209
4. Grow cells for 72 h at 30 °C at 320 rpm until an OD 600 of ∼10
is typically reached. Store 96-deep-well plates at 4 °C for several days to regrow plasmids in step 11.
5. Optional: Spin down 200 μL cell suspension and resuspend
cells in fresh PA-5052 autoinduction medium, but with no
trace metals (see Note 6).
6. Transfer 100 μL aliquots of E. coli cell cultures from half a
96-well plate (48 samples) and dilute twofold in 100 μL autolysis medium and incubate for 90 min at 30 °C with agitation
at 200 rpm.
7. Transfer 10 μL aliquots of cell lysates and duplicate into separate halves of a black 96-well plate filled with 140 μL protease
assay buffer.
8. Supplement all of the duplicates with 1 μL of 1 U/μL thrombin to cleave the linker connecting TVMV to its AI-domain
and incubate for 30 min.
9. Initiate the reaction by adding 50 μL of 20 μM TVMV peptide
substrate solution giving rise to 5 μM TVMV peptide substrate
in the final reaction.
10. Monitor the reaction progress in a 96-well-plate fluorescence
reader by measuring the release of the quenched fluorophore
7-methoxycoumarinyl-4-acetyl at 405 nm following excitation
at 330 nm.
11. Evaluate the induction of TVMV protease activities by comparing the time-course of the reaction in the presence and
absence of thrombin.
12. Regrow E. coli from step 3 that code for protease transducer
mutants that display a high induction ratio in step 9 in 5 mL
LB medium supplemented with 100 μg/mL carbenicillin.
13. Purify and sequence plasmid DNA that codes for the protease
transducer mutants with the desired properties.
To characterize engineered protease switches biochemically under
defined reaction conditions, it is recommended to purify individual protease switches to homogeneity by means of affinity
chromatography.
1. Transform plasmids coding for the desired synthetic protease
switch into chemically competent BL21(DE3)-RIL cells, plate
onto LB agar plates supplemented with 100 μg/mL carbenicillin, and 34 μg/mL chloramphenicol and incubate overnight at
37 °C.
2. Inoculate 250 mL TB-based autoinduction medium supplemented with 100 μg/mL carbenicillin and 34 μg/mL chloramphenicol with a single colony.
3.3 Purifying
Synthetic Protease
Switches
Engineering Synthetic Protease Switches
4. Grow cells for 72 h at 30 °C at 320 rpm until an OD 600 of ∼10
is typically reached. Store 96-deep-well plates at 4 °C for several days to regrow plasmids in step 11.
5. Optional: Spin down 200 μL cell suspension and resuspend
cells in fresh PA-5052 autoinduction medium, but with no
trace metals (see Note 6).
6. Transfer 100 μL aliquots of E. coli cell cultures from half a
96-well plate (48 samples) and dilute twofold in 100 μL autolysis medium and incubate for 90 min at 30 °C with agitation
at 200 rpm.
7. Transfer 10 μL aliquots of cell lysates and duplicate into separate halves of a black 96-well plate filled with 140 μL protease
assay buffer.
8. Supplement all of the duplicates with 1 μL of 1 U/μL thrombin to cleave the linker connecting TVMV to its AI-domain
and incubate for 30 min.
9. Initiate the reaction by adding 50 μL of 20 μM TVMV peptide
substrate solution giving rise to 5 μM TVMV peptide substrate
in the final reaction.
10. Monitor the reaction progress in a 96-well-plate fluorescence
reader by measuring the release of the quenched fluorophore
7-methoxycoumarinyl-4-acetyl at 405 nm following excitation
at 330 nm.
11. Evaluate the induction of TVMV protease activities by comparing the time-course of the reaction in the presence and
absence of thrombin.
12. Regrow E. coli from step 3 that code for protease transducer
mutants that display a high induction ratio in step 9 in 5 mL
LB medium supplemented with 100 μg/mL carbenicillin.
13. Purify and sequence plasmid DNA that codes for the protease
transducer mutants with the desired properties.
To characterize engineered protease switches biochemically under
defined reaction conditions, it is recommended to purify individual protease switches to homogeneity by means of affinity
chromatography.
1. Transform plasmids coding for the desired synthetic protease
switch into chemically competent BL21(DE3)-RIL cells, plate
onto LB agar plates supplemented with 100 μg/mL carbenicillin, and 34 μg/mL chloramphenicol and incubate overnight at
37 °C.
2. Inoculate 250 mL TB-based autoinduction medium supplemented with 100 μg/mL carbenicillin and 34 μg/mL chloramphenicol with a single colony.
3.3 Purifying
Synthetic Protease
Switches
Engineering Synthetic Protease Switches