The use of antibiotic selection and positive and negative controls are important elements of interpreting data from a bacterial transformation. In this lesson, you will learn how antibiotic selection results in colony formation and how controls help pinpoint the cause of experimental problems.
Review of Bacterial Transformation and Genetic Engineering
We have been considering the steps necessary to produce genetically engineered bacteria capable of producing human insulin. First, we inserted the human insulin gene into a bacterial plasmid using restriction enzymes and ligase. Then, we inserted this recombinant plasmid into the bacteria E. coli using a transformation protocol that featured calcium chloride and heat shock. But, what exactly would successful experimental results look like?
The kanamycin resistance gene in the plasmid makes it resistant to the kanamycin antibiotic.
Recall that even though we're taking steps to increase the probability that a transformation event will occur, bacterial transformation remains a relatively infrequent event. To distinguish the few transformed cells from the millions of untransformed ones, we use antibiotic selection. Since the plasmid housing the human insulin gene also contains an antibiotic resistance gene, say the kanamycin resistance gene, bacteria carrying our recombinant insulin plasmid will be resistant to the antibiotic kanamycin.
Bacterial colonies in a petri dish
E. coli cells that were not transformed with the recombinant plasmid will be killed by this antibiotic. The handful of cells that remain will grow into bacterial colonies. A bacterial colony is an aggregate of bacterial cells that grew from a single progenitor cell. Consider for a moment what the plate would look like if we could see all the microscopic E. coli cells on the plate immediately following our transformation protocol.
Although there are thousands or millions of E. coli on the plate, most are going to be killed by the antibiotic in the plate. The remaining recombinant cells will undergo many rounds of cell division. As more and more copies of the original recombinant cell are added, a colony that is visible to the naked eye is formed.
It is important to note that each colony consists of clones of the starting recombinant cell. That is, barring a rare spontaneous mutation event, the cells of each colony should be identical. Since spontaneous mutation is relatively rare under experimental conditions, every cell in a bacterial colony is usually identical. This means that any biological product produced by these clones will be identical as well. This is particularly important both to a researcher who wishes to limit variables affecting his or her experiment and to a pharmaceutical manufacturer who wishes to guarantee patients that the medicine provided is safe.
When conducting an experiment, it's always important to consider all the possible outcomes.
It would be fantastic if every transformation experiment always worked perfectly, but unfortunately we live in an imperfect world, so we should probably be prepared in case we get less-than-perfect results. Previously, we learned that a control is a means of ensuring that only one factor is being tested at a time. Typically, this involves setting up two groups to compare: the experimental group, which has been altered by a single variable, and the control group, which remains unchanged.
For instance, say I want to test new fertilizer. If I didn't include a control group (that is, plants that were not treated with the experimental fertilizer), I would have no way of knowing if the fertilizer only appeared to work poorly. If the seeds were too old or the soil was too dry, we might incorrectly conclude that the new fertilizer doesn't work. By comparing the growth of the experimental group to an untreated control group, we'd notice that neither group of plants grew and draw a completely different conclusion than we might have without the control.
Okay, let's apply that logic to our transformation experiment. One control we could perform is testing the growth of bacteria on non-antibiotic media. If no bacterial cells grow under these conditions, we know that something was wrong with the bacterial strain we used. Since all bacterial cells are capable of growing under these conditions, a lawn is formed instead of individual colonies. This control is known as a positive control with respect to bacterial growth because it is a test in which the affected result can be predicted. We expect the bacteria to grow on a regular agar plate.
Expected bacterial growth under various controls
A second control that would help is testing the effect of the antibiotic on untransformed bacteria.
If any untransformed bacteria can grow on an antibiotic plate, something may be wrong with the antibiotic. This is known as a negative control with respect to bacterial growth because it is a test in which a change in the system is not predicted. We don't expect the bacteria to grow on an antibiotic plate.
Optimally, a bacterial lawn should grow on the positive control plate, nothing should grow on the negative control plate, and bacterial colonies should grow on the experimental plate. However, when optimal results aren't achieved, scientists can use the results from the controls to quickly determine and correct the most likely cause of the error. For instance, if the bacteria grew on the antibiotic plate, it's probably time to open a new bottle of antibiotics because the old one may be inactive.
Significance of Bacterial Transformation
The ultimate goal of bacterial transformation is to genetically modify bacteria for research or manufacturing purposes. This procedure continues to be a critical part of any molecular biology toolbox. Bacteria are commonly used by scientists to store genetically engineered DNA, typically in plasmids.
Scientists can also use bacteria bearing recombinant DNA as biological factories to mass-produce protein or even more of the recombinant DNA itself. As we have discussed, this has been valuable not only from a research standpoint but also from a medical standpoint. Pharmaceutical companies can grow vast quantities of insulin-producing bacteria and then harvest the insulin produced by those bacterial cells for clinical use.
In summary, a bacterial colony is an aggregate of bacterial cells that grew from a single progenitor cell. A positive control is a test in which the affected result can be predicted. In a bacterial transformation experiment, growing untransformed bacteria on a regular growth plate is considered a positive control with respect to growth because we expect the bacterial cells to grow. A negative control is a test in which a change in the system is not predicted. In a bacterial transformation experiment, attempting to grow untransformed bacteria on an antibiotic plate is considered a negative control with respect to growth because we do not expect any growth.
At the end of this lesson, you'll be able to:
- Explain how bacterial transformation works and its importance
- Define positive and negative controls with respect to bacterial transformation experiments