Transgenic Microorganisms

The process by which scientists introduce new genetic material into a microorganism is called molecular or gene cloning. It involves the isolation of DNA from a source other than the microorganism itself. Source organisms span the world of living things, from microbes to plants to animals, including humans. Scientists obtain source DNA in several different ways: by disrupting cells of the target microbe (or plant or animal) and fragmenting it into small pieces, by synthesizing it from an RNA template using an enzyme called reverse transcriptase, or by knowing the specific gene sequence and synthesizing it directly in the laboratory.

Once obtained, the pieces of DNA are inserted into a small genetic component that has the ability to make copies of itself (replicate) independently from the microbial genome. This self-replicating unit is called a cloning vector. Although these genetic elements exist naturally in the form of plasmids and bacterial viruses, many of the ones used today have been altered to improve their properties for transferring genes. Restriction enzymes, which nick the donor DNA and the cloning vector at specific sites, and DNA ligase, which attaches the donor DNA to the cloning vector, allow the source genes of interest to be inserted into the cloning vector without disrupting its ability to replicate.

The next step in the process is the introduction of the cloning vector with its segment of new DNA into a living cell. Bacteria have the ability to transport DNA into their cells in a process called transformation, and this ability is commonly exploited to achieve this goal. Getting the DNA into the cell, however, is only the beginning. No transformation is 100 percent efficient, and so the bacteria that receive the gene(s) of interest must be separated from those that did not. One of the best studied and most commonly used cloning vectors, pBR322, is especially useful for this purpose, as it contains several genes for antibiotic resistance. Hence, any cell transformed with DNA containing pBR322 will be antibiotic resistant, and thus can be isolated from similar cells that have not be so transformed by merely growing them in the presence of the appropriate drugs. All that remains is to identify bacteria that are producing the product of the desired gene(s), and cloning is a success.

The introduction of human genes into bacteria has several complicating wrinkles that make cloning them even more challenging. For example, a bacterial gene codes for a protein from start to finish in one long string of nucleotides, whereas human cells have stretches of noncoding nucleotides called introns within their genes. Bacteria do not have the same ability as human cells to remove these introns when producing proteins from the gene, and if the introns are not removed, the intended protein cannot be produced. This, along with other complications, has been overcome using many of the tools of genetic engineering.