290
increments of three nucleotides such as to truncate the template
construct in the register of nucleotide triplets which corresponds
to single amino acids at the protein level. The resultant linearized
plasmid fragments are phosphorylated and religated to produce a
library of circular plasmids. Optionally, remnants of the template
construct can be depleted from the library by restriction digest.
Plasmid libraries are then transformed and screened for favorable
phenotypes (see Subheading 3.2).
We applied PATCHY to the fusion of the BsYtvA LOV photosensor and the BjFixL effector module that previously yielded the
blue-light-repressed histidine kinase YF1 [8], see above. To facilitate library screening, hybrid gene libraries were constructed in the
context of the pDusk-DsRed reporter plasmid [16] which encodes
YF1, the cognate response regulator BjFixJ and the DsRed fluorescent reporter under control of the BjFixK2 promotor. In the dark,
YF1 phosphorylates BjFixJ which in turn binds to the BjFixK2
promotor and thereby ramps up the expression of the reporter
gene. Under blue light, YF1 acts as a net phosphatase on BjFixJ
which in turn results in about 10–15 fold decreased DsRed expression compared to in the dark (Fig. 3). Fluorescence-based screening (see Subheading 3.3.1) of the PATCHY library revealed that
photoreceptor activity and regulation by light are by and large
determined by linker length with a pronounced heptad (sevenresidue) periodicity. Insertion of single residues sufficed to invert
the response to blue light. An inversion of the light response of
YF1 can not only be effected via linker-length modifications but
Gene A
Gene B
L
B
L
A
1.
2.
3.
Fig. 2 The PATCHY template construct contains a tandem fusion of the longest
desired fragments of genes A and B on a circular plasmid (step 1). PATCHY uses
staggered primers to create linear DNA molecules bearing versions of genes A
and B that are terminally truncated at certain positions corresponding to the
primer annealing sites (step 2). The PATCHY hybrid gene library is created through
phosphorylation and blunt-end ligation to yield circular plasmids (step 3)
Robert Stabel et al.
increments of three nucleotides such as to truncate the template
construct in the register of nucleotide triplets which corresponds
to single amino acids at the protein level. The resultant linearized
plasmid fragments are phosphorylated and religated to produce a
library of circular plasmids. Optionally, remnants of the template
construct can be depleted from the library by restriction digest.
Plasmid libraries are then transformed and screened for favorable
phenotypes (see Subheading 3.2).
We applied PATCHY to the fusion of the BsYtvA LOV photosensor and the BjFixL effector module that previously yielded the
blue-light-repressed histidine kinase YF1 [8], see above. To facilitate library screening, hybrid gene libraries were constructed in the
context of the pDusk-DsRed reporter plasmid [16] which encodes
YF1, the cognate response regulator BjFixJ and the DsRed fluorescent reporter under control of the BjFixK2 promotor. In the dark,
YF1 phosphorylates BjFixJ which in turn binds to the BjFixK2
promotor and thereby ramps up the expression of the reporter
gene. Under blue light, YF1 acts as a net phosphatase on BjFixJ
which in turn results in about 10–15 fold decreased DsRed expression compared to in the dark (Fig. 3). Fluorescence-based screening (see Subheading 3.3.1) of the PATCHY library revealed that
photoreceptor activity and regulation by light are by and large
determined by linker length with a pronounced heptad (sevenresidue) periodicity. Insertion of single residues sufficed to invert
the response to blue light. An inversion of the light response of
YF1 can not only be effected via linker-length modifications but
Gene A
Gene B
L
B
L
A
1.
2.
3.
Fig. 2 The PATCHY template construct contains a tandem fusion of the longest
desired fragments of genes A and B on a circular plasmid (step 1). PATCHY uses
staggered primers to create linear DNA molecules bearing versions of genes A
and B that are terminally truncated at certain positions corresponding to the
primer annealing sites (step 2). The PATCHY hybrid gene library is created through
phosphorylation and blunt-end ligation to yield circular plasmids (step 3)
Robert Stabel et al.