Genetic engineering is responsible for medicines, pest-resistance foods, and even denim. In this lesson, you’ll explore the process of bacterial transformation, one way in which scientists use genetic engineering to move genes from organism to organism.
Genes and Technology
They’re nicknamed ‘Frankenfoods.’ Geneticists have taken pieces of DNA from organisms like bacteria and introduced them into the genetic makeup of corn, soy, and other foods. Some consumers are wary of the effects that eating GMOs (genetically modified organisms) might have on our bodies.
But products made by genetic engineering, which is any human-created change in an organism’s DNA, are not only in our foods – they’re almost everywhere.One of the most common genetic engineering techniques is called bacterial transformation, the process in which a circular piece of DNA is induced into a bacterial host. Such pieces of circular DNA are called plasmids. You can think of plasmids as ‘extra’ pieces of DNA – they’re not part of the bacteria’s main genome. They do, however, sometimes have genes that are useful in helping the bacteria survive.For example, some plasmids have genes that confer antibiotic resistance, or the ability to survive in the presence of an antibiotic that would normally destroy it. This is very helpful for the bacteria, but not so great for us when we’re fighting an infection with antibiotics!Bacteria pass these plasmids between them naturally.
This can happen via infection by a virus or, sometimes, through two bacteria physically touching to swap genetic information, which is called conjugation. (By the way, this gene swapping is one reason why we’re having difficulties with bacteria that are resistant to a lot of common antibiotics.)In this lab, we’ll see how a plasmid that confers antibiotic resistance is moved into bacteria by scientists via transformation – and how we can tell if we’ve been successful or not.
Transforming and Plating E. coli
Let’s start by meeting our host organism. Escherichia coli, commonly abbreviated E. coli, is a common rod-shaped bacterium present, among other places, in the human digestive system.
Normally, these bacteria are sensitive to the antibiotic ampicillin — that is, when exposed to this drug, they don’t multiply very effectively or are destroyed completely.We’ll take two microcentrifuge tubes – one with the plasmid (labeled +) and one without (labeled -) – and fill them with 250 microliters of calcium chloride. Calcium chloride will bind to both plasmid DNA (which has a negative charge because of its phosphate backbone) and the positively charged part of the bacterial cell membrane.Next, using sterile technique, we’ll add some bacteria cells to each tube and mix well. Then we’ll add solution that contains suspended plasmids into the (+) tube, and let them ‘chill out’ on ice for at least ten minutes.
This is important because it sets up the next step – heat shocking.During heat shocking, cells at a very cold temperature (on ice) are suddenly plunged into a hot water bath that raises their temperature to about 42 degrees C, and then rapidly placed back on ice. This method encourages the cell membranes to become permeable to the plasmid DNA so that it’s taken into the cell.
At this point, hopefully, the plasmid has been taken up by the bacteria in the tube labeled (+).In science, though, hope is not enough. We need evidence – but how should we get it?We could expose the bacteria in each tube to antibiotics to see if they are resistant and can survive, or are not and will die. But bacteria are notoriously crabby about taking their medicine. So, we’ll create four agar plates.
Two will contain just a growth medium (agar) and a substance called luria broth (nutrients), and two will have both luria broth and ampicillin. We’ll label the plates, we’ll carefully inoculate each plate with bacteria from the proper tube, and then we’ll incubate them at 37 degrees Celsius for 24 to 48 hours.
Before we examine the plates, think about the following questions. What do you expect to see on each plate? What was the purpose of each plate?Remember, we began with a single E. coli culture. The first thing we need to determine is if the bacteria from that original culture were able to withstand our manipulations – that is, did it survive the heat shock protocol? Bacteria of all sorts grow readily on LB plates.
Does it matter if those bacteria have resistance to ampicillin? Nope. If we’ve done everything correctly, we should expect to see growth on both LB (only) plates. These appear as colonies, or circular areas of growth on the agar plate. Or, sometimes, if there’s a lot of bacteria, what’s called a lawn will likely occur; this is where the entire plate is covered.How about the LB/AMP plates? Normally, ampicillin interferes with a bacteria’s ability to make a structurally sound cell wall, preventing colonies from growing.
In short, no plasmid, no growth. If we performed the transformation correctly, we should see growth on the LB/AMP (+) plate, but not the LB/AMP (-) plate.We can also complete a simple calculation to estimate transformation efficiency, the approximate percentage of cells transformed in the LB/AMP (+) tube. This can be done by using the formula:Transformation efficiency = total number of colonies on a plate/the amount of DNA transferred in microgramsTo use this formula, all we have to do is count the colonies on a plate, and divide that number by the amount of DNA that we placed on each plate.
Let’s briefly recap this lesson. Bacterial transformation, the process in which a plasmid is induced into a bacterial host, is one example of genetic engineering, which is any human-created changes in an organism’s DNA. In this lab, we completed a protocol that allowed us to place a plasmid that conferred resistance to the antibiotic ampicillin into the bacterial host cell E.
coli.After completing this protocol, we were able to determine if the transformation was successful by growing bacteria on plates that either contained just nutrients or had both nutrients and ampicillin. Bacteria were able to grow on both plates that contained just the nutrients, but only the bacteria that had been transformed with the plasmid were able to grow into colonies on the plate that had the ampicillin. This is because the plasmid contained a gene for antibiotic resistance.
After you are finished with this lesson, you should be able to:
- Recall the role of plasmids in genetic engineering
- Discuss the process of testing for and proving bacterial transformation
- Estimate a transformation’s efficiency by performing an easy calculation