One-Step Affinity Purification of MarathonRT Reverse Transcriptase for RNA Sequencing Applications

Materials and reagents

Note: Equivalent materials and reagents may be used as substitutes.

Biological materials

1. Escherichia coli Rosetta 2(DE3)pLysS chemically competent cells, prepared in-house (Merck, Novagen, catalog number: 71403)

2. Plasmid pLJSRSF7-T7-6xHis-SUMO-MarathonRT-CBD (MRT-CBD) (Addgene, catalog number: 253350) originally described in [1] or pET-6xHis-SUMO-MarathonRT (MRT) (Addgene, catalog number: 109029) originally described in [14]

3. Plasmid for RNA template generation (Addgene, catalog number: 101156) originally described in [24]

4. Reverse transcription primer (5′-TCACTGCATACGACGATTCTG-3′) used in the activity assay

Reagents

Antibiotics

1. Kanamycin sulfate (Thermo Fisher Scientific, Fisher Scientific, Fisher BioReagents, catalog number: BP906-5)

2. Chloramphenicol (Thermo Fisher Scientific, Fisher Scientific, Fisher BioReagents, catalog number: BP904-100)

Inducers and reducing agents

3. Isopropyl-B-D-thiogalactopyranoside (IPTG) (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: R0392)

4. Dithiothreitol (DTT) (Thermo Fisher Scientific, Fisher Scientific, Fisher BioReagents, catalog number: BP172-25)

5. β-Mercaptoethanol (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific Acros, catalog number: 125472500)

Buffers, acids, and bases

6. HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (Thermo Fisher Scientific, Fisher Scientific, Fisher BioReagents, catalog number: BP310-1)

7. Tris base [Tris(hydroxymethyl)aminomethane] (Thermo Fisher Scientific, Fisher Scientific, Fisher BioReagents, catalog number: BP152-1)

8. Hydrochloric acid (HCl) for pH adjustment (any)

9. Sulfuric acid (Thermo Fisher Scientific, Fisher Scientific, Fisher Chemicals, catalog number: S/9240/PB15)

10. Sodium hydroxide (NaOH) (Thermo Fisher Scientific, Fisher Scientific, Fisher Chemical, catalog number: S/4920/60)

11. Potassium hydroxide (KOH) (Thermo Fisher Scientific, Fisher Scientific, Fisher Chemical, catalog number: P/5560/60)

Salts

12. Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, Fisher Chemical, catalog number: S/3120/63)

13. Potassium chloride (KCl) (Thermo Fisher Scientific, Fisher Scientific, Fisher Chemical, catalog number: P/4240/60)

14. Magnesium chloride (MgCl₂) (Thermo Fisher Scientific, Fisher Scientific, Fisher BioReagents, catalog number: 10386743)

15. Sodium phosphate monobasic anhydrous (NaH₂PO₄) (Thermo Fisher Scientific, Fisher Scientific, Fisher BioReagents, catalog number: BP329500)

16. Sodium phosphate dibasic anhydrous (Na₂HPO₄) (Thermo Fisher Scientific, Fisher Scientific, Fisher BioReagents, catalog number: BP332-500)

17. Potassium phosphate, monobasic (KH₂PO₄) (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific Acros, catalog number: 205925000)

Amino acids/protein components

18. BSA (albumin, bovine) (Avantor, VWR Life Science, catalog number: 0332-100G)

Organic compounds and solvents

19. Glycerol, ≥99%, analytical reagent grade (Thermo Fisher Scientific, Fisher Scientific, Fisher Chemical, catalog number: G/0650/08)

20. Ethanol (EtOH), AA grade (Anora Group Oyj, Anora Industrial, Etax Aa, catalog number: 1025874)

21. Tween 20 (Thermo Fisher Scientific, Fisher Scientific, Fisher BioReagents, catalog number: BP337-500)

22. Orthophosphoric acid, >85%, extra pure, SLR 500 mL (Thermo Fisher Scientific, Fisher Scientific, Fisher Chemical, catalog number: 10122010)

23. Ethylene glycol diacetate (EGDA) (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific Acros, catalog number: 10022400)

Chaotropic agents and denaturants

24. Guanidine thiocyanate (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific Chemicals, catalog number: 411112500)

