12
IIS restriction enzymes. These cut outside their recognition motif
in a sequence-independent fashion to create tailored singlestranded DNA extensions. Crucially, unlike conventional restriction enzymes, the resulting single-stranded extensions are
non-palindromic and thus facilitate the assembly of multiple DNA
fragments in a directional manner.
Golden Gate cloning constitutes one of the most widely used
DNA assembly methods based on type IIS restriction enzymes
allowing for the directional and seamless assembly of multiple
DNA fragments [109]. In the context of engineering synthetic
protein switches, distinct structural motifs, linker elements, and
functional domains are first amplified by PCR using synthetic oligonucleotides that introduce tailored DNA overhangs. These
overhangs code for a type IIS restriction site and a short recombination motif that guide the ligation of multiple DNA fragments
with complementary extension motifs. Individual DNA fragments
are then fused following the combined action of a type IIS restriction enzyme and a DNA ligase. One key disadvantage of type IIS
restriction enzyme-dependent cloning strategies is the need to
remove any potential restriction sites in the coding sequence.
While this does not pose a concern for synthetic DNA fragments,
where restriction sites can be specifically omitted, this is not the
case with genomic sequences and DNA constructs that are already
available in the plasmid database of a lab.
Alternatively, USER Enzyme can be employed to create short
single-stranded 3′ DNA extensions [110–114]. Here, singlestranded 3′ DNA extensions are created through the excision of
uracil residues that are introduced via synthetic oligonucleotides at
the PCR amplification step. The resulting 3′ DNA extensions subsequently guide the DNA ligase-dependent fusion of two or more
DNA fragments. Scar sites are minimal as the only sequence
requirement is a pair of A and T residues spaced apart by approximately two to six nucleotides. Similar to type IIS restriction sites,
the single-stranded extensions of USER enzyme can be nonpalindromic to enable the directional assembly of multiple DNA
fragments.
Ultimately, the preferred DNA assembly procedure will be
determined by a number of factors: This includes the architecture
of a specific protein switch (e.g., whether it is modularly organized
or integrated), the source of DNA (e.g., whether it is of genomic
or synthetic origin), as well as any idiosyncrasies associated with the
construction of a particular protein switch (e.g., whether linker
regions feature repeat regions, secondary structures, or high GC
content). In addition, the potential for automation and the use of
commercial DNA synthesis and cloning services plays an increasingly important consideration in devising cost-effective and efficient DNA assembly processes and needs to be assessed individually
for different types of synthetic protein switches.
Viktor Stein
IIS restriction enzymes. These cut outside their recognition motif
in a sequence-independent fashion to create tailored singlestranded DNA extensions. Crucially, unlike conventional restriction enzymes, the resulting single-stranded extensions are
non-palindromic and thus facilitate the assembly of multiple DNA
fragments in a directional manner.
Golden Gate cloning constitutes one of the most widely used
DNA assembly methods based on type IIS restriction enzymes
allowing for the directional and seamless assembly of multiple
DNA fragments [109]. In the context of engineering synthetic
protein switches, distinct structural motifs, linker elements, and
functional domains are first amplified by PCR using synthetic oligonucleotides that introduce tailored DNA overhangs. These
overhangs code for a type IIS restriction site and a short recombination motif that guide the ligation of multiple DNA fragments
with complementary extension motifs. Individual DNA fragments
are then fused following the combined action of a type IIS restriction enzyme and a DNA ligase. One key disadvantage of type IIS
restriction enzyme-dependent cloning strategies is the need to
remove any potential restriction sites in the coding sequence.
While this does not pose a concern for synthetic DNA fragments,
where restriction sites can be specifically omitted, this is not the
case with genomic sequences and DNA constructs that are already
available in the plasmid database of a lab.
Alternatively, USER Enzyme can be employed to create short
single-stranded 3′ DNA extensions [110–114]. Here, singlestranded 3′ DNA extensions are created through the excision of
uracil residues that are introduced via synthetic oligonucleotides at
the PCR amplification step. The resulting 3′ DNA extensions subsequently guide the DNA ligase-dependent fusion of two or more
DNA fragments. Scar sites are minimal as the only sequence
requirement is a pair of A and T residues spaced apart by approximately two to six nucleotides. Similar to type IIS restriction sites,
the single-stranded extensions of USER enzyme can be nonpalindromic to enable the directional assembly of multiple DNA
fragments.
Ultimately, the preferred DNA assembly procedure will be
determined by a number of factors: This includes the architecture
of a specific protein switch (e.g., whether it is modularly organized
or integrated), the source of DNA (e.g., whether it is of genomic
or synthetic origin), as well as any idiosyncrasies associated with the
construction of a particular protein switch (e.g., whether linker
regions feature repeat regions, secondary structures, or high GC
content). In addition, the potential for automation and the use of
commercial DNA synthesis and cloning services plays an increasingly important consideration in devising cost-effective and efficient DNA assembly processes and needs to be assessed individually
for different types of synthetic protein switches.
Viktor Stein