202
4. LB medium: 10 g peptone, 5 g yeast extract, and 10 g NaCl in
1 L dH 2 O.
5. LB agar plates: 10 g peptone, 5 g yeast extract, 10 g NaCl, and
15 g agar in 1 L dH 2 O.
1. Chemically competent Top10 E. coli cells (or equivalent) for
DNA cloning.
2. Pfu C X DNA polymerase (Agilent).
3. 10× Pfu C x reaction buffer.
4. 10 mM dNTPs each.
5. Primer-For: 5′-GCTGAAGTCTTACGAGGAAGAGTTGGC- 3′
(T M = 70.5 °C).
6. Primer-C-Ter:
5′-ACGGTUTCGCGACCTACACCG-3′
(T M = 72.1 °C).
7. Primer-Library: 5′-AACCGUGCGCTTTNNNNNNGGAA
GCACCCACCACCATCAT- 3′ (T M = 70.3 °C).
8. Primer-Rev.: 5′-CGTTGTAAAACGACGGCCAGTG-3′ (T M
= 70.3 °C).
9. Vector for the expression of recombinant NIa proteases with
maltose binding protein (MBP): e.g., pRK793 based on
pMAL-C2.
10. 10× T4 DNA ligase buffer (NEB).
11. 400 U/μL T4 DNA ligase (NEB).
12. 1 U/μL USER Enzyme Mix (NEB).
13. Restriction enzymes NcoI and BamHI to digest the pRK793
vector backbone.
14. Gibson Assembly Kit.
15. Wizard SV Gel and PCR Clean-Up System.
16. Miniprep plasmid DNA purification system.
1. Chemically competent BL21(DE3)-RIL E. coli cells (or equivalent) for protein expression.
2. Plasmid 05665 featuring the bacteriophage λ SRRz autolysis
cassette [8, 9] under the control of the tetracycline inducible
promoter.
3. 96-deep-well-plates to grow E. coli in high throughput.
4. 50× 5052 medium: 25% glycerol (w/v), 2.5% glucose (w/v)
and 10% α-lactose (w/v) [10].
5. 20× NPS: 0.5 M (NH 4 ) 2 SO 4 , 1 M KH 2 PO 4 and 1 M Na 2 HPO 4
pH 6.75.
6. 1 M MgSO 4 .
2.2 Construction
of DNA Libraries
with USER Enzyme
2.3 High-Throughput
Expression
of Synthetic Protease
Receptors
Viktor Stein and Kirill Alexandrov
4. LB medium: 10 g peptone, 5 g yeast extract, and 10 g NaCl in
1 L dH 2 O.
5. LB agar plates: 10 g peptone, 5 g yeast extract, 10 g NaCl, and
15 g agar in 1 L dH 2 O.
1. Chemically competent Top10 E. coli cells (or equivalent) for
DNA cloning.
2. Pfu C X DNA polymerase (Agilent).
3. 10× Pfu C x reaction buffer.
4. 10 mM dNTPs each.
5. Primer-For: 5′-GCTGAAGTCTTACGAGGAAGAGTTGGC- 3′
(T M = 70.5 °C).
6. Primer-C-Ter:
5′-ACGGTUTCGCGACCTACACCG-3′
(T M = 72.1 °C).
7. Primer-Library: 5′-AACCGUGCGCTTTNNNNNNGGAA
GCACCCACCACCATCAT- 3′ (T M = 70.3 °C).
8. Primer-Rev.: 5′-CGTTGTAAAACGACGGCCAGTG-3′ (T M
= 70.3 °C).
9. Vector for the expression of recombinant NIa proteases with
maltose binding protein (MBP): e.g., pRK793 based on
pMAL-C2.
10. 10× T4 DNA ligase buffer (NEB).
11. 400 U/μL T4 DNA ligase (NEB).
12. 1 U/μL USER Enzyme Mix (NEB).
13. Restriction enzymes NcoI and BamHI to digest the pRK793
vector backbone.
14. Gibson Assembly Kit.
15. Wizard SV Gel and PCR Clean-Up System.
16. Miniprep plasmid DNA purification system.
1. Chemically competent BL21(DE3)-RIL E. coli cells (or equivalent) for protein expression.
2. Plasmid 05665 featuring the bacteriophage λ SRRz autolysis
cassette [8, 9] under the control of the tetracycline inducible
promoter.
3. 96-deep-well-plates to grow E. coli in high throughput.
4. 50× 5052 medium: 25% glycerol (w/v), 2.5% glucose (w/v)
and 10% α-lactose (w/v) [10].
5. 20× NPS: 0.5 M (NH 4 ) 2 SO 4 , 1 M KH 2 PO 4 and 1 M Na 2 HPO 4
pH 6.75.
6. 1 M MgSO 4 .
2.2 Construction
of DNA Libraries
with USER Enzyme
2.3 High-Throughput
Expression
of Synthetic Protease
Receptors
Viktor Stein and Kirill Alexandrov