Thermus thermophilus CRISPR Cas6 Heterologous Expression and Purification

Materials and reagents

Biological materials

1. Thermus thermophilus HB27 (ATCC, catalog number: BAA-163; extract gDNA according to Wei et al. [3])

2. Escherichia coli DH5α (Thermo Scientific, catalog number: 18265017, stored at -80 °C)

3. Escherichia coli BL21(DE3) (Thermo Scientific, catalog number: EC0114, stored at -80 °C)

Reagents

1. pET28a-HRV3C (Addgene ID: 241018, store at -20 °C)

2. pGEX-6p-1 (Addgene ID: 241019, store at -20 °C)

3. NcoI (Thermo Scientific, catalog number: FD0574, store at -20 °C)

4. XhoI (Thermo Scientific, catalog number: FD0694, store at -20 °C)

5. BamHI (Thermo Scientific, catalog number: FD0054, store at -20 °C)

6. SalI (Thermo Scientific, catalog number: FD0644, store at -20 °C)

7. Phanta Max Super-Fidelity DNA polymerase (Vazyme, catalog number: P505-d3-AA, store at -20 °C)

8. DpnI (Thermo Scientific, catalog number: FD1404, store at -20 °C)

9. ABclonal MultiF Seamless Assembly Mix (ABclonal, catalog number: RK21020, store at -20 °C)

10. Nuclease-free water (Sigma-Aldrich, CAS number: 7732-18-5)

11. 2× Phanta Max buffer (Vazyme, catalog number: P505-d3-Ab, store at -20 °C)

12. dNTP mix (10 mM each) (Vazyme, catalog number: P505-d3-Ac, store at -20 °C)

13. Tryptone (OXOID, catalog number: LP0042B)

14. Yeast extract (OXOID, catalog number: LP0021B)

15. Agar (BioFroxx, catalog number: 8211GR500)

16. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I6758-5G)

17. Sodium chloride (NaCl) (Sigma-Aldrich, CAS number: 7558-79-4)

18. HEPES (Sigma-Aldrich, catalog number: H3375)

19. Glycerol (Sigma-Aldrich, catalog number: G5516)

20. Imidazole (Sigma-Aldrich, catalog number: I2399)

21. Reduced glutathione (Sigma-Aldrich, catalog number: G4251)

22. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541)

23. Dibasic sodium phosphate (Na₂HPO₄) (Sigma-Aldrich, catalog number: S3225)

24. Monobasic potassium phosphate (KH₂PO₄) (Sigma-Aldrich, catalog number: P9791)

25. Kanamycin (Sigma-Aldrich, catalog number: K1377)

26. Ampicillin (Sigma-Aldrich, catalog number: A5354-10ML)

27. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626)

28. Sodium dodecyl sulfate (SDS) (BioFRoxx, catalog number: 3250GR500)

29. Tris base (2-Amino-2-(hydroxymethyl)-propane-1,3-diol,Tris base) (BioFRoxx, CAS number: 77-86-1)

30. Glycine (BioFRoxx, CAS number: 56-40-6)

31. Ammonium persulfate (APS) (SCR, CAS number: 7727-54-0)

32. Acryl/Bio 30% solution (37.5:1) (Sanggon Biotech, catalog number: B546018-0500, store at 2–8 °C)

33. TEMED (Sigma, CAS number: 110-12-9)

34. Disodium EDTA dihydrate (SCR, CAS number: 6381-92-6)

35. Sodium hydroxide (NaOH) (SCR, CAS number: 1310-73-2)

36. Glacial acetic acid (SCR, CAS number: 64-19-7)

37. Agarose (Singke, catalog number: TSJ001)

38. Goldview (GL Biotech, catalog number: H003-1)

Solutions

1. LB medium (LB) (see Recipes)

2. Kanamycin stock solution (30 mg/mL) (see Recipes)

3. IPTG stock solution (100 mM) (see Recipes)

4. His-lysis buffer (see Recipes)

5. 5 M Imidazole stock solution (see Recipes)

6. His-washing buffer (see Recipes)

7. 100 mM Imidazole His-elution buffer (see Recipes)