Imidazole derivatives

25. Imidazole (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific Acros, catalog number: 122025000)

Colorimetric reagents

26. Coomassie Blue G250 (Thermo Fisher Scientific, Fisher Scientific, Fisher BioReagents, catalog number: C/P541/46)

27. Malachite green (oxalate) (Thermo Fisher Scientific, Fisher Scientific, Fisher Chemical, catalog number: M/1245/46)

28. Ammonium molybdate (IV) tetrahydrate (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific Acros, catalog number: 205851000)

Media components

29. Tryptone (Neogen, catalog number: NCM0120A)

30. Yeast extract (Neogen, catalog number: NCM0218)

31. Agar (Neogen, catalog number: NCM0238A)

Enzymes

32. EcoRI (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: FD0274)

33. FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: EF0654)

34. T7 polymerase (20 U/μL) (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: EP0111)

35. RQ1 RNase-free DNase (Promega, catalog number: M6101)

36. RNasin Plus ribonuclease inhibitor 10,000 U (Promega, catalog number: N2615)

37. Pyrophosphatase, inorganic (0.1 U/μL) (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: EF0221)

Nucleotides

38. dNTP set, 100 mM solutions (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: R0181)

39. NTP set, 100 mM solutions (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: R0481)

Gel electrophoresis

40. SDS-PAGE 10% gels, Tris-Glycine buffering system for proteins (any)

41. Urea-PAA 5% gel, TBE buffering system for RNA (any)

42. Agarose gel 1% gel, TBE or TAE buffering system for DNA (any)

43. Midori Green Advance (Nippon Genetics, catalog number: MG04)

44. SYBR Gold nucleic acid gel stain (10,000× concentrate in DMSO) (Thermo Fisher Scientific, Fisher Scientific, Invitrogen, catalog number: S11494)

45. InstantBlue Coomassie protein stain (ISB1L) (Abcam, catalog number: ab119211)

46. PageRuler Plus Prestained protein ladder, 10–250 kDa (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: 26619)

47. ssRNA ladder (New England Biolabs, catalog number: N0362S)

48. GeneRuler 1 kb DNA ladder (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: SM0311)

Specialized materials and kits:

49. Liquid nitrogen

50. NucleoSpin Gel and PCR Clean-up XS (Macherey-Nagel, catalog number: 740611.250)

51. NucleoSpin RNA Columns for RNA purification (Macherey-Nagel, catalog number: 740955.50S)

52. Qubit RNA Broad Range (BR) Assay kit (Thermo Fisher Scientific, Fisher Scientific, Invitrogen, catalog number: Q10211)

Solutions

1. Kanamycin, 50 mg/mL (see Recipes)

2. Chloramphenicol, 35 mg/mL (see Recipes)

3. IPTG, 1 M (see Recipes)

4. Glycerol, 80% (see Recipes)

5. Glycerol, 90% (see Recipes)

6. Imidazole, 1 M (see Recipes)

7. NaOH, 5 M (see Recipes)

8. KOH, 5 M (see Recipes)

9. NaCl, 5 M (see Recipes)

10. KCl, 3 M (see Recipes)

11. MgCl₂, 4 M (see Recipes)

12. DTT, 1 M (see Recipes)

13. K-HEPES pH 7.5, 1 M (see Recipes)

14. Na-HEPES pH 7.5, 1 M (see Recipes)

15. Tris-HCl pH 8.3, 1 M (see Recipes)

16. Tris-HCl pH 7.5, 1 M (see Recipes)

17. Tris-HCl pH 7.0, 1 M (see Recipes)

18. BSA 10 μg/μL (see Recipes)

19. BSA 0.1 μg/μL (see Recipes)

20. Guanidine thiocyanate, 5 M (see Recipes)

21. Solutions for measuring MRT activity

a. Inorganic phosphate solution (Pi), 100 mM (see Recipes)

b. Malachite green reagent, 0.12% in 3 M H₂SO₄ (see Recipes)

c. Ammonium molybdate, 7.5% (w/v) (see Recipes)

d. Tween 20, 10% (v/v) (see Recipes)

22. Buffers for MRT expression

a. LB Miller broth (see Recipes)

b. LB Miller agar (see Recipes)

c. PBS, 10× (see Recipes)

d. PBS, 1× (see Recipes)

23. Buffers for MRT purification

a. Buffer A (see Recipes)

b. Buffer B (see Recipes)

c. Buffer C (see Recipes)

d. Buffer D (see Recipes)

e. Buffer G (-glycerol) (see Recipes)

f. Buffer G (+glycerol)/storage buffer (see Recipes)

