1: Activity 1-1 - Protein Extraction Using Bacterial Cell Lysis

Project 1a - Protein Extraction Using Bacterial Cell Lysis

Cell lysis via Lysozyme and Freeze-Thaw Cycle

Background:

The Bacterial Cell Lysis Kit is designed for the efficient extraction of soluble proteins from bacterial cells using an enzyme-assisted chemical lysis approach. The primary method of cell disruption relies on lysozyme digestion of the bacterial cell wall. Lysozyme specifically cleaves the β-1,4-glycosidic linkages in the peptidoglycan cell wall, thereby weakening the integrity of the bacterial wall, particularly in Gram-negative species like E. coli. This enzymatic step is enhanced with non-ionic detergent, which is also part of the buffer, which helps disrupt the cytoplasmic membrane. The buffer also contains a mixture of organic buffers and salts that stabilize proteins and reduce aggregation or denaturation of proteins during extraction.

Before applying the lysis buffer, you will subject the bacterial pellet to three freeze-thaw cycles. This step involves repeatedly freezing the cell suspension (typically at −20°C or −80°C) and then thawing it at room temperature or 37°C. Freeze-thawing helps initiate cell lysis by forming ice crystals that puncture cellular membranes and disrupt internal structures. Repeating this process three times significantly weakens the cell envelope, making the cells more susceptible to enzymatic and detergent-mediated lysis. It also facilitates better access of lysozyme and detergent to the peptidoglycan layer and inner membrane, improving the efficiency and completeness of protein extraction. Importantly, this method is gentle and preserves protein integrity for downstream functional or structural analysis.

After lysis, the mixture is clarified by low-speed centrifugation, allowing for the separation of the soluble cell-free extract (CFE) from insoluble components such as cell debris and inclusion bodies. The resulting supernatant is suitable for various downstream applications, including affinity chromatography, SDS-PAGE, protein quantification, purification, and enzyme kinetic and drug studies.

Preparation (Before Class)

1. Prepare four (4) sterile 1L LB broths with 100 µg/mL ampicillin (LB + Amp).
2. Inoculate three flasks with pT7BM3 E. coli into three of the LB + Amp flasks.
3. Inoculate one flask with pET3a E. coli into the remaining LB + Amp flask.
4. Label each flask clearly with the Name and date.
5. Incubate all four flasks at 32°C with shaking overnight.
6. After ~24 hours of incubation, store the cultures at 4°C in the Lab fridge until use (Can be stored up to a week)

Materials

• (From preparation step) E. coli cultures (BL21) with pT7BM3 or pET3a plasmids
• Disinfectant spray, biohazard tip waste, Biohazard liquid waste container
• Centrifuge with 50 mL rotor capacity
• 50 mL conical tubes (Corning™352070 or similar)
• -80°C freezer or dry ice
• 37°C shaking incubator
• Vortex mixer
• Bacterial Cell Lysis Kit (GoldBio #GB-176)
  • Lysis Buffer (stored at 4°C)
  • Lysozyme (stored at -20°C)
• Ice bucket (keep filled with ice)

Important Reminder: Keep all enzyme-containing tubes on ice throughout the experiment unless directed otherwise. Cold temperatures help maintain enzyme activity and prevent denaturation. You’ll be given an individual ice bucket to use at your bench.

Collecting Cell Pellet

1. Label and weigh four 50 mL conical tubes. Record the weight:

• Sample A: ______ g
• Sample B#1: ______ g
• Sample B#2: ______ g
• Sample B#3: ______ g

2. Pour the corresponding cultured bacteria into each labeled tube. Ensure that final tube weights match (±0.5g) by adjusting volumes so they’re roughly equal (balance is important for centrifugation).

3. Centrifuge tubes at 5000 rpm for 7 minutes at 4°C.

4. Carefully remove the supernatant without disturbing the pellet.

5. Repeat steps 2–4 five more times.

Freeze-Thaw Lysis (Pre-Lysis Step)

1. Place cell pellets in dry ice or -80°C freezer for ~10 minutes (until fully frozen). (If you have a -20°C freezer, then leave it in for 20 minutes)
2. Thaw samples in a 37°C shaking incubator for 10 minutes.
3. Repeat the freeze-thaw cycle three times in total.

Cell Lysis

1. Add 5x volume of lysis buffer based on pellet weight
   ➤ Example: your conical tube pre-weigh was 50 g, and your new weight is 51 g, and thus, you have 1g of pellet. → Add 5 mL lysis buffer.

2. Record the pellet weights and buffer volumes added:
   • A: ___ g → ___ mL buffer
   • B#1: ___ g → ___ mL buffer
   • B#2: ___ g → ___ mL buffer
   • B#3: ___ g → ___ mL buffer
3. Pipette up and down gently to resuspend each pellet. ⚠️ Avoid bubbles! Use fresh tips if clogged and pipette slowly.
4. Add 10 µL lysozyme for every 1 mL of lysis buffer added.
   • A: ___ µL lysozyme
   • B#1: ___ µL lysozyme
   • B#2: ___ µL lysozyme
   • B#3: ___ µL lysozyme
5. Place tubes in a 37°C shaking incubator for 1 hour to allow enzymatic lysis.
6. Vortex each lysate for 30 seconds to help homogenize the lysed solution.

Collect the Soluble Extract (CFE)

1. Centrifuge tubes at 5000 rpm for 30 minutes at 4°C. Make sure tubes are balanced in the centrifuge!
2. Carefully collect the supernatant (the liquid portions that contain your soluble proteins).
3. Combine all the CFE-B supernatents, and then aliquot into fresh labeled tubes:
   1. 1 mL → Label as “Bradford and Beer - CFE - A” or “Bradford and Beer - CFE - B
   2. Split the remaining supernatant in half, and label "Part 1 - CFE-A or B" and the other half "Part 2 - CFE-A or B"
4. Record and compare the color or appearance of each sample.