11
earliest homology-dependent recombination methods [101, 102]
and has recently been applied to engineer defined linker libraries
for light-activated histidine protein kinase switches [103]. Here, a
small number of DNA templates with overlapping homologous
sequences prime each other during every reannealing step to
recombine two DNA fragments. Recombination by means of
OE-PCR can, however, prove technically challenging considering
the relatively low efficiency of recombination between two larger
single-stranded DNA fragments. This is further aggravated by the
exponential nature of PCR amplification, which potentially renders
OE-PCR susceptible to nonspecific DNA amplification products
and limits the number of DNA fragments that can be simultaneously recombined.
More recently, Gibson assembly has originated as a powerful,
homology-dependent cloning strategy relying on the combined
action of a dsDNA 5′ to 3′ exonuclease, a thermostable DNA ligase
and a thermostable DNA polymerase [104]. Reactions are typically
conducted at 50 °C and initiated by the exonuclease-dependent
chew back of the 5′ end. This results in the formation of singlestranded 3′ DNA extensions that guide the reannealing of homologous DNA sequences that are subsequently extended and filled by
the DNA polymerase and eventually sealed by the DNA ligase.
Unlike OE-PCR, Gibson Assembly occurs at a constant temperature without the need for thermal cycling coordinating successive
reannealing and amplification steps. This significantly increases the
efficiency of recombination, enables the simultaneous assembly of
multiple DNA fragments, and prevents any bias that may arise
through successive reannealing and amplification cycles. While
technically easy, the efficiency of Gibson assembly can be reduced
by secondary structures, repeat regions and GC-rich regions as they
frequently occur in the glycine- and serine-rich polypeptide linkers
as is applicable in the construction of synthetic protein switches.
Beyond Gibson assembly, a number of alternative methods
have been devised that rely on similar principles such as sequence
and ligase-independent cloning (SliC) [105], circular polymerase
extension cloning (CPEC) [106], seamless ligation cloning extract
(SLICE) [107], or AQUA [108] where an exonuclease, DNA
polymerase and DNA ligase function are included either as part of
cell extract or within a cell.
While OE-PCR and Gibson assembly enable the seamless assembly
of DNA sequences independent of restriction sites, both methods
rely on homologous DNA sequences of 20–50 bp. This generally
restricts the reuse of DNA coding for common receptor, actuator,
and linker elements from existing, sequence verified DNA constructs and libraries. In addition, the longer the overhangs, the
more expensive the synthesis of tailored oligonucleotides becomes.
Alternatively, cloning strategies have been devised based on type
3.3 LigationDependent Assembly
Strategies
Engineering Synthetic Protein Switches
earliest homology-dependent recombination methods [101, 102]
and has recently been applied to engineer defined linker libraries
for light-activated histidine protein kinase switches [103]. Here, a
small number of DNA templates with overlapping homologous
sequences prime each other during every reannealing step to
recombine two DNA fragments. Recombination by means of
OE-PCR can, however, prove technically challenging considering
the relatively low efficiency of recombination between two larger
single-stranded DNA fragments. This is further aggravated by the
exponential nature of PCR amplification, which potentially renders
OE-PCR susceptible to nonspecific DNA amplification products
and limits the number of DNA fragments that can be simultaneously recombined.
More recently, Gibson assembly has originated as a powerful,
homology-dependent cloning strategy relying on the combined
action of a dsDNA 5′ to 3′ exonuclease, a thermostable DNA ligase
and a thermostable DNA polymerase [104]. Reactions are typically
conducted at 50 °C and initiated by the exonuclease-dependent
chew back of the 5′ end. This results in the formation of singlestranded 3′ DNA extensions that guide the reannealing of homologous DNA sequences that are subsequently extended and filled by
the DNA polymerase and eventually sealed by the DNA ligase.
Unlike OE-PCR, Gibson Assembly occurs at a constant temperature without the need for thermal cycling coordinating successive
reannealing and amplification steps. This significantly increases the
efficiency of recombination, enables the simultaneous assembly of
multiple DNA fragments, and prevents any bias that may arise
through successive reannealing and amplification cycles. While
technically easy, the efficiency of Gibson assembly can be reduced
by secondary structures, repeat regions and GC-rich regions as they
frequently occur in the glycine- and serine-rich polypeptide linkers
as is applicable in the construction of synthetic protein switches.
Beyond Gibson assembly, a number of alternative methods
have been devised that rely on similar principles such as sequence
and ligase-independent cloning (SliC) [105], circular polymerase
extension cloning (CPEC) [106], seamless ligation cloning extract
(SLICE) [107], or AQUA [108] where an exonuclease, DNA
polymerase and DNA ligase function are included either as part of
cell extract or within a cell.
While OE-PCR and Gibson assembly enable the seamless assembly
of DNA sequences independent of restriction sites, both methods
rely on homologous DNA sequences of 20–50 bp. This generally
restricts the reuse of DNA coding for common receptor, actuator,
and linker elements from existing, sequence verified DNA constructs and libraries. In addition, the longer the overhangs, the
more expensive the synthesis of tailored oligonucleotides becomes.
Alternatively, cloning strategies have been devised based on type
3.3 LigationDependent Assembly
Strategies
Engineering Synthetic Protein Switches