8. 200 mM Imidazole His-elution buffer (see Recipes)

9. 500 mM Imidazole His-elution buffer (see Recipes)

10. 1 M Imidazole His-elution buffer (see Recipes)

11. Phosphate buffer (see Recipes)

12. GST-elution buffer (see Recipes)

13. SEC buffer (see Recipes)

14. 10% SDS solution (see Recipes)

15. 1.5 M Tris-HCl (pH 8.8) stock solution (see Recipes)

16. 1.0 M Tris-HCl (pH 6.8) stock solution (see Recipes)

17. 2.5 M glycine stock solution (see Recipes)

18. 10% Ammonium persulfate (APS) (see Recipes)

19. SDS-PAGE running buffer (10× Tris-Glycine-SDS, pH 8.3) (see Recipes)

20. SDS-PAGE resolving gel (12% polyacrylamide) (see Recipes)

21. SDS-PAGE stacking gel (5% polyacrylamide) (see Recipes)

22. 0.5 M EDTA (pH 8.0) stock solution (see Recipes)

23. 50× TAE buffer (see Recipes)

24 1.5 % agarose gel (see Recipes)

Recipes

1. LB medium

Dissolve reagents in approximately 900 mL of water. Adjust pH to 7.0 if necessary. Bring the volume to 1 L with water. For plate media, add agar to a final concentration of 1.5% (15 g/L). Sterilize by autoclaving at 121 °C for 20 min.

2. Kanamycin stock solution (30 mg/mL)

Dissolve 3 g of kanamycin in ddH₂O to a final volume of 100 mL. Filter sterilize using a 0.22 μm filter. Store at -20 °C for long-term storage or 4 °C for short-term storage. This 1000-fold concentrated stock solution is designed to be added at 1 mL per 1 L of LB medium to achieve a final kanamycin concentration of 30 μg/mL.

Critical: Add kanamycin after the LB medium has cooled down to below 50 °C following autoclaving to prevent antibiotic degradation.

3. IPTG stock solution (100 mM)

Dissolve 2.383 g of IPTG in ddH₂O to a final volume of 100 mL. Filter sterilize using a 0.22 μm filter. Store at -20 °C for long-term storage or 4 °C for short-term storage. This stock solution is designed to be added at 5 mL per 1 L of culture to achieve a final IPTG concentration of 0.5 mM.

4. His-lysis buffer (Base buffer)

Dissolve HEPES in approximately 700 mL of Milli-Q water. HEPES dissolves best at neutral pH. Adjust the pH to 7.5 using a pH meter at room temperature. Then add the other components. Bring the final volume to 1 L with Milli-Q water. Filter using a 0.22 μm filter. Store at 4 °C for 1 week.

5. 5 M Imidazole stock solution

Dissolve imidazole in Milli-Q water to a final volume of 100 mL. Adjust pH to 7.5 with HCl (imidazole dissolves best at neutral pH). Filter using a 0.22 μm filter. Store at -20 °C for long-term storage or 4 °C for short-term storage.

6. His-washing buffer

7. 100 mM Imidazole His-elution buffer

8. 200 mM Imidazole His-elution buffer

9. 500 mM Imidazole His-elution buffer

10. 1 M Imidazole His-elution buffer

11. Phosphate buffer (pH 7.4)

Dissolve salts in approximately 800 mL of Milli-Q water and stir until fully dissolved. Adjust pH to 7.4. Bring the final volume to 1 L with Milli-Q water. Filter using a 0.22 μm filter. Store the buffer at 4 °C for up to 1 month.
Critical: Use anhydrous Na₂HPO₄ (141.96 g/mol) for accurate molarity. If using hydrated forms (Na₂HPO₄·7H₂O, MW 268.07), adjust required mass = (10 mM × 268.07 g/mol) = 2.68 g/L.

12. GST-elution buffer (pH 8.0)

Dissolve in 40 mL of phosphate buffer.

Critical: GSH dissolves slowly; stir gently at room temperature for 10–15 min. Adjust pH to 8.0 with 1 M NaOH (typically requires ~1–2 mL).