24. Buffers for Bradford Assay

a. Bradford reagent (see Recipes)

25. Buffers for RNA template purification [25]

a. Lysis/binding buffer (LBB) (see Recipes)

b. Chaotropic wash (CW) (see Recipes)

c. Ethanol wash (EW) (see Recipes)

26. Buffers for MarathonRT unit definition

a. MRT 2× reaction buffer (see Recipes)

b. Pi dilutions (for standard curve) (see Recipes)

c. Malachite green detection solution (DS) (see Recipes)

Recipes

1. Kanamycin, 50 mg/mL (25 mL)

Weigh 1.25 g of kanamycin into a 50 mL conical centrifuge tube. Add nuclease-free water up to 20 mL and dissolve kanamycin using an end-over-end mixer. Adjust the volume to 25 mL and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Aliquot 1 mL into 1.5 mL microfuge tubes. Store at -20 °C indefinitely.

2. Chloramphenicol, 35 mg/mL (25 mL)

Weigh 0.88 g of chloramphenicol into a 50 mL conical centrifuge tube. Add nuclease-free water up to 20 mL and dissolve chloramphenicol using an end-over-end mixer. Adjust the volume to 25 mL and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Aliquot 1 mL into 1.5 mL microfuge tubes. Store at -20 °C indefinitely.

3. IPTG, 1 M (20 mL)

Weigh 4.77 g of IPTG into a 50 mL conical centrifuge tube. Add nuclease-free water up to 15 mL and dissolve IPTG using an end-over-end mixer. Adjust the volume to 20 mL in a volumetric bottle or cylinder and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Aliquot 1 mL into 1.5 mL microfuge tubes. Store at -20 °C for up to 2 years.

4. Glycerol, 80% (80 mL)

Weigh 64 g of glycerol into a 100 mL glass bottle. Add 16 g of nuclease-free water. Mix over the cap 5–10×. Autoclave in liquid cycle. Store at room temperature indefinitely.

5. Glycerol, 90% (80 mL)

Weigh 72 g of glycerol into a 100 mL glass bottle. Add 8 g of nuclease-free water. Mix over the cap 5–10×. Autoclave in liquid cycle. Store at room temperature indefinitely.

6. Imidazole, 1 M (100 mL)

Weigh 6.81 g of imidazole into a 100 mL beaker. Add nuclease-free water up to ~80 mL and dissolve imidazole using a magnetic stirrer. Adjust the volume to 100 mL in a volumetric bottle or cylinder and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Store at 4 °C protected from light for up to 2 years.

7. NaOH, 5 M (100 mL)

Weigh 20 g of NaOH pellets. Add NaOH slowly to water (do not add water to pellets) and stir in a 250–500 mL beaker with cooling if needed. Start with ~80 mL of nuclease-free water, dissolve, then adjust to 100 mL in a volumetric bottle or cylinder. Aliquot into 50 mL conical centrifuge tubes. Store at room temperature indefinitely.

8. KOH, 5 M (100 mL)

Weigh 28.06 g of KOH pellets. Add KOH slowly to water (do not add water to pellets) and stir in a 250–500 mL beaker with cooling if needed. Start with ~80 mL of nuclease-free water, dissolve, then adjust to 100 mL in a volumetric bottle or cylinder. Aliquot into 50 mL conical centrifuge tubes. Store at room temperature indefinitely.

9. NaCl, 5 M (100 mL)

Weigh 29.22 g of NaCl into a 250 mL beaker. Add nuclease-free water up to 80 mL and dissolve NaCl using a magnetic stirrer. Adjust the volume to 100 mL in a volumetric bottle or cylinder and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Store at room temperature indefinitely.

10. KCl, 3 M (100 mL)

Weigh 22.36 g of KCl into a 250 mL beaker. Add nuclease-free water up to 80 mL and dissolve KCl using a magnetic stirrer. Adjust the volume to 100 mL in a volumetric bottle or cylinder and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Store at room temperature indefinitely.

11. MgCl₂, 4 M (100 mL)

Weigh 50.34 g of MgCl₂ into a 250 mL beaker. Add ~80 mL of nuclease-free water. Use a magnetic mixer to dissolve MgCl₂. Set the final volume to 100 mL with nuclease-free water using a volumetric bottle or a cylinder and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Store at room temperature indefinitely.

