Protein Production: Pre-Culture, Inoculation, Lysis and Incubation with Beads

Pre-Culture

• Pick a single colony from the plate and grow it overnight in an appropriate volume of LB broth at 37°C.

Inoculation

• Dilute the overnight culture at a 1:100 ratio in LB medium with the appropriate antibiotic (final concentration of antibiotic: 1:1000).
• Grow at 37°C until the OD₆₀₀ reaches 0.6 (monitor growth by measuring OD₆₀₀ approximately 2 hours after inoculation).

Lysis and Incubation with Beads

1. Cell Collection

• Collect 1 mL of the cell suspension and measure its OD₆₀₀.

2. Centrifugation

• Centrifuge cultures at 4000 rpm for 20 minutes at 4°C. Store the pellets at -30°C.

3. Lysis of Bacteria

• Preparation of Lysis Buffer (10 mL total):
  • 9.4 mL PBS (50 mM PB + 150 mM NaCl)
  • 50 μL MgCl₂ (from a 1 M stock)
  • 50 μL DNase (from a 5 mg/mL stock)
  • 100 μL PMSF (from a 100 mM stock in EtOH)
• On ice, gently resuspend the cell pellet in the lysis buffer (5 mL for <1 L culture, 10-20 mL for >1 L).
• Sonicate in 50 mL Falcon tubes for 5 minutes at 20% amplitude (3 seconds on, 1 second off). Successful lysis is indicated by a color change in the solution.

4. DNA Digestion

• Incubate on ice for 2 hours on a shaking platform.
• Centrifuge at 9290 g for 1 hour at 4°C and recover the supernatant.

5. Ni-NTA Bead Preparation

• Prepare 2 mL of Ni-NTA beads for every liter of culture media. Wash each 1 mL of beads three times with 1 mL of water and then three times with 1 mL of equilibration buffer (centrifuge for 3 minutes at 3000 rpm and 4°C each time).
• Incubate the supernatant with the washed Ni-NTA beads overnight in a cold room with slow, continuous rotation.

Column Purification Buffers (pH 7.4)

• Equilibration: 50 mL PBS solution
• Wash 1: 150 mL PBS + 10 mM Imidazole (34 mg)
• Wash 2: 50 mL PBS + 20 mM Imidazole (204 mg)
• Elution: 50 mL PBS + 0.5 M Imidazole (1702 mg)

Purification

1. Preparation

• Label three 15 mL Falcon tubes: one for flow-through, one for the first wash, and one for the second wash.
• Prepare 12 labeled 1.7 mL Eppendorf tubes.
• Wash the purification column with water and then with 10 mL of equilibration buffer.

2. Loading and Washing

• Pack the column with the SN + Ni-NTA (total volume: 10 mL).
• Keep the flow-through.
• Wash the column with 10 mL of Wash 1 buffer and collect this in the corresponding Falcon tube.
• Repeat with 10 mL of Wash 2 buffer, keeping the collected solution.

3. Elution

• Elute with 10 mL of elution buffer into the 1.7 mL Eppendorf tubes (fill each tube to about 800 μL).
• Measure protein concentration using a NanoDrop.

MoClo Assembly Protocol

Level 0 Plasmid Construction

Step 1: Preparing the Tubes and Starting the Reaction

The assembly reaction of the DNA fragments is carried out in 20 μL: 14 μL H₂O + plasmids/PCR and 6 μL of the mix below (Excel to calculate necessary volumes of each component is provided as supplementary information to Crozet et al.).

Step 2: Preparing the Reaction

• Assembly Reaction Setup: The assembly of DNA fragments is performed in a total volume of 20 μL, consisting of:
  • 14 μL H₂O
  • Plasmids/PCR products
  • 6 μL of the assembly mix (volumes of each component can be calculated using the provided Excel file).


• Recipient Plasmid: We utilize pICH41308 from the MoClo kit as the recipient plasmid.
• Thermocycler Program: The assembly reaction is conducted in a thermocycler using a program that alternates between optimal temperatures for digestion and ligation, lasting approximately 2 hours and 15 minutes. The BbsI sites remain intact during this assembly, allowing for multiple cycles of digestion and ligation.
• Storage: The MoClo assembly can be stored at 4°C for short-term use or at -20°C for long-term storage.

