Introduction

Golden Gate Assembly is a widely used DNA assembly method that enables the insertion of multiple DNA fragments into a vector in a single, one-pot reaction.^[1] In this strategy, DNA parts from so called donor vectors are inserted in an acceptor vector, making it especially useful for assembling modular systems.^[2]

This technique relies on Type IIS restriction enzymes, such as BsaI, which recognize a specific DNA sequence but cut a defined number of nucleotides away from it. This property allows the design of custom single-stranded overhangs. In a single restriction–ligation reaction with DNA ligase, excised fragments from donor vectors flanked by these overhangs can be assembled in a predefined order into a compatible backbone of an acceptor vector. Because the recognition sites are removed during assembly, the final recombinant plasmid contains none of these restriction sites anymore, enabling efficient cloning of multiple fragments in one step (Fig. 1)^[2][3].

Components necessary for the Golden Gate Assembly reaction and their functions:^[3]

• Type IIS Restriction Enzyme – recognizes specific DNA sequences but cut outside of them

• DNA Ligase – seals the remaining nicks, generating a continuous DNA molecule

Schematic Workflow

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Applications in the NRPieceS Project

We applied Golden Gate cloning to mix and match NRPieceS donor modules from our library, which contain BsaI recognition sites, into native clusters serving as acceptor vectors. This allowed us to construct unique NRPS that could potentially produce new-to-nature peptides.To learn more about how we applied this principle in building our library, visit our Engineering Page, or explore our experimental outcomes on the Wet Lab Results Page.