KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Bacterial Strains | ||
| Competent E. coli for cloning (e.g. NEB 10-beta) | NEB | C3019H |
| E. coli proline auxotroph (e.g. CAG18515; see Note 1) | CGSC | 7331 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Restriction enzymes (e.g. EcoRI-HF & BamHI-HF) | NEB | R3101; R3136 |
| T4 DNA ligase | NEB | M0202 |
| Q5 High-Fidelity 2x Master Mix | NEB | M0492 |
| repliQa HiFi Assembly Mix | Quantabio | 95190 |
| Glu-C protease (or other appropriate protease; see Note 5) | Promega | V1651 |
| M9 Medium Components | ||
| 20 proteinogenic amino acids | Sigma-Aldrich | various |
| Sodium chloride (NaCl, CAS: 7647-14-5) | Sigma-Aldrich | S7653 |
| Ammonium chloride (NH4Cl, CAS: 12125-02-9) | Sigma-Aldrich | A9434 |
| Potassium phosphate, monobasic (KH2PO4, CAS: 7778-77-0) | Sigma-Aldrich | P5655 |
| Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O, CAS: 7782-85-6) | Sigma-Aldrich | S9390 |
| D-Glucose (CAS: 50-99-7) | Sigma-Aldrich | G7021 |
| Biotin (CAS: 58-85-5) | Sigma-Aldrich | B4639 |
| Thiamine hydrochloride (CAS: 67-03-8) | Sigma-Aldrich | T1270 |
| Iron(II) sulfate heptahydrate (FeSO4·7H2O, CAS:7782-63-0) | Sigma-Aldrich | F8633 |
| Magnesium sulfate hepahydrate (MgSO4·7H2O, CAS: 10034-99-8) | Sigma-Aldrich | M2773 |
| Calcium chloride dihydrate (CaCl2·2H2O, CAS: 10035-04-8) | Sigma-Aldrich | C7902 |
| Copper(II) sulfate pentahydrate (CuSO4·5H2O, CAS: 7758-99-8) | Sigma-Aldrich | C8027 |
| Manganese(II) chloride tetrahydrate (MnCl2·4H2O, CAS: 13446-34-9) | Sigma-Aldrich | 63535 |
| Zinc sulfate heptahydrate (ZnSO4·7H2O, CAS: 7446-20-0) | Sigma-Aldrich | Z0251 |
| Ammonium molybdate tetrahydrate [(NH4)6Mo7O24·4H2O, CAS: 12054-85-2] | Sigma-Aldrich | M1019 |
| Commercial Kits | ||
| Plasmid Miniprep Kit | Zymo Research | D4015 |
| Gel DNA Recovery Kit | Zymo Research | D4001 |
| Oligonucleotides | ||
| proS-ins-fwd: 5’- GTGAGAATCCAAGCTAGCTCAGCCTTTAATCT GTTTCACCAG-3’ | IDT | |
| proS-ins-rev: 5’- CGTATAATATTTGCCCATGGATTCACGCCCTT CT CTTTTGAC -3’ | IDT | |
| proS-vec-fwd: 5’- GAGAAGGGCGTGAATCCATGGGCAAATATTA TACGCAAG -3’ | IDT | |
| proS-vec-rev: 5’- GTGAAACAGATTAAAGGCTGAGCTAGCTTGG ATTCTCACC-3’ | IDT | |
| Recombinant DNA | ||
| pQE-80L | Qiagen | |
| Gene encoding POI | IDT | gBlock gene fragment |