In molecular biology, some antibiotics have a specialized role distinct from their use in treating infections. These “selection antibiotics” are used as a tool in the laboratory to identify cells that have successfully taken up foreign DNA. This process is a foundational technique in genetic engineering, allowing scientists to isolate and grow only the specific cells they have experimentally modified.
The Role of Selectable Markers
At the heart of this selection process is a selectable marker, which is a gene that provides resistance to a specific antibiotic. Scientists strategically bundle this antibiotic resistance gene with another gene they wish to study, called the “gene of interest.” Both genes are inserted together into a small, circular piece of DNA called a plasmid, which acts as a vehicle to carry the genetic bundle into a host cell, such as a bacterium.
By linking the resistance gene to the study gene on the same plasmid, the survival of a cell in the presence of an antibiotic is a direct indicator that it has received the gene of interest. Without this marker, it would be nearly impossible to distinguish the tiny fraction of successfully modified cells from the vast majority that were not.
The plasmid, containing both the marker and the gene of interest, is then introduced into a population of host cells. The constant pressure from the antibiotic encourages the bacteria to keep the plasmid as they divide. Since making copies of the extra plasmid DNA can slow down cell division, bacteria might otherwise discard it over time.
The Selection Process in the Lab
The practical application of selection antibiotics begins with a procedure called transformation, where scientists introduce the engineered plasmids into a large population of host cells, like E. coli. This is often accomplished by making the bacterial cell membranes temporarily permeable through a rapid temperature change known as a heat shock. This process is not perfectly efficient, meaning only a small fraction of the bacterial cells will successfully take up a plasmid.
Following transformation, the entire population of bacteria is spread onto a nutrient-rich agar plate that is infused with a specific selection antibiotic. This step is where the “selection” occurs, as the antibiotic acts as a filter, eliminating any bacterium that did not successfully incorporate the plasmid.
Bacteria that failed to take up the plasmid lack the resistance gene and are killed by the antibiotic. In contrast, the few cells that did receive the plasmid can neutralize the antibiotic, allowing them to survive and multiply. These cells form visible clusters called colonies, and each colony is a population of identical cells that all contain the plasmid with the desired gene.
Common Types and Their Mechanisms
Scientists use a variety of selection antibiotics, each with a distinct method of action that targets processes unique to bacteria. A frequently used antibiotic is ampicillin, which works by preventing bacteria from building their cell walls correctly by inhibiting the synthesis of a material called peptidoglycan. Without a stable cell wall, the bacterial cell cannot withstand internal pressure and ultimately ruptures.
Another common selection agent is kanamycin, which targets the protein-making machinery of the cell, known as ribosomes. It binds to a specific part of the bacterial ribosome and disrupts translation, leading to the production of nonfunctional proteins or halting protein synthesis altogether. The resistance gene paired with kanamycin often produces an enzyme that modifies the antibiotic, rendering it harmless.
Tetracycline also targets protein synthesis but in a different way. It binds to the ribosome and blocks the attachment of the molecule that delivers amino acids. This blockage effectively stops the protein chain from growing, and its resistance gene typically produces a pump that actively pushes tetracycline molecules out of the cell.
Applications in Biotechnology and Research
The ability to select for genetically modified cells is fundamental to numerous advancements in biotechnology and scientific research. One of the most significant applications is in the production of pharmaceuticals. Scientists can insert the gene for a human protein, such as insulin or human growth hormone, into bacteria and use antibiotic selection to cultivate large quantities of these engineered bacteria.
This technique is also used for basic research aimed at understanding the function of genes. By inserting a specific gene into a model organism like a bacterium or yeast, researchers can study its effects in a controlled system. The selection process ensures that they are observing a pure population of cells that carry the gene, allowing for accurate conclusions about its role.
In the field of agricultural biotechnology, selection antibiotics are a tool in the development of genetically modified plants. Scientists introduce genes for desirable traits, such as pest resistance or drought tolerance, into plant cells. Antibiotic selection is then used to identify the plant cells that have successfully integrated the new genes before they are grown into full plants.