How can a plasmid be inserted into a bacterial cell? How can transformed bacteria carrying a recombinant plasmid be distinguished from untransformed counterparts? These questions and more will be answered in this lesson.
Genetic Engineering and Bacterial Transformation
We have been discussing one method of creating human insulin for diabetic patients. To this point, our efforts have been focused on creating a recombinant DNA plasmid that will be able to express human insulin. But, if you recall, our ultimate plan is to get bacterial cells to make insulin. That means we need to get our human insulin plasmid into a bacterial cell. Is that even feasible?
Well, lucky for us, there are a number of ways to genetically modify bacterial cells. One of the most common procedures used in laboratories and classrooms alike is known as transformation. Transformation is the process in which the genetic makeup of a cell is changed by the introduction of DNA from the surrounding environment.
Scientists commonly use the bacteria found in our gut in bacterial transformation experiments. This bacteria is known as Escherichia coli, or E. coli for short. E. coli is one of the most commonly used organisms in scientific experiments.
Bacterial cells that are actively growing are most amenable to bacterial transformation. However, they must undergo a series of treatments before they are competent for transformation. There are several ways to make cells competent for transformation, but we will consider the simplest version, which only requires calcium chloride, ice, and heat.
Recall that a cell is surrounded by a cell membrane composed of phospholipid molecules. The phospholipids are organized into a lipid bilayer in which the hydrophobic tails point inward and the hydrophilic heads point outward. Since the hydrophilic head possesses an electronegative phosphate group, the outside of the bacterial membrane is negatively charged.
We have also learned that DNA is charged. The phosphate group in DNA molecules gives DNA a negative charge as well.
Uh, does anyone see a problem with this? We want to push the DNA into the cell, right? But, if DNA and the cell membrane have the same charge, this is going to be like trying to push the negative side of two magnets together. The DNA and cell membrane are going to repel each other!
Bathing the bacterial cells in the salt solution calcium chloride solves this problem. The calcium ions are attracted to both the negatively charged cell membrane and DNA molecules in the solution.
The close proximity of the positively charged ions to these negative charges effectively neutralizes the charge on both the cell membrane and the DNA molecules. This enables the DNA plasmid to move toward the cell membrane without being repelled.
Well, getting the DNA plasmid near the cell membrane is just half the story. Somehow, we need to move the plasmid into the cell. Scientists determined that a sudden increase in temperature could propel a plasmid into a bacterial cell. This treatment is called a heat shock.
While the cells and DNA are incubated with the calcium chloride solution, the entire solution is chilled on ice. Then, the cell/DNA solution is removed from the ice bath and briefly heated to 42 degrees Celsius.
This heat pulse induces some plasmids to enter bacterial cells. It is important to note that chilling the cell/DNA solution is crucial for transformation to occur because the difference between room temperature and 42 degrees Celsius is insufficient.
Scientists aren’t exactly sure how transformation works. One theory is that the heat shock treatment effectively opens pores in the cell membrane through which a plasmid molecule can travel.
It’s also believed that the heat differential between the outside of the cell and the inside of the cell may provide the force that pushes the plasmid into the cell. A plasmid is carried along as the warm water rushes through the pore to equalize temperature. You can think of it like the front door to your heated house on a cold winter day. If you leave the door open, the door may slam as the warm air inside the house rushes outside.
Few cells ever undergo transformation during this process. That’s one reason using bacteria as a host is so useful.
Optimal Growth and Recovery
Because E. coli reproduce so quickly, even one transformed cell can quickly be grown into millions of cells bearing the recombinant plasmid. Now, you may wonder, ‘If 42 degrees is pretty inefficient, then why not increase the temperature or lengthen the heat shock?’
Remember that we’re dealing with living cells. And recall that E. coli naturally occur in our gut. That means that optimal growth temperature for them is human body temperature, or 37 degrees Celsius. Higher heat shock temperatures or longer heat shocks simply kill the E. coli.
E. coli cells are typically given time to recover in a nutrient broth following the heat shock step. This gives the cells time to recover before the next step in the process. However, the recovery step of bacterial transformation is a bit more complicated than simply giving the cells time to rest. We’ll revisit the real reason for the recovery step in a minute.
Since the transformation process is inefficient, we need a way of distinguishing the few transformed cells from a lot of untransformed cells. Luckily, most plasmids used in transformation experiments include a selectable marker.
When the host cell in a transformation experiment is bacterial, like E. coli, selection is achieved by using an antibiotic, such as kanamycin. Recall that our human insulin plasmid also contains an antibiotic resistance gene. That means that growing the bacterial cells in the presence of that specific antibiotic, let’s say kanamycin, will select for the transformed cells.
Selection of transformed bacteria is typically performed on a solid medium called agar. Agar is a less-refined version of agarose, the polysaccharide isolated from seaweed that enabled us to separate DNA based on size during gel electrophoresis. Like agarose, it also possesses gelatin-like properties. It is typically formed into discs in petri dishes for bacterial growth.
Only bacteria that have been transformed with the human insulin plasmid will grow under the antibiotic conditions because they express a protein that confers antibiotic resistance to kanamycin. This antibiotic resistance protein is the main reason for the recovery step.
Consider that most antibiotics, like kanamycin, simply kill bacteria. To survive in the antibiotic environment of this selection step, a bacterial cell must already have the antibiotic resistance protein on hand. However, since this protein is not needed under normal growth conditions, it’s not produced until needed. Therefore, the recovery step gives the genetically engineered bacterial cells the time necessary to produce the antibiotic resistance protein that will protect them from the antibiotic selection.
In summary, transformation is the process in which the genetic makeup of a cell is changed by introduction of DNA from the surrounding environment. To make bacterial cells competent for transformation, the cells are incubated in chilled calcium chloride.
Heat shock is a sudden increase in temperature used to propel a plasmid into a bacterial cell.
The recovery step of a bacterial transformation experiment gives genetically engineered bacteria time to produce antibiotic resistance protein.
In a bacterial transformation experiment, antibiotic selection is used to isolate transformed cells by eliminating the untransformed ones.
At the conclusion of this video, you will be able to explain the steps involved in bacterial transformation, including the importance of heat shock and antibiotic selection.