Golden Gate Assembly of Transcriptional Unit Libraries into a Rearrangeable Gene Cluster

Both regulatory sequences and genome organization contribute to the production of diverse transcript isoforms, which can influence how genes, or sets of genes, are expressed. An efficient, modular approach is needed to generate the combinatorial complexity required to empirically test many combinations of different regulatory sequences and different gene orders. Golden Gate assembly provides such a tool for seamless one-pot cleavage and ligation, by using type IIS restriction enzymes, which cleave outside of their recognition site. In addition to reducing the number of steps, this one-pot reaction can improve correct assemblies by the continued cleavage of self-ligation products that retain the recognition site. Switching the specific restriction enzyme used between steps allows for modular assembly of several units. A protocol to perform modular assemblies with two type IIS restriction enzymes, namely BsaI-v2-HF and BsmBI-v2, is described here. This protocol includes a description for generating destination vectors that add loxPsym sites between transcriptional units, allowing for diversification of gene order, orientation, and spacing.