Overlap-directed DNA assembly methods allow
multiple DNA parts to be assembled together in
one reaction. These methods, which rely on
sequence homology between the ends of DNA
parts, have become widely adopted in synthetic
biology, despite being incompatible with a key principle
of engineering: modularity. To answer this, we
present MODAL: a Modular Overlap-Directed
Assembly with Linkers strategy that brings modularity
to overlap-directed methods, allowing assembly
of an initial set of DNA parts into a variety of
arrangements in one-pot reactions. MODAL is
accompanied by a custom software tool that
designs overlap linkers to guide assembly,
allowing parts to be assembled in any specified
order and orientation. The in silico design of synthetic
orthogonal overlapping junctions allows for
much greater efficiency in DNA assembly for a
variety of different methods compared with using
non-designed sequence. In tests with three different
assembly technologies, the MODAL strategy gives
assembly of both yeast and bacterial plasmids,
composed of up to five DNA parts in the kilobase
range with efficiencies of between 75 and 100%.
It also seamlessly allows mutagenesis to be
performed on any specified DNA parts during
the process, allowing the one-step creation of construct
libraries valuable for synthetic biology
applications.