The plant Golden Gate toolkit: compatibility, interoperability, and harmonized assembly in plant synthetic biology

In plant synthetic biology, the emergence of Golden Gate (GG) cloning has led to the rapid development of numerous cloning kits that enable the standardized assembly of genetic modules and transcriptional units through a single, one-pot reaction. However, this rapid and somewhat uncoordinated expansion has introduced challenges, particularly regarding compatibility between cloning kits, which can hinder adoption and routine use in new laboratories. To address these recurring issues, this review provides a comprehensive overview of more than 25 well-established plant-specific GG cloning kits, along with an in-depth examination of the compatibility of their available modules, with the goal of improving interoperability across systems. To support a more integrated and forward-looking development of future GG-based efforts, we present key approaches for increasing system harmonization, including several domestication strategies, methylase-assisted hierarchical DNA assembly, and the inclusion of multiple cloning sites. As a whole, this review aims to streamline cloning workflows and reduce technical barriers for new plant synthetic biologists in pursuit of increasing the throughput of their experiments.