Emily Keator received her B.S. in Biology from Davidson College and currently does neuroscience research.
Genes are popularly referred to as “the blueprints for life.” Studying these blueprints has led to leaps for mankind ranging from the Human Genome Project (completed in 2003), to identifying risk factors for deadly and debilitating genetic diseases (as well as potential treatments), to improved methods of genetic testing, to the use of genetic technologies to produce hardier plants and cheaper hormone therapies, and so much more. But in order to fully understand these blueprints and the structures they encode, scientists must conduct deep studies of specific genes. They need to investigate how genes are translated (i.e., how the cell takes a genetic blueprint and turns it into a protein, the basis for cellular structures), how they interact with other genes, and how changing the amount or level of expression of a gene affects how cells work. Such research requires manipulating genes of interest to generate new cell lines for testing, create gene libraries, or producing large quantities of a gene’s encoded protein. To do so, scientists need to essentially copy a gene and paste it into different biological constructs.
But despite the simple blueprint metaphor, a gene cannot be run through a photocopier.
To copy a gene, researchers often employ a method known as bacterial cloning. This process places a gene of interest into a bacterial cell’s genome. The mention of bacteria might throw up a red flag, but not all bacteria are dangerous to humans, and they are often used in carefully controlled experiments. In the case of genetic cloning, bacteria are extremely useful due to their small sub-sections of their genomes, called plasmids, and their exponential growth rate, which facilitate rapid copy of genetic material. When bacterial cells divide and multiply, the gene of interest is copied and pasted into every new bacterial cell.
The first step of bacterial cloning is to get the gene of interest into the bacterial genome. Bacteria are one-celled, prokaryotic organisms and have simpler cellular structures than humans, who are complex and multi-cellular eukaryotes. The genomes of each are stored slightly differently: both rely on large, long structures of DNA called chromosomes, but while you can think of a bacterial genome as one large, single circle of DNA twisted up like a convoluted rubber band and free-floating in the bacterial cell, the human chromosome structure (26 chromosomes in total) is a long linear string of DNA twisted up and tucked away neatly into a sub-packet of the cell called the nucleus—think what happens to your headphones when you put them in your pocket. In addition to their larger, circular chromosome, bacteria have other small, circular, free-floating pieces of DNA called plasmids. These plasmids are maintained and copied separately from the larger circular chromosome. Because bacteria can pick up new plasmids from their surroundings in a process called transformation, plasmids allow bacteria to pick up new genes and adapt to the environment.
Researchers use these plasmids to get around the effort of working with the whole bacterial genome. Commercially developed plasmids, specially designed for all different types of research experiments, are available from many different species of bacteria. Researchers take these specially designed plasmids and combine them with many copies of the gene of interest within a test tube. In order to add the new piece of DNA to the plasmid, researchers add to the tube a special enzyme called a restriction enzyme. These enzymes recognize specific sequences of DNA, a two-stranded, ladder-like molecule, and induce a double-stranded break at that sequence. This means that the enzyme breaks both sides of the ladder. A restriction enzyme will cut only one specific sequence, the same way a key only turns a specific lock. The ends of the DNA containing the gene of interest are cut by the same restriction enzyme to match.
From there, molecules assemble like pieces of a jigsaw puzzle. The restriction enzyme has come along and cut specific sequences through each piece, at the places marked on the blueprint, and only the ends that complement each other can fit together again.
Researchers later add something called DNA ligase, a biological glue gun, to the test tube of DNA. The ligase zips DNA back together. Since the new gene was cut to match, some plasmids will incorporate the new piece and therefore incorporate the gene of interest as their own.
The next step in bacterial cloning transforms the bacteria; now that a vector has been generated to deliver a gene of interest into an organism, the recipient organism need to be induced to take up the plasmid. The newly constructed plasmids are placed in a tube with bacterial cells and put through a process called heat shock, which encourages along the process of transformation. The rapid transfer from ice to a hot water bath and quickly back to ice causes bacterial cells to loosen their membranes (the barrier between bacteria and their environment) and take up the plasmid. Plating these bacteria onto a nutrient-rich plate allows colonies to grow up overnight in a warm incubator.
Once the processes that introduce a plasmid to living cells are complete, the focus of the researchers turns to identifying the bacterial cells that actually contain the gene of interest. This can be done in a number of ways, but one of the simplest is to use reporter methods that cause the bacteria carrying the gene of interest to self-identify. Specially modified plasmids often used in cloning experiments contain genes for resistance to an antibiotic (such as ampicillin), and some other kind of reporter gene. Quite simply, the bacteria are plated onto agar mixed with ampicillin, and only those cells that took up a plasmid can grow in the presence of the antibiotic. This screens against any bacterial cells that no longer pertain to the experiment.
Furthermore, a cleverly-designed reporter gene system produces some kind of measurable sign that indicated the presence or absence of the gene of interest in a bacterial colony. Recall that in order to add a gene of interest to a plasmid, researchers cut the plasmid and pasted the gene in the middle of that break. If the cut occurs in the middle of a reporter gene, and the gene of interest gets pasted into that break, then whatever the reporter gene encodes cannot be properly translated—with only half the blueprint available, you can’t build a house. You can’t even start. If DNA ligase instead resealed the cut ends of the plasmid back together, the reported gene remains intact and functional, and some signal is produced. However, if the gene of interest was pasted into the middle of the reporter gene, it disrupts the function of the reporter gene.
A classic example of a biological reporter system is called the LacZ reporter gene system. When left intact, a gene known as LacZ produces a protein that interacts with a sugar called X-Galactosidase (“X-Gal”) to produce the color blue (left). If the gene of interest has been added to a plasmid, it fits right into the middle of the LacZ gene. When bacteria grow on a plate that also contains X-Gal, any colonies of bacteria containing the gene of interest will be white—the LacZ gene is disrupted and therefore no blue product appears. Researchers can discount any blue colonies, because they reflect the absence of the gene of interest.
Researchers can finally pick the right colonies and grow them in broth to create a solution of bacteria containing millions of copies of their gene of interest. This form of storage is isolated, simple to maintain, and quickly reproducible. From their new stock of bacteria containing the gene of interest, researchers can perform any number of experiments. For instance, a human protein can be produced in large quantities form the bacteria, isolated, and studied. Or, experiments that would be difficult to perform on a human gene in a human or an animal can be performed in the bacterial cells. The gene could also be further isolated from the bacterial colonies, and transferred into other cell lines from there for further experiments. It may not be as fast as a photocopier, but in modern molecular medicine, bacterial cloning is a vital and productive method to copying genes for further research.