Activity 2-4 - Bacterial Transformation with Engineered Plasmid

Competency

For transformation to occur, bacterial cells must be in a special state known as competence, where their cell membranes are temporarily more permeable to DNA. While some bacteria like Bacillus subtilis can naturally take up DNA from the environment, most laboratory strains of E. coli are not naturally competent. Therefore, they must be artificially induced into a competent state, usually by chemical or electrical methods.

In this experiment, you are using the calcium chloride (CaCl₂) method to induce competency. Treating E. coli cells with ice-cold calcium chloride makes the cell wall more permeable, possibly by neutralizing the negative charges on both the DNA and the cell membrane, reducing electrostatic repulsion. After this treatment, cells are kept on ice to stabilize them in a competent state until they are ready for transformation.

The actual transformation process begins by mixing the plasmid DNA (in this case, your P450 plasmid) with the competent E. coli cells. This is followed by a brief but critical step known as heat shock, typically at 42°C for 30–45 seconds. The sudden temperature change is thought to create a thermal imbalance across the membrane, forming transient pores through which the plasmid DNA can enter the bacterial cytoplasm. Not all cells will take up the plasmid, but those that do have the potential to express the genes it carries. Without the heat shock, transformation efficiency is drastically reduced, although some DNA may still be taken up passively during the incubation on ice. This highlights the importance of the heat shock step in increasing transformation success.

Immediately after heat shock, the transformed cells are incubated with a nutrient-rich medium (like LB broth) at 37°C for 30–60 minutes. This recovery phase serves several critical purposes. It allows the cells to repair their membranes. It gives time for gene expression to begin—especially the expression of the β-lactamase enzyme, encoded by the Ampicillin resistance gene on your plasmid. This enzyme hydrolyzes ampicillin, neutralizing its bactericidal effect. Without this recovery time, even successfully transformed cells may not survive antibiotic selection because they haven’t had a chance to produce enough β-lactamase. This is a classic example of how gene expression timing impacts cell survival in selective environments.

After recovery, the transformed cells are plated on two types of agar plates:

• LB + Ampicillin Plate: This is the selective plate, designed to allow only the bacteria that have successfully taken up the plasmid to grow. If a colony forms here, it suggests the cell has incorporated the plasmid and is producing β-lactamase.
• LB Only Plate: This is a control plate. All bacteria, transformed or not, can grow here because there is no antibiotic to inhibit them. This plate helps verify that the cells were viable post-transformation and that any lack of growth on the Ampicillin plate is not due to cell death or poor handling.

This comparison is key to understanding the role of selectable markers in molecular cloning. Only transformed cells that express the Ampicillin Resistance gene will survive on the Ampicillin plate, allowing for easy screening of successful transformations.

When you observe the plates in the next class, you should expect:

• Numerous colonies on the LB-only plate, representing all viable cells (both transformed and untransformed).
• Fewer colonies on the LB + Ampicillin plate, representing only those cells that were successfully transformed with the P450 plasmid.

The presence of colonies on the selective plate confirms plasmid uptake. If no colonies are seen on the Ampicillin plate but are present on the LB-only plate, this indicates that transformation was unsuccessful or recovery time was insufficient for the expression of the resistance gene.

In this experiment, your plasmid encodes a cytochrome P450 enzyme, which is often used in drug metabolism studies or biocatalysis research. By transforming this plasmid into E. coli, you're essentially programming the bacteria to produce the P450 enzyme. If you later induce the expression of this gene (e.g., using IPTG if under a lac promoter), you can purify the enzyme for experiments like:

• Substrate metabolism assays
• Spectrophotometric P450 activity measurements
• Structural or kinetic studies

Materials:

• Plasmid DNA (isolated in the previous lab)
• Competent E. coli cells (prepared using 0.1 M CaCl₂)
• Micropipettes and sterile tips
• LB broth (Luria-Bertani media) – 1 tube per group
• LB agar plates – 2 per group (without antibiotics)
• LB agar + Ampicillin (100 µg/mL) plates – 2 per group
• 42°C water bath with a thermometer (temperature must be exact)
• 37°C shaking incubator and 37°C still incubator
• Sterile inoculation loop or bacterial spreader
• Bunsen burner with sparker (for sterilization)
• 70% ethanol and bleach (for surface disinfection)
• 0.1 M Calcium Chloride solution (Carolina #851840)

Transformation Protocol

1. Transfer 50 µL of your competent bacteria into a 1.5mL centrifuge tube.
   • (Optional) Mix in 50µL of 0.1M CaCl2 to your bacteria
2. Place the tube on ice and let it sit for at least 2 minutes. 
3. Add 10 µL of your P450 plasmid DNA (Not Digested) into the tube with the competent cells. Gently mix via pipetting—do not cause bubbles. Make sure all liquid is at the bottom of the tube. Place the tube back on ice for another 2 minutes.
4. Carefully place the tube in a 42°C water bath for exactly 45 seconds. This creates a thermal imbalance that helps the plasmid enter the cells. Remove the tube promptly and return it to ice for 1 minutes.
5. (Optional) Add 500 µL of LB broth (room temperature) to the tube. This provides nutrients for the cells to begin recovering and expressing the plasmid genes.
   • Place the tube in a 37°C shaking incubator (or water bath shaker) and incubate for 30 minutes. This allows the cells to begin producing the β-lactamase enzyme, which will protect them from ampicillin.
6. Using a pipette, transfer the half of the mixture onto one LB agar + Ampicillin plate and the other half to an LB-only plate (Optional). Use a sterile spreader or gently shake the plate side to side to spread the liquid evenly.
7. Let the liquid absorb into the plate for about 1 minutes. Then invert the plate (agar side up) and place it in a 37°C incubator overnight.
8. During the next lab session, examine both the LB-only plate and the LB + Ampicillin plate:
   • Count the number of bacterial colonies on each.
   • Note differences in growth between the plates.
   • Observe and record any differences in colony color or appearance on the Ampicillin plate, and compare your results with your classmates.
   • Record all your findings in your lab notebook.