216
inhibited at higher NaCl concentrations and should be kept at
100 mM NaCl or less.
2. For the reliable determination of enzyme kinetics, the protease
substrate needs to be fully dissolved at high substrate concentrations. If a protease substrate is not fully soluble at the desired
substrate concentration and the substrate solution appears
cloudy, its solubility can be improved by appending negatively
charged residues.
3. In the construction of DNA libraries, it is recommended to
purify all DNA fragments for the DNA assembly reaction by
means of agarose gel electrophoresis. In our experience, the
Wizard SV Gel and PCR Clean-Up System kit provides the
highest quality of DNA fragments free of impurities that can
potentially interfere with subsequent enzymatic or transformation steps. If necessary, individual DNA fragments can be additionally cleaned by means of ethanol precipitation.
4. Alternatively, suitable vector backbones can be prepared by
means of PCR independent of any restriction site. Due to the
large size of the pRK793 backbone and the difficulties associated with PCR amplification and susceptibility to point mutations, preparing the vector backbone by restriction enzyme
digest is preferred in this case.
5. Plasmid 05665 features a bacteriophage λ derived SRRz autolysis cassette [9, 17] under the control of a tetracycline inducible
promoter. This allows releasing the recombinantly expressed
protease switches into the lysate under relatively mild conditions. The use of detergent-based cell lysis procedures is not
recommended as it interferes with the protease assay.
6. The induction of autolysis by means of the SRRz autolysis cassette with tetracycline is most efficient in fresh PA-5052 autoinduction medium. In addition, the presence of glucose, which
inhibits the expression of genes under the control of the LacO
promoter, in fresh PA-5052 autoinduction medium is irrelevant at this stage as the protease transducer is already expressed.
Depending on the type of protease transducer, including trace
metals may interfere with protease function and should therefore be omitted.
7. Alternatively, proteins can also be expressed by means of
Isopropyl β-D-1-thiogalactopyranoside (IPTG). In this case,
cells are grown to an OD 600 of 0.6–0.8 before being induced
with 0.1–1 mM IPTG and expressed for 3–12 h at 30 °C.
Optimal induction conditions need to be determined empirically for each type of protease sensor.
8. Depending on the synthetic protease sensor, it is critical to
include the correct amount of NaCl in the storage buffer.
Notably, the allosteric protease receptors are not stable in the
absence of NaCl, but form a precipitate.
Viktor Stein and Kirill Alexandrov
inhibited at higher NaCl concentrations and should be kept at
100 mM NaCl or less.
2. For the reliable determination of enzyme kinetics, the protease
substrate needs to be fully dissolved at high substrate concentrations. If a protease substrate is not fully soluble at the desired
substrate concentration and the substrate solution appears
cloudy, its solubility can be improved by appending negatively
charged residues.
3. In the construction of DNA libraries, it is recommended to
purify all DNA fragments for the DNA assembly reaction by
means of agarose gel electrophoresis. In our experience, the
Wizard SV Gel and PCR Clean-Up System kit provides the
highest quality of DNA fragments free of impurities that can
potentially interfere with subsequent enzymatic or transformation steps. If necessary, individual DNA fragments can be additionally cleaned by means of ethanol precipitation.
4. Alternatively, suitable vector backbones can be prepared by
means of PCR independent of any restriction site. Due to the
large size of the pRK793 backbone and the difficulties associated with PCR amplification and susceptibility to point mutations, preparing the vector backbone by restriction enzyme
digest is preferred in this case.
5. Plasmid 05665 features a bacteriophage λ derived SRRz autolysis cassette [9, 17] under the control of a tetracycline inducible
promoter. This allows releasing the recombinantly expressed
protease switches into the lysate under relatively mild conditions. The use of detergent-based cell lysis procedures is not
recommended as it interferes with the protease assay.
6. The induction of autolysis by means of the SRRz autolysis cassette with tetracycline is most efficient in fresh PA-5052 autoinduction medium. In addition, the presence of glucose, which
inhibits the expression of genes under the control of the LacO
promoter, in fresh PA-5052 autoinduction medium is irrelevant at this stage as the protease transducer is already expressed.
Depending on the type of protease transducer, including trace
metals may interfere with protease function and should therefore be omitted.
7. Alternatively, proteins can also be expressed by means of
Isopropyl β-D-1-thiogalactopyranoside (IPTG). In this case,
cells are grown to an OD 600 of 0.6–0.8 before being induced
with 0.1–1 mM IPTG and expressed for 3–12 h at 30 °C.
Optimal induction conditions need to be determined empirically for each type of protease sensor.
8. Depending on the synthetic protease sensor, it is critical to
include the correct amount of NaCl in the storage buffer.
Notably, the allosteric protease receptors are not stable in the
absence of NaCl, but form a precipitate.
Viktor Stein and Kirill Alexandrov