Recursive Cloning for the Assembly of Biosynthetic Pathways and Artificial CRISPR Arrays

Advances in the field of Synthetic Biology have given rise to a multitude of new molecular cloning techniques with improved flexibility, efficiency and accuracy. While these methods have enabled multi-part assembly of complex constructs, they are often lacking in several respects: How can additional parts be added after assembly? What about standardisation to custom specifications? Here we present Protected Oligonucleotide Duplex Assisted Cloning (PODAC), a novel cloning technique that eschews large, one-off multi-part assemblies in favour of a more iterative workflow.

PODAC is a Golden Gate derivative whereby a cloning site for future iterations is included together with the genetic part to be cloned. To protect the new cloning site from being destroyed itself during the one-pot restriction and ligation reaction, it is supplied in the form of a synthetic oligonucleotide duplex carrying methylated cytosine residues. Passage through E. coli reactivates these protected sites, resulting in a construct that can accept another part during a next cycle of PODAC.   

Each cycle is performed using the same endonuclease and sticky ends. This not only reduces overall complexity but also maximises the reusability of part storage vectors. Because the restriction enzyme used (BsaI) cleaves DNA outside of its recognition site, the sequence of the sticky ends and resulting assembly scars can be chosen at will. This allows for easy standardisation to the specifications imposed by the experimental context. As a first proof-of-principle, PODAC was used to assemble the violacein biosynthesis pathway.

Moreover, because PODAC does not rely on PCR or long sequence homologies, it can safely be applied to designs containing repeated sequences. One application where this is the case is the creation of artificial CRISPR arrays. To demonstrate its broad applicability, PODAC was used to construct guide RNA arrays that target various catabolic pathways in E. coli for dCas9 mediated transcriptional roadblock silencing.

In conclusion, we believe that the wide applicability and amenability to standardisation make PODAC a useful addition to the existing Synthetic Biology toolkit.