Step 3: Selection and Validation of Assemblies

Transformation

• 1 tube of 50 μL NEB 10-beta competent E. coli (stored at -80°C) with 3-4 μL of the MoClo assembly.

Procedure

• Remove a tube of bacteria from -80°C for each assembly and control, and place on ice.
• Once thawed, add 3-4 μL of the assembly/control to the tube.
• Heat shock the mixture at 42°C for 45 seconds, then immediately return to ice.
• After 2-3 minutes, add 250 μL of sterile LB broth (without antibiotic) and incubate at 37°C with shaking at 500 rpm for a minimum of 20 minutes (maximum 30 minutes to avoid excessive bacterial growth).
• Plate the entire suspension on LB agar plates containing X-Gal and Spectinomycin.
• Once the plates are dry, invert them and incubate at 37°C overnight. Alternatively, plates can be incubated at 30°C for up to 2-3 days (e.g., over the weekend).

Step 4: Mini Prep and Validation

1. Mini Prep

• Perform a mini prep using a suitable kit to isolate plasmid DNA.

2. Restriction Digest

• Digest the plasmids using restriction enzymes as follows:
  • NEB T4 DNA Ligase Buffer (10x): 1 μL
  • BsaI: 0.2 μL
  • Plasmid DNA: 200 ng
  • H₂O: adjust to a total volume of 10 μL
• Incubate for 30 minutes to 1 hour at 37°C.

3. Gel Preparation

• Prepare a 1% agarose gel.
• Load the entire digestion reaction onto the gel.

4. Visualization

• Run the gel and visualize the bands using a gel imager or reader.

SDS-PAGE Gel Preparation Protocol

1. Prepare Tubes and Stands

Set up and label the necessary tubes and stands.

2. ThermoBlock Setup

Turn on the ThermoBlock and set it to 70ºC.

3. Add PBS

Calculate and add PBS to the labeled tubes according to your experimental design.

4. Add Samples

Introduce samples and reducing buffer to each tube.

5. Centrifuge

Briefly centrifuge the tubes for 5 seconds to mix.

6. Heat Samples

Place the tubes in the ThermoBlock and heat for 10 minutes.

7. Set Up the SDS Mini Gel Tank

Assemble the electrodes and frames for the SDS Mini Gel Tank.

8. Prepare Running Buffer

If needed, prepare the running buffer.

9. Fill the Tank

Fill the tank to approximately three-quarters full with the running buffer.

10. Install the Gel

Carefully remove the 12 or 15 well SDS gel from its packaging, drain any excess buffer, and gently slide the gel into the tank without fully submerging it.

11. Remove the Comb

Gently remove the comb from the gel. Wash the gel and the wells with additional running buffer already present in the tank.

12. Load Samples

Add your samples and protein ladders into the gel according to the predetermined layout.

13. Submerge the Gel

Add more running buffer to gently submerge the gel fully by sliding it down the frame.

14. Cover and Start

Cover the tank (with wires connected) and switch on the machine. It should ramp up to 200V, and small bubbles should be visible rising in the gel near the electrode.

15. Run the Gel

Allow the gel to run for approximately 30 minutes or until the blue dye front reaches the bottom of the gel.

16. Crack the Gel Casing

Using a spatula, gently crack the gel casing and discard it. Cut off the wells just above the blue line.

17. Transfer to Tray #1

Move the gel to Tray #1 containing distilled water.

18. Wash the Gel

Wash the gel in distilled water for about 5 minutes in Tray #1.

19. Prepare Staining Tray (Tray #2)

In a separate tray (Tray #2), add 20 mL of Page Blue stain.

20. Microwave Stain

Add the gel to Tray #2 and microwave for approximately 30 seconds (do not boil).

21. Shake Staining Tray

Set Tray #2 to shake for 60 minutes.

22. Transfer Gel

Gently remove the gel and transfer it back to Tray #1 or to Tray #3 with distilled water.

23. Further Washing

Continue shaking and transfer the gel to fresh distilled water every 15 minutes, or leave it overnight after a couple of washes for optimal clarity.