12. DTT, 1 M (25 mL)

Weigh 3.86 g of DTT into a 50 mL conical centrifuge tube. Add nuclease-free water up to 20 mL and dissolve DTT using an end-over-end mixer. Adjust the volume to 25 mL and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Aliquot 1 mL into 1.5 mL microfuge tubes. Store at -20 °C up to 2 years.

13. K-HEPES pH 7.5, 1 M (100 mL)

Weigh 23.83 g of HEPES into a 250 mL beaker. Add ~80 mL of nuclease-free water. Use a magnetic mixer to dissolve HEPES. Once dissolved, adjust the pH with 5 M KOH solution. Set the final volume to 100 mL with nuclease-free water using a volumetric bottle or a cylinder and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Aliquot 10 mL into 15 mL centrifuge tubes and store at -20 °C for up to 2 years.

Critical: HEPES buffers are light- and temperature-sensitive. HEPES solution is stable at 4 °C for up to a month when protected from light.

14. Na-HEPES pH 7.5, 1 M (100 mL)

Weigh 23.83 g of HEPES into a 250 mL beaker. Add ~80 mL of nuclease-free water. Use a magnetic mixer to dissolve HEPES. Once dissolved, adjust the pH with 5 M NaOH solution. Set the final volume to 100 mL with nuclease-free water using a volumetric bottle or a cylinder and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Aliquot 10 mL into 15 mL centrifuge tubes and store at -20 °C for up to 2 years.

Critical: HEPES buffers are light- and temperature-sensitive. HEPES solution is stable at 4 °C for up to a month when protected from light.

15. Tris-HCl pH 8.3, 1 M (100 mL)

Weigh 12.11 g of Tris base into a 250 mL beaker. Add ~80 mL of nuclease-free water. Use a magnetic mixer to dissolve Tris base. Once dissolved, adjust the pH with HCl solution. Set the final volume to 100 mL with nuclease-free water using a volumetric bottle or a cylinder and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Store at room temperature for up to 1 year.

16. Tris-HCl pH 7.5, 1 M (100 mL)

Weigh 12.11 g of Tris base into a 250 mL beaker. Add ~80 mL of nuclease-free water. Use a magnetic mixer to dissolve Tris base. Once dissolved, adjust the pH with HCl solution. Set the final volume to 100 mL with nuclease-free water using a volumetric bottle or a cylinder and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Store at room temperature for up to 1 year.

17. Tris-HCl pH 7.0, 1 M (100 mL)

Weigh 12.11 g of Tris base into a 250 mL beaker. Add ~80 mL of nuclease-free water. Use a magnetic mixer to dissolve Tris base. Once dissolved, adjust the pH with HCl solution. Set the final volume to 100 mL with nuclease-free water using a volumetric bottle or a cylinder and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Store at room temperature for up to 1 year.

18. BSA 10 μg/L (10 mL)

Weigh 0.1 g of BSA into a 50 mL tube. Add ~8 mL of nuclease-free water. Use an end-over-end mixer to dissolve BSA. Avoid foam. Set the final volume to 10 mL with nuclease-free water using a volumetric bottle or a cylinder and aliquot into microfuge tubes. Store at -20 °C for years. Avoid freeze-thaws.

19. BSA 0.1 μg/μL (1 mL)

Pipette 10 μL of 10 μg/μL BSA into 990 μL of nuclease-free water. Mix by pipetting up and down. Use immediately.

20. Guanidine thiocyanate, 5 M (10 mL)

Weigh 5.91 g of guanidine thiocyanate into a 50 mL tube. Add ~8 mL of nuclease-free water. Use a magnetic mixer with heating to dissolve it. Set the final volume to 10 mL with nuclease-free water using a volumetric bottle or a cylinder and transfer into a 15 mL tube. Store at room temperature protected from light for years.

21. Solutions for measuring MRT activity

Critical: Use only sterile plasticware for preparation of these solutions, except for malachite green reagent. Nuclease-free water must be taken directly from the water purification device into plasticware. Glassware might contain trace amounts of phosphate, which will interfere with the assay. Do not sterilize these solutions.

a. Inorganic phosphate solution (P_i), 100 mM (10 mL)

Dissolve 0.12 g of NaH₂PO₄ (119.98 g/mol) in 7–8 mL of nuclease-free water in a 15 mL tube. Adjust the final volume to 10 mL with nuclease-free water in the same tube. Do not filter-sterilize. Store at 4 °C for 1–2 months.

b. Malachite green reagent, 0.12% in 3 M H₂SO₄ (180 mL)

