Creating competent cells, especially BL21 strains, is a fundamental technique in molecular biology. Competent cells are bacteria that can take up foreign DNA, a crucial step in cloning and protein expression. BL21 strains are commonly used for protein production due to their deficient ion protease, which prevents the degradation of expressed proteins. In this guide, we will walk you through a straightforward method to prepare highly efficient BL21 competent cells in your lab. Whether you're a seasoned researcher or just starting, mastering this protocol will significantly enhance your molecular biology toolkit. Let's dive into the nitty-gritty details to ensure your success in making these essential cells.

Understanding Competent Cells

Before we jump into the protocol, let's understand why competent cells are essential and what makes them 'competent.' Competent cells are bacterial cells that have been treated to increase their ability to take up external DNA. This is achieved by altering the cell membrane to make it more permeable. The process involves chemical treatments, such as using calcium chloride or electroporation, which uses electrical pulses to create temporary pores in the cell membrane. The efficiency of competent cells is measured by their transformation efficiency, which indicates how many colony-forming units (CFUs) are produced per microgram of DNA. High-efficiency competent cells are crucial for cloning experiments where you need to introduce plasmids into bacteria for replication or protein expression. BL21 strains, derived from Escherichia coli, are specifically engineered for protein production. They lack certain proteases that can degrade the target protein, making them ideal hosts for expressing recombinant proteins. By making your own competent cells, you can save costs and have greater control over the quality and efficiency of your cells.

Materials Needed

To prepare BL21 competent cells, gather the following materials:

• BL21 E. coli strain: Obtain a fresh stock of BL21 cells.
• LB Broth: Use Luria-Bertani (LB) broth for growing the cells.
• Sterile Culture Tubes and Flasks: Ensure all containers are sterile to avoid contamination.
• Ice-cold Calcium Chloride (CaCl2) solution: Prepare a sterile solution of 50-100 mM CaCl2.
• Glycerol: Use sterile glycerol for creating a cryoprotective agent.
• Sterile Microcentrifuge Tubes: For aliquoting and storing the competent cells.
• Ice Bucket: To keep the cells cold during the procedure.
• Centrifuge: A refrigerated centrifuge is preferable to maintain low temperatures.
• Spectrophotometer: To measure the optical density (OD) of the cell culture.
• Optional: Dimethyl sulfoxide (DMSO) can also be used as a cryoprotectant.

Having all these materials ready before starting the protocol will streamline the process and minimize the risk of errors. Make sure all solutions are sterile to prevent contamination, which can significantly reduce the efficiency of your competent cells. Preparing competent cells is a meticulous process, so attention to detail is key.

Step-by-Step Protocol

Follow these steps to create high-quality BL21 competent cells:

1. Inoculate and Grow the Culture

Start by inoculating a single colony of BL21 E. coli into 5 mL of LB broth. Grow this overnight at 37°C with shaking at 200 rpm. The next day, dilute this overnight culture 1:100 into 50 mL of fresh LB broth in a sterile flask. Continue to grow the culture at 37°C with shaking until the optical density (OD600) reaches between 0.4 and 0.6. This is the optimal growth phase for making competent cells, as the cells are actively dividing and more receptive to transformation. Monitoring the OD600 is crucial because cells that are too dense or not dense enough will yield lower competence. Ensure the flask is well-aerated to promote healthy growth. Overcrowding can lead to stress and reduce the cells' ability to become competent.

2. Chill the Cells

Once the OD600 reaches the desired range (0.4-0.6), quickly chill the culture on ice for 10-20 minutes. This step is critical to slow down cellular processes and prepare the cells for the subsequent washing steps. Cold temperatures help stabilize the cell membrane, making it more amenable to the chemical treatments that induce competence. Avoid any sudden temperature changes to prevent shocking the cells. Keep the culture undisturbed on ice to minimize stress. Chilling the cells properly is a crucial step in ensuring high transformation efficiency.

3. Wash with Cold CaCl2

Centrifuge the chilled culture at 4°C and 4000 rpm for 10 minutes to pellet the cells. Carefully discard the supernatant, being sure not to disrupt the pellet. Resuspend the cell pellet in 25 mL of ice-cold 50-100 mM CaCl2 solution. Gently swirl to ensure the pellet is completely resuspended. Incubate the cells on ice for 20 minutes. This step is crucial as CaCl2 helps neutralize the negative charge of the DNA and the cell membrane, facilitating DNA binding. Centrifuge again at 4°C and 4000 rpm for 10 minutes. Discard the supernatant and resuspend the pellet in 2.5 mL of ice-cold CaCl2 solution. This concentration step is vital for increasing the efficiency of the competent cells. The cold temperature helps to maintain the integrity of the cell membrane during this process.

