Transformation (biology)
Transformation in biology refers to the process by which bacteria take up exogenous genetic material from their environment and incorporate it into their own genomes. This process can occur naturally or be induced artificially, enabling scientists to conduct various genetic experiments. The concept gained prominence through early 20th-century research, notably the work of Frederick Griffith, who discovered that a non-virulent bacterial strain could become pathogenic when exposed to heat-killed virulent bacteria. Subsequent research identified DNA as the key transformative agent.
In laboratory settings, transformation typically involves making bacterial cells competent to uptake DNA through methods such as incubation with calcium chloride or electroporation, which uses an electrical field to increase cell membrane permeability. Competent cells are then able to integrate plasmids—circular DNA molecules that act as vectors for gene transfer. This technology allows researchers to manipulate genetic material to study gene functions, explore disease pathways, and produce proteins, such as insulin. Transformation is a critical tool in molecular biology, facilitating advances in genetic engineering and biotechnology.
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Transformation (biology)
Bacteria are amazing organisms that can do anything from making us healthy by living in our intestines to cellular machines that can be used to produce substances that can be used to treat disease (e.g., insulin). In the latter case, an additional copy of circular DNA called a plasmid, allows scientists to perform many different genetic tests and experiments about cell function. These bacteria are ideal organisms to ask questions about the function of a cell, test hypotheses about foreign DNA of genes (animal or human), and produce specific proteins that can be used to further knowledge of the function of the human body.
At the beginning of the twentieth century, scientists learned how to isolate certain compounds from a substance and were able to study disease at the molecular level. With the advent of identifying DNA and RNA (genetic material of life), many questions were answered using bacteria, including how a disease works at the genetic level.
Brief History
A landmark experiment that introduced the concept of transformation occurred in 1928 when a British bacteriologist, Frederick Griffith, noted that a non-virulent (non-pathogenic) strain of Streptococcus pneumonia became virulent (pathogenic) after it was grown in the same petri dish as a heat-killed virulent strain. Griffith reasoned that there must have been some "transforming principle" that conferred the pathogenic property to the non-virulent strain of bacteria.
In 1944 three Rockefeller Institute scientists, Oswald Avery, Colin MacLeod, and Macyln McCarty, demonstrated that deoxyribonucleic acid (DNA) is the "transforming principle" through further experiments with pneumococcal bacteria. They proved this through extracting the DNA from the bacterial culture and exposing it with both a protein-digesting enzyme and a DNA-digesting enzyme. Only the DNA-digesting enzyme was effective. This result showed that the transforming material was genetic in nature, and the process was called "transformation." Until the genetic material DNA and RNA were better understood, the idea of transformation was cautiously assimilated into the scientific community. In 1953, the discovery of genetic markers and bacterial conjugation and transduction helped researchers to better understand the process of transferring genetic material.
By 1970, bacteria such as Escherichia coli, were commonly being used in the laboratory. During this time it was believed that transformation could not be performed in E. coli without the help of a bacteriophage, which is a virus that infects bacteria and reproduces inside of it. However, it was discovered that using calcium chloride in the transformation reaction could help facilitate transformation. Calcium chloride aids in transformation by making the cell membrane more porous to DNA. Two years later, Stanley Cohen and colleagues showed that calcium chloride could also work for plasmid DNA.
Building upon the knowledge that a cell wall could be made more permeable chemically, an alternative method of aiding DNA through the cell wall was discovered in the late 1980s, called electroporation. In electroporation, cells to be transformed are placed in a chamber on a glass slide in an electrical field of 10-20 kV/cm. This leads to a change in the voltage across the membrane, which induces a transient permeability that allows a sufficient amount of plasmid DNA to cross the membrane and into the cell plasma.
Overview
Transformation is the process of transferring exogenous (outside the organism) genetic material into a bacterial cell. It is one of several techniques that transfer exogenous genetic material, which occurs either artificially in the laboratory or in nature. Other types of genetic transfer include bacterial conjugation (with the use of sex pili), transduction (using a virus), and transfection (using virus to infect eukaryotic cells). The term also describes the change that takes place in an animal cell after invasion of a tumor-inducing virus.
Transformation occurs naturally in bacteria when exogenous genetic material (DNA) is taken up directly from the environment through the cell membrane. Approximately one percent of bacterial species are able to receive and recombine exogenous DNA. In bacteria, there are several genes that express certain proteins that facilitate the process. On the molecular level, it is a complex, energy-requiring process where the bacterium must be competent, or in a physiological state to be able to take up the DNA. In the bacterium Bacillus subtilis, achieving a state of competence requires at least 40 genes. Also, the DNA that is taken up by the host bacterium is usually from bacteria of the same species. Competence is achieved by inducing DNA-damaging conditions.
A cell can become artificially competent in a laboratory setting by exposing it to conditions that would not normally occur in nature. The cell is usually incubated in a cold solution that contains calcium chloride or another divalent cation. After the incubation period, the cells are exposed to a heat shock (about 45 seconds) in a water bath, and then placed back on ice for about two minutes. Calcium chloride enables the cell membrane to become more permeable to the DNA whereas the heat shock helps the DNA cross the membrane. Specifically, DNA enters the cell membrane of E. coli through channels called zones of adhesion, or Bayer’s junction. A typical bacterial cell can have up to 400 of these zones.
Artificially inducing competence in bacteria such as E. coli enables scientists to manipulate DNA to ask questions about genes involved in cell function, test hypotheses about genes’ roles in disease pathways, and to express proteins (e.g., insulin). A plasmid, or circular DNA found in some species of bacteria, is used in transformation reactions. This plasmid is used as a vector (instrument of transfer) by genetically modifying a desired gene that has been inserted into the plasmid. An important requirement for a plasmid is that it must contain an origin of replication. This allows the DNA to be replicated in the bacterial cell independent of the replication of the bacteria’s non-plasmid chromosomes. This technique is often used to clone DNA, or obtain multiple copies of the same DNA, using bacteria. In some cases, the bacteria can use the DNA to produce a desired protein that is harvested from culture medium after a specific time period.
Bibliography
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