process. We constructed a set of standard BsaI-based MetClo vectors for flexible DNA assembly that is backward compatible with existing part libraries
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