4. Add Cryoprotectant

Add glycerol to a final concentration of 10-15% (e.g., add 0.25-0.375 mL of sterile glycerol to 2.5 mL of cell suspension). Glycerol acts as a cryoprotectant, preventing the formation of ice crystals that can damage the cells during freezing. Gently mix the solution to ensure even distribution of the glycerol. Alternatively, you can use DMSO at a similar concentration. However, glycerol is often preferred due to its lower toxicity. The cryoprotectant step is crucial for long-term storage of the competent cells without significant loss of efficiency.

5. Aliquot and Freeze

Aliquot the competent cells into sterile microcentrifuge tubes in volumes suitable for your experiments (e.g., 50-100 µL per tube). Immediately snap-freeze the aliquots in liquid nitrogen or a dry ice-ethanol bath. This rapid freezing ensures that the cells are preserved quickly, minimizing the formation of damaging ice crystals. Store the frozen competent cells at -80°C for long-term storage. Avoid repeated freeze-thaw cycles, as they can significantly reduce the efficiency of the cells. Label each tube clearly with the date and cell type for easy identification.

Testing Competency

After preparing your BL21 competent cells, it's essential to test their competency. This ensures that your cells are indeed capable of taking up DNA efficiently. Here's how you can do it:

Transformation Procedure

1. Thaw one aliquot of your competent cells on ice.
2. Add 1-5 µL of a test plasmid (e.g., pUC19) to the thawed cells. The plasmid should be at a concentration of 0.1-1 ng/µL.
3. Incubate the mixture on ice for 20-30 minutes. This allows the DNA to bind to the cells.
4. Heat shock the cells by placing them in a 42°C water bath for 30-60 seconds. This step facilitates the entry of DNA into the cells.
5. Immediately place the cells back on ice for 2 minutes.
6. Add 200-500 µL of LB broth (without antibiotics) to the cells.
7. Incubate the cells at 37°C for 1 hour with shaking (around 200 rpm). This allows the cells to recover and express the antibiotic resistance gene encoded on the plasmid.

Plating and Colony Counting

1. Spread appropriate dilutions of the transformed cells (e.g., 10 µL, 100 µL) onto LB agar plates containing the appropriate antibiotic (e.g., ampicillin for pUC19).

2. Incubate the plates overnight at 37°C.

3. Count the number of colonies on each plate. Calculate the transformation efficiency using the formula:

   Transformation Efficiency = (Number of Colonies / Amount of DNA Used in µg) x (Final Volume After Recovery / Volume Plated)

   A good batch of competent cells should have a transformation efficiency of at least 10^6 CFU/µg of DNA. Higher efficiencies (e.g., 10^7 - 10^8 CFU/µg) are desirable for more demanding cloning applications. If your transformation efficiency is low, review each step of the protocol to identify potential issues, such as contamination, incorrect OD600, or improper handling of the cells.

Troubleshooting Tips

Making competent cells can sometimes be tricky. Here are some common issues and how to address them:

• Low Transformation Efficiency: This is the most common problem. Ensure your cells are grown to the correct OD600 (0.4-0.6). Overgrown or undergrown cultures will have reduced competence. Also, check the quality of your CaCl2 solution and ensure it is sterile. Contamination can drastically reduce efficiency. Finally, make sure your heat shock is performed correctly and that you are using a high-quality test plasmid.
• Contamination: If you see colonies on your control plates (plates without antibiotic), your cells or solutions are likely contaminated. Always use sterile techniques and filter-sterilize all solutions.
• Poor Cell Growth: If your BL21 cells are not growing well, check the quality of your LB broth and ensure that your incubator is set to the correct temperature and shaking speed. Old or poorly stored LB broth can inhibit growth.
• Inconsistent Results: If you are getting inconsistent results between batches, ensure that you are following the protocol exactly each time. Small variations in the procedure can lead to significant differences in competence. Keep detailed records of each batch to help identify any variations.
• Cell Death During Freezing: If your cells are not surviving the freezing process, ensure that you are using the correct concentration of cryoprotectant (10-15% glycerol or DMSO) and that you are snap-freezing the cells quickly in liquid nitrogen or a dry ice-ethanol bath. Slow freezing can cause ice crystals to form, which damage the cells.

Conclusion

Mastering the art of creating competent cells, particularly BL21 strains, is an invaluable skill for any molecular biologist. By following this detailed protocol, you can produce high-quality competent cells in your own lab, saving time and resources. Remember to pay close attention to each step, especially the growth phase, chilling process, and cryopreservation. Regularly testing your cells will ensure they meet the required efficiency for your experiments. With practice and attention to detail, you'll be able to consistently generate competent BL21 cells that are ready for any cloning or protein expression challenge. Good luck, and happy experimenting!