298
single clone. For long-term storage, 200 μL of the saturated
cultures is mixed with 200 μL 50% (w/v) glycerol and frozen
at −80 °C.
8. Quantitative characterization of isolated hybrid variants: Using
suitable functional assays, the biological activity of the hybrid
variants isolated in Subheading 3.2 step 5 should be characterized in more detail. In the example case, replicates of each
clone were inoculated in 5 mL LB/Kanamycin and grown at
37 °C for 16 h in darkness or constant blue light (470 nm, 100
μW/cm
2
) (see Note 8). Saturated cultures were diluted tenfold
in ddH 2 O, and OD 600 was measured in clear microtiter plates
with a microplate reader. Samples were further diluted fivefold
in ddH 2 O, and DsRed fluorescence was measured in black
microtiter plates with settings of 554 ± 9 nm for excitation and
591 ± 20 nm for emission, respectively. Fluorescence readings
were normalized to the OD 600 of the corresponding sample.
9. Sequence analysis of isolated hybrid variants: Clones that
showed desirable properties in the previous step are analyzed
by Sanger DNA sequencing (see Note 9).
We applied PATCHY to better characterize the underlying design
principles and signal-transduction mechanisms in the chimeric
photoreceptor YF1 [8]. The original YF1 receptor originated from
the fusion of the blue light-sensitive light–oxygen–voltage photosensor module of BsYtvA to the effector module of the BjFixL
histidine kinase. Notably, in YF1 the linker between these modules
essentially derived from the BjFixL parental protein, i.e., i = 3 and
j = 25 (see Subheading 3.1 step 1). Sparse sampling of linker
composition had earlier identified linker length as the main determinant for activity and regulation of the hybrid receptors [8]. A
seven-residue periodicity of the dependence of activity and regulatory properties on linker length resulted from the coiled-coil conformation of the linker, as later evidenced in the high-resolution
structure of dark-adapted YF1 [9]. Notably, only a tiny fraction of
many conceivable hybrid variants were studied at this point [8].
Despite these biochemical and structural data, the mechanism
by which signals are transduced from the LOV photosenor to the
histidine kinase effector remained unclear. We reasoned that comprehensive interrogation of linker sequence space could yield additional mechanistic insight. The linkers between the respective
sensor and effector modules in the parental receptors BsYtvA and
BjFixL comprise 23 and 27 residues, respectively. Provided that
fusions between the parental proteins are restricted to these linker
regions, there are (23 + 1) ∙ (27 + 1) = 672 different ways to
recombine the BsYtvA LOV photosensor with the BjFixL effector
(see Figs. 1 and 2). Using PATCHY as described in Subheading
3.1, we generated a construct library that theoretically contains
3.3 PATCHY Case
Studies
3.3.1 Linker
Libraries of YF1
Robert Stabel et al.
single clone. For long-term storage, 200 μL of the saturated
cultures is mixed with 200 μL 50% (w/v) glycerol and frozen
at −80 °C.
8. Quantitative characterization of isolated hybrid variants: Using
suitable functional assays, the biological activity of the hybrid
variants isolated in Subheading 3.2 step 5 should be characterized in more detail. In the example case, replicates of each
clone were inoculated in 5 mL LB/Kanamycin and grown at
37 °C for 16 h in darkness or constant blue light (470 nm, 100
μW/cm
2
) (see Note 8). Saturated cultures were diluted tenfold
in ddH 2 O, and OD 600 was measured in clear microtiter plates
with a microplate reader. Samples were further diluted fivefold
in ddH 2 O, and DsRed fluorescence was measured in black
microtiter plates with settings of 554 ± 9 nm for excitation and
591 ± 20 nm for emission, respectively. Fluorescence readings
were normalized to the OD 600 of the corresponding sample.
9. Sequence analysis of isolated hybrid variants: Clones that
showed desirable properties in the previous step are analyzed
by Sanger DNA sequencing (see Note 9).
We applied PATCHY to better characterize the underlying design
principles and signal-transduction mechanisms in the chimeric
photoreceptor YF1 [8]. The original YF1 receptor originated from
the fusion of the blue light-sensitive light–oxygen–voltage photosensor module of BsYtvA to the effector module of the BjFixL
histidine kinase. Notably, in YF1 the linker between these modules
essentially derived from the BjFixL parental protein, i.e., i = 3 and
j = 25 (see Subheading 3.1 step 1). Sparse sampling of linker
composition had earlier identified linker length as the main determinant for activity and regulation of the hybrid receptors [8]. A
seven-residue periodicity of the dependence of activity and regulatory properties on linker length resulted from the coiled-coil conformation of the linker, as later evidenced in the high-resolution
structure of dark-adapted YF1 [9]. Notably, only a tiny fraction of
many conceivable hybrid variants were studied at this point [8].
Despite these biochemical and structural data, the mechanism
by which signals are transduced from the LOV photosenor to the
histidine kinase effector remained unclear. We reasoned that comprehensive interrogation of linker sequence space could yield additional mechanistic insight. The linkers between the respective
sensor and effector modules in the parental receptors BsYtvA and
BjFixL comprise 23 and 27 residues, respectively. Provided that
fusions between the parental proteins are restricted to these linker
regions, there are (23 + 1) ∙ (27 + 1) = 672 different ways to
recombine the BsYtvA LOV photosensor with the BjFixL effector
(see Figs. 1 and 2). Using PATCHY as described in Subheading
3.1, we generated a construct library that theoretically contains
3.3 PATCHY Case
Studies
3.3.1 Linker
Libraries of YF1
Robert Stabel et al.