302
structure of said linker. Insertion of single residues in the linker
can lead to inversion of the receptor response to blue light [10].
Recent mechanistic investigations [19, 20] reveal light-induced
left-handed supercoiling of the linker and thus provide the structural rationale for signal inversion via linker-length variations. In
the context of the residue exchanges D21V and H22P, each of
which induces signal inversion in YF1, light-regulated function is
only sustained by linkers of discrete lengths.
As a method, PATCHY is generally suitable for the construction and mechanistic interrogation of hybrid gene libraries. In
addition to the engineering of signal receptors, PATCHY also
appears well suited to the construction of fluorescent reporters.
4 Notes
1. The computer script is available from Github at https://github.
com/vrylr/PATCHY.git. To execute it, a Python interpreter is
required which can be obtained free of charge from http://
www.python.org or https://winpython.github.io.
2. We recommend to initially pool all forward primers at equimolar ratio and use them at a total concentration of 10 μM.
The reverse primers should be prepared accordingly. Given
that the individual primers differ in their sequences, they may
well possess different annealing and amplification efficiencies
which would result in a biased distribution of hybrid genes.
Adjustment of the relative primer concentrations may be a
remedy for this problem. In principle, one can employ nonequal concentrations within each primer pool to deliberately
predispose the PATCHY PCR reaction toward the generation
of certain sets of hybrid variants. Alternatively, the PATCHY
protocol can be rerun with subsets of the forward and reverse
pools that entirely lack certain primers.
3. One can carry on with the subsequent steps, even if less DNA
has been obtained at this stage.
4. We found it useful to implement several steps for removal of
the initial template construct from the PATCHY library. (a) In
many cases, the linear PATCHY PCR products can effectively
be separated from the circular template via gel extraction (see
Subheading 3.1 step 6). (b) Following phosphorylation and
circularization of the PATCHY PCR products, the template
can selectively be degraded via restriction digest with DpnI or
the specific endonuclease chosen in the template design (see
Subheading 3.1 steps 9 and 10). (c) Lastly, a deliberate frameshift can be introduced into the template construct to disable
it. Functional analysis of the PATCHY library, in particular by
Robert Stabel et al.
structure of said linker. Insertion of single residues in the linker
can lead to inversion of the receptor response to blue light [10].
Recent mechanistic investigations [19, 20] reveal light-induced
left-handed supercoiling of the linker and thus provide the structural rationale for signal inversion via linker-length variations. In
the context of the residue exchanges D21V and H22P, each of
which induces signal inversion in YF1, light-regulated function is
only sustained by linkers of discrete lengths.
As a method, PATCHY is generally suitable for the construction and mechanistic interrogation of hybrid gene libraries. In
addition to the engineering of signal receptors, PATCHY also
appears well suited to the construction of fluorescent reporters.
4 Notes
1. The computer script is available from Github at https://github.
com/vrylr/PATCHY.git. To execute it, a Python interpreter is
required which can be obtained free of charge from http://
www.python.org or https://winpython.github.io.
2. We recommend to initially pool all forward primers at equimolar ratio and use them at a total concentration of 10 μM.
The reverse primers should be prepared accordingly. Given
that the individual primers differ in their sequences, they may
well possess different annealing and amplification efficiencies
which would result in a biased distribution of hybrid genes.
Adjustment of the relative primer concentrations may be a
remedy for this problem. In principle, one can employ nonequal concentrations within each primer pool to deliberately
predispose the PATCHY PCR reaction toward the generation
of certain sets of hybrid variants. Alternatively, the PATCHY
protocol can be rerun with subsets of the forward and reverse
pools that entirely lack certain primers.
3. One can carry on with the subsequent steps, even if less DNA
has been obtained at this stage.
4. We found it useful to implement several steps for removal of
the initial template construct from the PATCHY library. (a) In
many cases, the linear PATCHY PCR products can effectively
be separated from the circular template via gel extraction (see
Subheading 3.1 step 6). (b) Following phosphorylation and
circularization of the PATCHY PCR products, the template
can selectively be degraded via restriction digest with DpnI or
the specific endonuclease chosen in the template design (see
Subheading 3.1 steps 9 and 10). (c) Lastly, a deliberate frameshift can be introduced into the template construct to disable
it. Functional analysis of the PATCHY library, in particular by
Robert Stabel et al.