Prepare 3 M sulfuric acid by diluting 30 mL of concentrated sulfuric acid in 150 mL of nuclease-free water in a well-rinsed glass bottle. Add 0.22 g of malachite green and mix well. This yields a 0.12% malachite green solution in 3 M (6 N) sulfuric acid. Do not filter-sterilize. The solution is stable at room temperature for up to 1 year. Always store the solution in glassware!

c. Ammonium molybdate, 7.5% (w/v) (10 mL)

Dissolve 0.75 g of ammonium molybdate tetrahydrate in 5 mL of nuclease-free water in a 15 mL tube. Once dissolved, adjust the volume to 10 mL with nuclease-free water in the same tube. Do not filter-sterilize. The solution is stable indefinitely if stored in a plastic bottle. However, the solution may occasionally precipitate. If this happens, prepare a new solution.

d. Tween 20, 10% (v/v) (10 mL)

Dilute 1 mL of concentrated Tween 20 in 5–6 mL of nuclease-free water in a 50 mL tube. Mix properly on an end-over-end rotator. Adjust the volume to 10 mL with nuclease-free water. Store at room temperature. Do not filter-sterilize.

22. Buffers for MRT expression

a. LB Miller broth (1 L)

Weigh all components into a 1 L beaker. Add RO water up to 900 mL and dissolve using a magnetic stirrer. Adjust the volume to 1 L in a volumetric cylinder or bottle. Dispense 500 mL into 1 L glass bottles and sterilize by autoclaving at 121 °C for 15 min. Store at room temperature for years.

b. LB Miller agar (250 mL)

Weigh all components into a 500 mL beaker. Add nuclease-free water up to 200 mL and dissolve using a magnetic stirrer. Adjust the volume to 250 L in a volumetric cylinder or bottle. Dispense 250 mL into 100 mL glass bottles and sterilize by autoclaving at 121 °C for 15 min. Store at room temperature for years.

c. PBS, 10× (100 mL)

Weigh all components into a 250 mL beaker. Add nuclease-free water up to 90 mL and dissolve using a magnetic stirrer. Adjust the volume to 100 mL in a volumetric bottle or cylinder and filter through a disposable 0.2 μm 150 mL filter into a sterile 100 mL glass bottle. Store at room temperature indefinitely.

d. PBS, 1× (100 mL)

Pipette 10 mL of 10× PBS stock into a volumetric bottle or cylinder. Bring the volume to 100 mL and transfer to a 100 mL glass bottle. Mix over the cap. Store at room temperature indefinitely.

23. Buffers for MRT purification

The buffer nomenclature is the same as in the original publication [13].

We recommend that all buffers be prepared a day before the purification of the protein and always stored at 4 °C. Sterilize all solutions before making the buffers. We recommend filter-sterilizing all self-prepared solutions made of powders and only autoclaving glycerol. Add β-Mercaptoethanol and DTT to the buffers just before use. These components are unstable and will render the buffers not usable the next day.

a. Buffer A

b. Buffer B

c. Buffer C

d. Buffer D

e. Buffer G (-glycerol)

f. Buffer G (+glycerol)/storage buffer

We recommend that the buffer be prepared fresh, aliquoted into 1.5 mL microfuge tubes, and stored at -20 °C. We have successfully used the buffer stored for 2 years.

24. Buffers for Bradford assay

a. Bradford reagent

Weigh 0.1 g of Coomassie Brilliant Blue G250. Use a magnetic mixer to dissolve it in 50 mL of ethanol in a 1 L beaker for 1 h. Add 100 mL of orthophosphoric acid and stir. Add 350 mL of nuclease-free water and stir. Aliquot into 50 mL glass bottles. Store at 4 °C protected from light.

25. Buffers for RNA template purification [25]

a. Lysis/binding buffer (LBB)

Weigh 5.91 g of guanidine thiocyanate into a 50 mL beaker. Add ~8 mL of nuclease-free water and 0.5 mL of Tris-HCl pH 7.0. Use a magnetic mixer with heating to dissolve guanidine thiocyanate. Once dissolved, adjust the final volume to 10 mL with nuclease-free water using a volumetric bottle or a cylinder. Transfer to a 15 mL tube and store at room temperature for up to 6 months.

b. Chaotropic wash (CW)

c. Ethanol wash (EW)

Pipette the components in the following order: water, Tris-HCl, NaCl, and EtOH to avoid salt precipitation in ethanol.

