171
1. Lysis and Binding buffer: 50 mM sodium phosphate, 300 mM
sodium chloride, 30 mM imidazole, pH 7.4.
2. Elution buffer: 50 mM sodium phosphate, 300 mM sodium
chloride, 500 mM imidazole, pH 7.4.
3. Sonicator or other means of cell lysis.
4. Syringes and 0.45 μm filter to clarify cell lysate.
5. 1 mL Nickel Sepharose His Trap column (GE Healthcare).
6. SDS-PAGE electrophoresis equipment to verify protein
expression and quality.
1. 96- or 384-well transparent bottom plate (Greiner Bio) for
absorbance reading. For alternative readouts based on fluorescence or luminescence, other types of plates are required.
2. Phosphate buffered saline (PBS) as diluent: 10 mM Na 2 HPO 4 ,
2 mM KH 2 PO 4 , 137 mM NaCl, and 2.7 mM KCl, pH 7.4.
3. 1 mM nitrocefin substrate stock solution: 1 mg nitrocefin
(Merck) dissolved in 100 μL DMSO and 1.9 mL PBS as per
manufacturer’s instructions.
4. Multi-well plate reader capable of reading OD at 492 nm.
Target protease or target protein binder whose cleavage or
binding site is present in the biosensor linker. By way of example, enterokinase (NEB), TEV protease, and anti-HA tag antibody have been successfully detected [13].
3 Methods
1. After the gene coding for Tem1-BLIP (see Note 3) has been
synthesized, it may be used as a standard template for inserting
suitable peptides as required. We generally use custom gene
synthesis to obtain the backbone, but the plasmid may also be
obtained from our lab upon request. For cloning, we typically
use inverse PCR with a reverse primer binding at the 3′ end
of the Tem1 ORF and a forward primer binding at the 5′ end
of the BLIP ORF while the 5′ tails encode the desired peptide
and linker amino acids. Furthermore, the 5′ fifteen bases of the
primers are complementary to each other to enable Infusion
cloning. Alternatively, if the sequence of the insert is too long,
the desired plasmid can be constructed by means of serial PCR
using overlapping primers. We frequently employ primers that
have been gel purified, especially if the oligonucleotide is
greater than 40 bases to avoid single-base dropouts and
frameshifts.
2. A typical PCR reaction based on Pfu Turbo polymerase typically
contains in a total volume of 50 μL (Pfu Turbo polymerase is
2.3 Purification
of Expressed Protein
2.4 Biosensor Assay
3.1 Plasmid
Construction
Sensing by Allosteric Derepression
1. Lysis and Binding buffer: 50 mM sodium phosphate, 300 mM
sodium chloride, 30 mM imidazole, pH 7.4.
2. Elution buffer: 50 mM sodium phosphate, 300 mM sodium
chloride, 500 mM imidazole, pH 7.4.
3. Sonicator or other means of cell lysis.
4. Syringes and 0.45 μm filter to clarify cell lysate.
5. 1 mL Nickel Sepharose His Trap column (GE Healthcare).
6. SDS-PAGE electrophoresis equipment to verify protein
expression and quality.
1. 96- or 384-well transparent bottom plate (Greiner Bio) for
absorbance reading. For alternative readouts based on fluorescence or luminescence, other types of plates are required.
2. Phosphate buffered saline (PBS) as diluent: 10 mM Na 2 HPO 4 ,
2 mM KH 2 PO 4 , 137 mM NaCl, and 2.7 mM KCl, pH 7.4.
3. 1 mM nitrocefin substrate stock solution: 1 mg nitrocefin
(Merck) dissolved in 100 μL DMSO and 1.9 mL PBS as per
manufacturer’s instructions.
4. Multi-well plate reader capable of reading OD at 492 nm.
Target protease or target protein binder whose cleavage or
binding site is present in the biosensor linker. By way of example, enterokinase (NEB), TEV protease, and anti-HA tag antibody have been successfully detected [13].
3 Methods
1. After the gene coding for Tem1-BLIP (see Note 3) has been
synthesized, it may be used as a standard template for inserting
suitable peptides as required. We generally use custom gene
synthesis to obtain the backbone, but the plasmid may also be
obtained from our lab upon request. For cloning, we typically
use inverse PCR with a reverse primer binding at the 3′ end
of the Tem1 ORF and a forward primer binding at the 5′ end
of the BLIP ORF while the 5′ tails encode the desired peptide
and linker amino acids. Furthermore, the 5′ fifteen bases of the
primers are complementary to each other to enable Infusion
cloning. Alternatively, if the sequence of the insert is too long,
the desired plasmid can be constructed by means of serial PCR
using overlapping primers. We frequently employ primers that
have been gel purified, especially if the oligonucleotide is
greater than 40 bases to avoid single-base dropouts and
frameshifts.
2. A typical PCR reaction based on Pfu Turbo polymerase typically
contains in a total volume of 50 μL (Pfu Turbo polymerase is
2.3 Purification
of Expressed Protein
2.4 Biosensor Assay
3.1 Plasmid
Construction
Sensing by Allosteric Derepression