Critical: GST elution is optimal at pH 8.0–8.5. Bring the final volume to 50 mL. Filter using a 0.22 μm filter. Store the buffer at 4 °C for up to 1 week (GSH oxidizes over time).

13. SEC buffer

14. 10% SDS Solution

Dissolve 50 g of sodium dodecyl sulfate (SDS) in Milli-Q water to a final volume of 500 mL with stirring. Store at room temperature for up to 6 months.

Caution: Wear a mask and gloves when handling SDS powder to avoid dust inhalation.

15. 1.5 M Tris-HCl (pH 8.8) (500 mL) stock solution

Dissolve 90.85 g of Tris base (MW 121.14 g/mol) in 300 mL of Milli-Q water with stirring. Adjust pH to 8.8 using concentrated HCl (add slowly, as Tris buffering capacity is high). Top up the volume to 500 mL. Store at 4 °C for up to 1 month.

Critical: Allow the solution to cool to room temperature before final pH adjustment, as Tris pH is temperature-sensitive.

16. 1.0 M Tris-HCl (pH 6.8) (50 mL) stock solution

Dissolve 6 g of Tris base (MW 121.14 g/mol) in 40 mL of Milli-Q water with stirring. Adjust pH to 6.8 using concentrated HCl. Top up the volume to 50 mL. Store at 4 °C for up to 1 month.

Critical: Recheck pH after mixing, as small deviations can affect gel stacking.

17. 2.5 M glycine (1 L) stock solution

Dissolve 187.7 g of glycine (MW 75.07 g/mol) in Milli-Q water to a final volume of 1 L with stirring. Store at room temperature for up to 6 months.

Critical: Ensure complete dissolution, as glycine can take time to dissolve fully.

18. 10% Ammonium persulfate (APS ) (1 mL)

Dissolve 0.1 g of APS in 1 mL of Milli-Q water immediately before use.

Critical: Do not store, as APS degrades rapidly in solution. Prepare fresh each day to ensure effective polymerization.

19. SDS-PAGE running buffer (10× Tris-Glycine-SDS, pH 8.3)

Bring the volume to 1 L with Milli-Q water and mix gently by stirring. Check pH (should be ⁓8.3). Store at room temperature for up to 6 months. To prepare 1× working buffer, dilute 100 mL of 10× with 900 mL of Milli-Q water within 1 week for optimal performance.

Critical: Do not adjust pH with NaOH, as sodium ions can interfere with electrophoresis.

20. SDS-PAGE resolving gel (12% polyacrylamide)

In a 50 mL conical flask, combine 3.3 mL of Milli-Q water, 2.5 mL of 1.5 M Tris-HCl (pH 8.8), 4.0 mL of 30% acrylamide/Bis, and 0.1 mL of 10% SDS. Mix gently by inversion. Then, add 0.1 mL of 10% APS and 4 μL of TEMED immediately before pouring to initiate polymerization. Pour into the gel cassette with a 5 mL micropipette, leaving ~1 cm space for the stacking gel. Overlay with isopropanol or water to ensure a flat interface. Allow polymerization for 30–45 min at room temperature.

Caution/Critical: Acrylamide is neurotoxic; handle with gloves in a fume hood. Prepare APS fresh daily. Polymerization time may vary with ambient temperature.

21. SDS-PAGE stacking gel (5% polyacrylamide)

In a 15 mL centrifuge tube, combine 2,062 μL of Milli-Q water, 375 μL of 1.0 M Tris-HCl (pH 6.8), 500 μL of 30% acrylamide/bis, and 30 μL of 10% SDS. Mix gently by inversion. Then, add 30 μL of 10% APS and 3 μL of TEMED immediately before pouring to initiate polymerization. Remove the isopropanol/water overlay from the polymerized resolving gel. Pour the stacking gel mixture on top with a micropipette and insert the comb. Allow polymerization for 20–30 min at room temperature.

Critical: Ensure the resolving gel is fully polymerized before pouring the stacking gel. Use fresh APS for consistent polymerization. Measure volumes precisely using calibrated micropipettes.

22. 0.5 M EDTA (pH 8.0) stock solution

Dissolve 18.61 g of disodium EDTA dihydrate in 80 mL of Milli-Q water. Stir vigorously using a magnetic stir plate.