26. Buffers for MarathonRT unit definition

a. MRT 2× reaction buffer

We recommend that the buffer be prepared fresh, aliquoted into 1.5 mL microfuge tubes, and stored at -20 °C. We have successfully used the buffer stored for 2 years.

Note: This original MRT reaction buffer with 2 mM MgCl₂ was used for establishing the assay. An optimized MRT reaction buffer with 4 mM MgCl₂ was subsequently released (uMRT RT kit protocol by RNAConnect, previously available from Kerafast). However, we retained the original buffer composition in order to compare the activity of all protein batches. A new user may find it beneficial to use the optimized buffer with a higher MgCl₂ concentration.

b. P_i dilutions (for standard curve)

Critical: P_i dilutions must be prepared just before use!

Prepare the P_i dilutions for the standard curve from the P_i stock (Recipe 21a). First, prepare 1 mM P_i by pipetting 990 μL of nuclease-free water into a 1.5 mL microfuge tube and adding 10 μL of 100 mM P_i. Mix by vortex and pulse-spin. Second, prepare 50 μM P_i by pipetting 950 μL of nuclease-free water into a 1.5 mL microfuge tube and adding 50 μL of 1 mM P_i from the previous step. Mix by vortex and pulse-spin. Next, prepare standard curve dilutions of the freshly prepared 50 μM P_i directly to the microplate. First, pipette ultrapure water; then, add P_i.

c. Malachite green detection solution (DS)

Critical: Malachite green DS must be prepared just before use!

Prepare the malachite green DS by mixing malachite green reagent (Recipe 21b) with 7.5% ammonium molybdate (Recipe 21c) and 10% Tween (Recipe 21d) into a 1.5 or 2 mL microfuge tube, as shown in the table above.

Critical: Always mix in the following order: malachite green, ammonium molybdate, and Tween-20. Otherwise, the solution will precipitate.

Laboratory supplies

1. MaxyClear Snaplock Microtubes, 1.5 mL (Thermo Fisher Scientific, Fisher Scientific, Axygen, catalog number: MCT150C) or equivalent

2. Screw cap tube, 15 mL, (L × Ø): 120 × 17 mm, PP, with print (Sarstedt, catalog number: 62.554.002) or equivalent

3. Screw cap tube, 50 mL, (L × Ø): 114 × 28 mm, PP, with print (Sarstedt, catalog number: 62.547.255) or equivalent

4. PCR tubes with 0.5 mL flat cap (Thermo Fisher Scientific, Fisher Scientific, Axygen, catalog number: PCR05C)

5. 0.2 mL PCR 8-well strips with single flat caps (Nippon Genetics, catalog number: FG-088WF)

6. 10 μL pipette tips nuclease-free (any)

7. 200 μL pipette tips nuclease-free (any)

8. 1 mL pipette tips nuclease-free (any)

9. Serological pipette, with tip, plugged, 10 mL, sterile, non-pyrogenic/endotoxin-free, non-cytotoxic, 1 piece(s)/blister (Sarstedt, catalog number: 86.1254.001) or equivalent

10. Serological pipette, with tip, plugged, 25 mL, sterile, non-pyrogenic/endotoxin-free, non-cytotoxic, 1 piece(s)/blister (Sarstedt, catalog number: 86.1685.001) or equivalent

11. Petri dishes (any)

12. Glass beads/cell spreader (any)

13. 250 mL Erlenmeyer flask (any)

14. 2 L Erlenmeyer flask (any)

15. Glass bottles for buffers and media (any)

16. Volumetric flasks for buffer preparation (any)

17. Glass beakers (any)

18. Sterile inoculation loops (any)

19. Disposable cuvettes PS semi-micro (Avantor, VWR, catalog number: 634-0676)

20. Fiberlite 1000 mL bottles (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: 010-1491) or Nalgene PPCO centrifuge bottles 500 mL (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: 3120-0500) or equivalent

21. Nalgene Oak Ridge Polysulfone centrifuge tubes w/sealing caps, 50 mL (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: 31370050) or equivalent

22. Pierce centrifuge columns, 10 mL (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: 89898)

23. Disposable PES bottle top filters 150 mL (Thermo Fisher Scientific, Fisher Scientific, Fisherbrand, catalog number: 15953307)

24. Disposable PES bottle top filters, 500 mL (Thermo Fisher Scientific, Fisher Scientific, Fisherbrand, catalog number: 15973307)

