291
also via the introduction of the single amino-acid exchanges D21V
or H22P within the LOV photosensor module [9, 17]. To obtain
a better mechanistic understanding of signal transduction and
modulation of the signal response, we applied PATCHY to the
systematic interrogation of linker determinants in the background
of either residue exchange (see Subheading 3.3.2). Similar to the
results for YF1, activity was only observed in discrete heptad registers, i.e., 7n for H22P, as well as 7n and 7n + 1 for D21V. In one
variant, elongation of the linker by three residues counteracted the
inversion of the signal response originally caused by the D21V
exchange.
2 Materials
1. 5× HF Buffer for PCR.
2. 2 U/μL Phusion High-Fidelity DNA Polymerase.
3. 10 mM dNTPs each.
4. 10 μM total forward primer pool for PATCHY.
5. 10 μM total reverse primer pool for PATCHY.
6. 10 U/μL NheI restriction enzyme.
7. 10 U/μL DpnI restriction enzyme.
8. 10 U/μL T4 Polynucleotide Kinase.
9. 50% PEG-4000 (w/v).
10. 0.5 mM ATP.
11. 30 U/μL T4 DNA Ligase.
12. 50× TAE buffer: 2 M Tris-Acetate, 50 mM EDTA, pH 8.5.
13. 1% Agarose in 1× TAE Buffer.
14. LB/Kanamycin medium: 10 g peptone, 5 g yeast extract and
10 g NaCl in 1 L dH 2 O supplemented with 50 mg/L
Kanamycin.
2.1 Chemicals
and Consumables
YF1
YF1
FixK2
FixJ
Ds Red
Fig. 3 The pDusk-DsRed reporter plasmid. In the absence of blue light, YF1 phosphorylates the response regulator BjFixJ which binds to the BjFixK2 promotor
and thereby upregulates reporter gene expression. Blue light inhibits expression
of DsRed by around 10–15-fold as it converts YF1 to a net phosphatase [16]
Generation of Hybrid Gene Libraries
also via the introduction of the single amino-acid exchanges D21V
or H22P within the LOV photosensor module [9, 17]. To obtain
a better mechanistic understanding of signal transduction and
modulation of the signal response, we applied PATCHY to the
systematic interrogation of linker determinants in the background
of either residue exchange (see Subheading 3.3.2). Similar to the
results for YF1, activity was only observed in discrete heptad registers, i.e., 7n for H22P, as well as 7n and 7n + 1 for D21V. In one
variant, elongation of the linker by three residues counteracted the
inversion of the signal response originally caused by the D21V
exchange.
2 Materials
1. 5× HF Buffer for PCR.
2. 2 U/μL Phusion High-Fidelity DNA Polymerase.
3. 10 mM dNTPs each.
4. 10 μM total forward primer pool for PATCHY.
5. 10 μM total reverse primer pool for PATCHY.
6. 10 U/μL NheI restriction enzyme.
7. 10 U/μL DpnI restriction enzyme.
8. 10 U/μL T4 Polynucleotide Kinase.
9. 50% PEG-4000 (w/v).
10. 0.5 mM ATP.
11. 30 U/μL T4 DNA Ligase.
12. 50× TAE buffer: 2 M Tris-Acetate, 50 mM EDTA, pH 8.5.
13. 1% Agarose in 1× TAE Buffer.
14. LB/Kanamycin medium: 10 g peptone, 5 g yeast extract and
10 g NaCl in 1 L dH 2 O supplemented with 50 mg/L
Kanamycin.
2.1 Chemicals
and Consumables
YF1
YF1
FixK2
FixJ
Ds Red
Fig. 3 The pDusk-DsRed reporter plasmid. In the absence of blue light, YF1 phosphorylates the response regulator BjFixJ which binds to the BjFixK2 promotor
and thereby upregulates reporter gene expression. Blue light inhibits expression
of DsRed by around 10–15-fold as it converts YF1 to a net phosphatase [16]
Generation of Hybrid Gene Libraries