EDTA will not dissolve fully until the pH is ~8.0. Add NaOH (either pellets or a concentrated solution, e.g., 10 M) gradually while monitoring pH with a calibrated pH meter. Typically, ~0.5–1 g of NaOH pellets or ~0.5–1 mL of 10 M NaOH is sufficient. Continue stirring until the EDTA dissolves completely.

23. 50× TAE buffer

Dissolve 242 g of Tris base in 700 mL of Milli-Q water with stirring until fully dissolved. Add 57.1 mL of glacial acetic acid and mix thoroughly. Add 100 mL of 0.5 M EDTA (pH 8.0) and mix. Bring the volume to 1 L with Milli-Q water. Stir gently. Verify the pH is ~8.0; adjust with acetic acid. Store at room temperature for up to 6 months. To prepare 1× TAE working buffer, dilute 20 mL of 10× with 980 mL of Milli-Q water.

Critical: Tris and EDTA solutions are pH-sensitive; ensure EDTA stock is at pH 8.0.

24. 1.5% agarose gel

Prepare a flask that can hold 2–4 times the volume of agarose you are preparing. Add 100 mL of the diluted buffer (1× TAE) to the flask, followed by 1.5 g of agarose. Heat the flask in the microwave in bursts of 30 s. Swirl after each burst. Cool it down to 40 °C. Add 10 μL of goldview. Pour in the tray. Add the combs.

Laboratory supplies

1. E.Z.N.A^® Cycle Pure Kit (omega BIO-TEK, catalog number: D6492-02)

2. E.Z.N.A.^® Plasmid DNA Mini Kit I (omega BIO-TEK, catalog number: D6943-02)

3. Bradford Protein Assay kit (Tiangen, catalog number: PA102)

4. Ni-charged Resin FF columns (GenScript, catalog number: L00666)

5. GST-Sefinose Resin 4FF columns (Sangon, catalog number: C600031)

6. Superdex 200 Increase 10/300 GL column (Cytiva, catalog number: 28990944)

7. Amicon Ultra centrifugal filter devices 10,000 MWCO (Millipore, catalog number: UFC901024)

8. Volumetric flasks, beakers, Erlenmeyer flasks

9. Pipettes (various volumes, calibrated) and pipette tips

10. Microcentrifuge tubes and centrifuge tubes (50 mL)

11. Petri dishes

12. Spreader for plating bacteria

13. Inoculation loops

14. Toothpicks (sterile, wooden)

15. Syringes

16. Syringe filters (0.22 μm, 0.45 μm)

Equipment

1. Thermocycler for PCR (Monad, model: Arhat 96)

2. Microcentrifuge (refrigerated) (Hunan Kecheng Instrument Equipment Co., model: H1-16KR)

3. Benchtop centrifuge (refrigerated) (Hunan Kecheng Instrument Equipment Co., model: H3-16KR)

4. Ultracentrifuge (refrigerated) (Beckman Coulter, model: optima xpn-100)

5. French Press (JNBIO, model: JNBIO-minipro)

6. NanoDrop (YoMin, model: Unano-1000)

7. Gel electrophoresis apparatus HET (Yeasen, catalog number: 80230ES01)

8. Power supply for agarose and SDS-PAGE EpPlus (Yeasen, catalog number: 80311ES05)

9. SDS-PAGE electrophoresis apparatus PET2 (Yeasen, catalog number: 80212ES05)

10. Gel documentation system (Clinx, model: GenoSens 2000)

11. pH meter (calibrated)

12. Magnetic stirrer and stir bars (Yeasen, model: MS400)

13. Rotary shaker/incubator (37 °C and 18 °C temperature control) (BLabotery, ZQPL-200)

14. Chromatography system (ÄKTA pure, catalog number: 21906560)

15. Autoclave (HefeiME, model: LX-B75L)

16. Static incubator (37 °C) (BLabotery, SPL-250)

17. Water bath

Software and datasets

1. SnapGene version 3.2.1 software was used for sequence analysis, primer design, PCR, and cloning stimulation

Procedure