25. Amicon Ultra centrifugal filter, 50 kDa MWCO (Merck, catalog number: UFC905024)

26. HisPur Ni-NTA resin (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: 88221)

27. Nunc 96-well microplate (Thermo Fisher Scientific, Fisher Scientific, Thermo Scientific, catalog number: 260836)

28. (Optional) TapeStation Screentapes for RNA (Agilent)

29. (Optional) Multichannel reagent reservoir, any

Equipment

1. 4 °C fridge

2. Cold room

3. -70 °C freezer

4. -20 °C freezer

5. Ice box

6. Direct 8 Milli-Q Direct Water Purification System with filter Biopak Polisher (Merck, catalog number: CDUFBI001)

7. QB Series Dry Block Heating Systems (Grant, model: QBD2) or equivalent

8. XT⁹⁶ Thermal Cycler XT96 Gradient (Avantor, VWR, catalog number: 732-3428) or equivalent

9. ThermoMixer C (Eppendorf, catalog number: 5382000015) or equivalent

10. Incubator shaker New Brunswick^TM Excella E25 Shaker (Eppendorf) or equivalent temperature-controlled shaker that can fit a 2 L flask

11. Microbiological incubator (any)

12. NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, Thermo Scientific, catalog number: ND-2000C) or equivalent

13. Superspeed Centrifuge Sorvall LYNX 4000 with rotors Fiberlite F20-12 × 50 LEX, Fiberlite F12-6 × 500 LEX or Fiberlite F10-4 × 1000 LEX (Thermo Fisher Scientific, Thermo Scientific, catalog number: 75006581) or equivalent

14. Centrifuge 5427 R with rotor FA-45-24-11 (Eppendorf, catalog number: 5429000010) or equivalent temperature-controlled centrifuge that fits 1.5/2 mL microfuge tubes

15. Centrifuge 5810 R with rotors fixed-angle FA-45-6-30 and optional: Swing-bucket rotor A-4-62 (Eppendorf, catalog number: 5811000015) or equivalent

16. Balance AB104-S/PH (Mettler Toledo, catalog number: 11135020) or equivalent

17. Top-loading balance (Sartorius, model: CP2202S) or equivalent

18. Stripettor Ultra Pipet Controller (Corning, catalog number: 4099) or equivalent

19. Electrophoresis Power Supply (CBS Scientific, model: EPS-600) or equivalent

20. Agarose gel electrophoresis equipment (any)

21. Open Air Rocker (Thermo Fisher Scientific, Fisher Scientific, Fisherbrand, catalog number: 88861026) or equivalent

22. ChemiDoc MP Imaging System (Bio-Rad, catalog number: 12003154) or equivalent

23. Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad, catalog number: 1658004) or equivalent

24. Dual Adjustable Vertical System for polyacrylamide gel electrophoresis (CBS Scientific, catalog number: DASG-250) or equivalent

25. (Optional) Automatic gel electrophoresis equipment for DNA and RNA 4150 TapeStation System (Agilent, catalog number: G2992AA)

26. Research Plus 0.5–10 μL pipette (Eppendorf, catalog number: EP3123000020) or equivalent

27. Research Plus 10–100 μL pipette (Eppendorf, catalog number: EP3123000047) or equivalent

28. Research Plus 100–1,000 μL pipette (Eppendorf, catalog number: EP3123000063) or equivalent

29. (Optional) Elite Variable Volume, multichannel pipette 10–100 μL (Thermo Fisher Scientific, Fisher Scientific, Fisherbrand, catalog number: 11825772)

30. Ultrasonic processor UP400S (400 W, 24 kHz) with Sonotrode H7 or H14 (Hielscher Ultrasonics)

31. Diaphragm Vacuum Pump LABOPORT (KNF, type: N 810.3 FT.18) or equivalent for filter sterilization

32. End-over-end rotator (any model)

33. Multiskan FC Microplate Photometer (Thermo Fisher Scientific, Thermo Scientific, catalog number: 51119000) or equivalent

34. Denovix QFX or equivalent fluorophore measuring device

35. 744 pH Meter (Metrohm, model: 744) or equivalent

36. Vortexer (any model)

37. Isotemp RT Advanced Hotplate Stirrer, 350 °C, Ceramic, Aluminum (Thermo Fisher Scientific, Fisher Scientific, Fisherbrand, catalog number: 15326607)

38. Spectrophotometer (any)

Procedure