The ability of a cell to absorb foreign genetic material from its environment is a fundamental process in molecular biology known as competence. Cells engineered or naturally adapted for this purpose are called competent cells, and their capacity to take up DNA is termed transformation. This process is a foundational technique in modern biotechnology, allowing scientists to introduce specific genetic instructions into microorganisms. By controlling this uptake, researchers can manipulate cells for applications ranging from basic genetic study to the industrial production of medicines.
The Definition of Competent Cells
A competent cell is a microorganism, typically a bacterium or yeast, that has been prepared to readily internalize extracellular DNA. This process, transformation, usually involves moving a specific piece of DNA, often a circular molecule called a plasmid, into the cell’s cytoplasm. Plasmids are engineered to carry a gene of interest, such as the human gene for insulin, along with genetic markers like antibiotic resistance genes for selection.
Once inside the bacterial cell, the foreign plasmid DNA is replicated and maintained independently of the host’s own chromosome. This genetic modification allows the cell to express the new gene. Transformed cells can then be grown in large cultures to produce significant quantities of the desired protein, or they can serve to clone and study the gene itself. The efficiency of this process is measured by the number of successful transformations per microgram of DNA, indicating how many cells successfully took up the genetic material.
Natural vs. Induced Competence
Competence exists in two forms: natural and induced. Natural competence is a genetically encoded trait that allows certain bacteria, such as Streptococcus pneumoniae, to actively take up free DNA from their surroundings. This is a survival mechanism, often triggered by environmental stress or high cell density, allowing the bacteria to acquire new traits or repair existing genetic damage.
Induced competence is a state created in the laboratory for organisms that are not naturally transformable, most commonly Escherichia coli. Researchers must physically alter the cell membranes to make them temporarily permeable. This artificial state allows for the reliable and controlled introduction of recombinant DNA into a host cell. The two primary methods for inducing this state rely on either chemical treatment combined with temperature shifts or the application of an electrical field.
Preparation Method 1 Chemical Treatment and Heat Shock
The chemical method is a widely used technique for creating artificially competent cells. The process begins by treating the cells with a cold solution containing divalent cations, most commonly calcium chloride (\(CaCl_2\)). The calcium ions neutralize the natural electrostatic repulsion between the negatively charged phosphate backbone of the DNA and the negatively charged components of the cell membrane.
The cells are kept on ice during this initial incubation. Following the cold incubation, the cells and DNA mixture are subjected to a rapid increase in temperature, known as a heat shock, typically to 42°C for 60 to 90 seconds. This sudden shift generates a thermal imbalance across the cell membrane, which is believed to momentarily disrupt the lipid bilayer and create temporary pores. The neutralized DNA then passes through these pores and into the cell’s interior, completing the transformation process.
Preparation Method 2 Electrical Pulses (Electroporation)
Electroporation uses a high-voltage electrical pulse to permeate the cell membrane. Cells are suspended in a low-salt solution, such as cold water or glycerol, and placed in a specialized cuvette with two aluminum electrodes. When the electrical pulse is delivered, it creates a temporary electric field across the cell membrane, causing transient openings called electropores to form.
The electric field drives the negatively charged DNA through the newly formed pores, resulting in high efficiency. Because high salt concentrations can cause the electrical current to arc and destroy the cells, preparing electrocompetent cells requires extensive washing to remove residual growth media and salts. This method is preferred for cell types resistant to chemical treatment and for applications requiring the highest possible transformation efficiency.