Recursive DNA Assembly Using Protected Oligonucleotide Duplex Assisted Cloning (PODAC)

A problem rarely tackled by current
DNA assembly methods is the
issue of cloning additional parts into an already assembled construct.
Costly PCR workflows are often hindered by repeated sequences, and
restriction based strategies impose design constraints for each enzyme
used. Here we present Protected Oligonucleotide Duplex Assisted Cloning
(PODAC), a novel technique that makes use of an oligonucleotide duplex
for iterative Golden Gate cloning using only one restriction enzyme.
Methylated bases confer protection from digestion during the assembly
reaction and are removed during replication in vivo, unveiling a new cloning site in the process. We used this method
to efficiently and accurately assemble a biosynthetic pathway and
demonstrated its robustness toward sequence repeats by constructing
artificial CRISPR arrays. As PODAC is readily amenable to standardization,
it would make a useful addition to the synthetic biology toolkit.