Bacterial transformation is a fundamental process in molecular biology where bacteria take up foreign genetic material from their environment. This genetic material, often plasmid DNA, integrates into the bacterial cell, allowing the bacterium to acquire new traits. This technique is a powerful tool for various scientific applications, such as studying gene function or producing large quantities of proteins like insulin.
Making Cells Ready for DNA
Preparing bacterial cells to accept external DNA involves making them “competent,” meaning their cell membranes are temporarily permeable. This state is not natural for bacteria and requires specific treatments to induce. The success of transformation, or yield, depends heavily on this preparatory step.
One common approach is chemical competence, which involves treating bacterial cells with a solution containing divalent cations, such as calcium chloride (CaCl2). This treatment neutralizes negative charges on both the DNA backbone and the bacterial cell membrane, reducing electrostatic repulsion. Cells are incubated in this cold CaCl2 solution, on ice, for 10 to 30 minutes, which further rigidifies the membrane.
Following cold incubation, a brief, controlled heat shock is applied. This involves quickly transferring cells to a higher temperature, often 42°C, for 30 to 90 seconds, before immediately returning them to ice. The rapid temperature change is thought to create transient pores in the cell membrane, allowing DNA to enter. This precise temperature shift and timing are important for maximizing DNA uptake.
An alternative method for preparing competent cells is electroporation, which uses an electrical pulse. For this method, cells must be washed extensively with deionized water or a low-salt solution to remove residual salts and impurities. The presence of salts can cause arcing during the electrical pulse, which can damage cells. These washed cells are then suspended in a non-conductive buffer, preparing them for the electric field. This electrical discharge generates temporary pores in the cell membrane, allowing the DNA to move into the cell.
Common Methods for DNA Delivery
Once bacterial cells are made competent, the next step involves introducing the DNA using specific delivery methods. Two widely used techniques are heat shock transformation and electroporation, each requiring distinct parameters for optimal results.
Heat shock transformation builds upon chemically competent cells by applying a sudden temperature shift. The prepared cells, kept cold on ice, are mixed with the DNA to be introduced, in a volume of 1 to 10 microliters. This mixture is then incubated on ice for 15 to 30 minutes, allowing the DNA to associate with the cell surface.
The heat shock involves rapidly transferring the tubes containing the cell-DNA mixture from ice to a water bath preheated to exactly 42°C. Cells are exposed to this temperature for a brief and specific duration, usually 30 to 90 seconds. Immediately after the heat shock, the tubes are returned to ice for 1 to 5 minutes to reseal the cell membranes.
Electroporation utilizes an electrical field to create temporary openings in the cell membrane. Competent cells are combined with the DNA (1 to 5 microliters) in a specialized cuvette with embedded electrodes. The cuvette is then placed into an electroporator.
A brief, high-voltage electrical pulse is delivered across the electrodes. The voltage and pulse duration vary depending on the bacterial strain and cuvette gap, with settings often ranging from 1.8 to 2.5 kilovolts for a 0.1-centimeter gap cuvette, and a pulse duration of a few milliseconds. Immediately after the pulse, a recovery medium is added to the cells, and they are transferred to a sterile tube.
Optimizing for Highest Yield
Beyond the fundamental steps of preparing competent cells and introducing DNA, several other factors influence the yield of transformed bacteria. Optimizing these variables can improve transformation success. These considerations involve the quality and quantity of the DNA, the conditions provided for cell recovery, and proper cell density during plating.
The quality of the DNA itself plays a substantial role in transformation efficiency. DNA should be pure, free from contaminants such as proteins, RNA, or salts, which can inhibit the transformation process. Supercoiled plasmid DNA, the naturally occurring compact form of circular DNA, transforms more efficiently than relaxed or linear DNA, as its compact structure is more readily taken up by the cells. The optimal quantity of DNA ranges from 1 nanogram to 100 nanograms per transformation; higher concentrations do not always increase yield due to saturation effects or toxicity.
Following DNA delivery, the recovery step is a period where cells can repair their membranes and begin expressing genes carried on the newly introduced plasmid, such as antibiotic resistance genes. This step is performed in a rich, non-selective medium like SOC broth (Super Optimal broth with Catabolite repression), which provides nutrients for cell growth and repair. Cells are incubated at 37°C for 30 to 60 minutes with gentle shaking, allowing them to recover from the stress of transformation and synthesize proteins.
After recovery, cells are plated onto selective agar media, often containing an antibiotic corresponding to a resistance gene on the plasmid. The density of cells plated can affect the visibility and enumeration of colonies; plating too many cells can result in confluent growth, making individual colonies indistinguishable, while too few may result in no colonies. Using an appropriate antibiotic concentration is also important; insufficient antibiotic will allow untransformed cells to grow, while excessively high concentrations may inhibit the growth of successfully transformed cells.
Confirming and Troubleshooting Results
After performing bacterial transformation, verifying the procedure’s success and addressing issues that lead to low or no yield are important. Confirmation ensures the desired genetic material has been taken up by the bacteria, while troubleshooting helps refine the protocol for future experiments.
The most direct way to confirm successful transformation is by observing colony growth on selective media. If the plasmid contains an antibiotic resistance gene, only bacteria that have successfully taken up the plasmid will grow on a medium containing that specific antibiotic. The presence of numerous colonies indicates successful transformation, whereas few or no colonies suggest a problem.
Further confirmation can involve isolating plasmid DNA from the transformed bacterial colonies using miniprep procedures. This isolated DNA can then be analyzed by gel electrophoresis to confirm its size and integrity, or it can be subjected to restriction enzyme digestion to verify specific recognition sites within the plasmid. Polymerase Chain Reaction (PCR) can also be used to amplify a specific gene or sequence from the transformed bacteria, providing molecular evidence of successful DNA uptake.
If transformation yields are low or absent, troubleshooting involves systematically reviewing the entire process. Issues often stem from the quality of competent cells, such as insufficient preparation or improper storage, leading to reduced permeability. The quality and quantity of the input DNA can also be a factor; degraded DNA or DNA contaminated with inhibitors will lead to poor results. Incorrect execution of protocol steps, such as imprecise heat shock temperatures or durations, or improper electroporation settings, are common issues that can be adjusted to improve future transformation yields.