208
for the elementary protease module (step 2), and primers
Primer-Library and Primer-Rev.
5. Mix equimolar amounts of the N- and C-terminal coding
DNA fragments generated in steps 3 and 4 together with 1 U
USER Enzyme per μg of total DNA and 200 U T4 DNA ligase
per μg DNA in 1× T4 DNA ligase buffer. Incubate for 60 min
at 37 °C.
6. Purify the assembly reaction using a suitable PCR purification kit.
7. Check the quality of USER-mediated enzyme DNA assembly
reaction by means of agarose gel electrophoresis. Typically,
one major band corresponding to the desired DNA assembly
product along with the two minor bands of the DNA assembly
substrates is visible.
8. Optional: Purify the desired DNA fragment by means of agarose gel electrophoresis (see Note 3).
9. Perform a Gibson Assembly reaction to recombine the DNA
insert library into a suitable expression vector as per manufacturer’s instructions. We typically use the vector pRK793 backbone based on the pMAL series cut with NcoI and BamHI
(see Note 4).
10. Transform 5 μL of the Gibson Assembly mix into 50 μL chemically competent E. coli cells and plate on LB agar plates supplemented with 100 μg/mL carbenicillin.
11. To check the quality of the library, inoculate single colonies in
5 mL LB medium supplemented with 100 μg/mL carbenicillin, purify plasmid DNA and sequence.
Once a DNA library has been generated, it needs to be screened
experimentally to identify mutants with the desired properties. The
following generic protocol can be used to screen the function of
autoinhibited protease modules, sensors, and switches aiming to
optimize the binding strength of the AI-domain, or the length and
structure of the connecting linkers.
1. Transform the library of autoinhibited TVMV mutants into
chemically competent BL21(DE3)-RIL cells hosting the
autolysis plasmid 05665 (see Note 5).
2. Plate transformed cells on LB agar plates supplemented with
100 μg/mL carbenicillin, 50 μg/mL kanamycin, and 34 μg/mL
chloramphenicol.
3. Following overnight incubation at 37 °C, inoculate single colonies into 96-deep-well plates filled with 1 mL minimal PA-5052
autoinduction medium [10] supplemented with 100 μg/mL
carbenicillin, 50 μg/mL kanamycin, and 34 μg/mL
chloramphenicol.
3.2 Engineering
Autoinhibited Protease
Modules: HighThroughput Screening
Viktor Stein and Kirill Alexandrov
for the elementary protease module (step 2), and primers
Primer-Library and Primer-Rev.
5. Mix equimolar amounts of the N- and C-terminal coding
DNA fragments generated in steps 3 and 4 together with 1 U
USER Enzyme per μg of total DNA and 200 U T4 DNA ligase
per μg DNA in 1× T4 DNA ligase buffer. Incubate for 60 min
at 37 °C.
6. Purify the assembly reaction using a suitable PCR purification kit.
7. Check the quality of USER-mediated enzyme DNA assembly
reaction by means of agarose gel electrophoresis. Typically,
one major band corresponding to the desired DNA assembly
product along with the two minor bands of the DNA assembly
substrates is visible.
8. Optional: Purify the desired DNA fragment by means of agarose gel electrophoresis (see Note 3).
9. Perform a Gibson Assembly reaction to recombine the DNA
insert library into a suitable expression vector as per manufacturer’s instructions. We typically use the vector pRK793 backbone based on the pMAL series cut with NcoI and BamHI
(see Note 4).
10. Transform 5 μL of the Gibson Assembly mix into 50 μL chemically competent E. coli cells and plate on LB agar plates supplemented with 100 μg/mL carbenicillin.
11. To check the quality of the library, inoculate single colonies in
5 mL LB medium supplemented with 100 μg/mL carbenicillin, purify plasmid DNA and sequence.
Once a DNA library has been generated, it needs to be screened
experimentally to identify mutants with the desired properties. The
following generic protocol can be used to screen the function of
autoinhibited protease modules, sensors, and switches aiming to
optimize the binding strength of the AI-domain, or the length and
structure of the connecting linkers.
1. Transform the library of autoinhibited TVMV mutants into
chemically competent BL21(DE3)-RIL cells hosting the
autolysis plasmid 05665 (see Note 5).
2. Plate transformed cells on LB agar plates supplemented with
100 μg/mL carbenicillin, 50 μg/mL kanamycin, and 34 μg/mL
chloramphenicol.
3. Following overnight incubation at 37 °C, inoculate single colonies into 96-deep-well plates filled with 1 mL minimal PA-5052
autoinduction medium [10] supplemented with 100 μg/mL
carbenicillin, 50 μg/mL kanamycin, and 34 μg/mL
chloramphenicol.
3.2 Engineering
Autoinhibited Protease
Modules: HighThroughput Screening
Viktor Stein and Kirill Alexandrov