Overview

Golden Gate [1, 2] is a powerful molecular biology technique that allows scarless assembly of a large number of DNA fragments. It makes use of type IIS restriction enzymes, such as BsaI, BsmBI, BbsI, SapI, etc., that have the peculiarity of having a recognition site outside their cutting site. This property gives several advantages during cloning:

• It allows scarless assembly: the cutting sites can be designed so that upon digestion and ligation, the final construct has only the desired sequence without the recognition sites.
• It allows assembly of a large number of fragments in a defined order: the cutting sites can be diverse and generate several overhangs after digestion that can be ligated easily and specifically, based on complementarity.
• It allows one pot digestion and ligation: the ligation is irreversible and the final DNA molecule will persist because there is no possibility of recreating the restriction sites. Thus, during the reaction, the final construct continues to accumulate, which increases the overall cloning efficiency.

Golden Gate cloning allows great freedom in design and can employed for building custom made DNA molecules. For these reasons it was adopted by the scientific community who recognised its potential even for developing standardized and modular cloning. Thus, several Golden Gate based tool kits were constructed both for prokaryotes and eukaryotes [3-7 for example]. The recently published Loop assembly system [8] brings Golden Gate cloning to a higher level of creativity and modularity as it allows recursive assembly of DNA fragments.

We welcome the iGEM initiative to fully support Type IIS parts that adhere to the MoClo/ PhytoBricks and Loop Type IIS assembly standards for the first time in the 2019 Competition (https://2019.igem.org/Competition/New/Type_IIS). In this framework, we designed a Loop assembly system dedicated to our chassis, the oleaginous yeast Yarrowia lipolytica.

A Type IIS RFC[10] Loop assembly system for Yarrowia lipolytica

The Loop assembly technique

Conclusions

References