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Genetic engineering, by its nature, requires that DNA be stored until needed and moved at will from the test-tube environment to a cellular environment or vice versa. A DNA plasmid makes it easier to accomplish this goal. A plasmid is a small circle of DNA, typically found in bacteria, that is separate from the majority of bacterial DNA located in the nucleoid. That might sound like a mouthful, but basically a plasmid is a vehicle for storing and studying genes.
Scientists use plasmids in laboratory experiments as a vector, or vehicle, for DNA of interest, typically a gene. Plasmids are usually represented by a simple circle with the important features noted. To be an effective tool for scientists, a plasmid typically possesses three basic features: a multiple cloning site, an origin of replication, and a selectable marker. Let's see how each of these plasmid features plays a key role in genetically engineering bacteria to produce human insulin.
If the main purpose of a plasmid is to serve as a vehicle for genes of interest, we need to be able to insert the human insulin gene into the plasmid. This is the purpose of a multiple cloning site. A multiple cloning site is the location in a plasmid where a sequence of DNA, typically a gene, can be inserted. We'll discuss in greater detail how a gene can be inserted into the multiple cloning site in another lesson.
Once a gene is inserted into a plasmid, you obviously don't want to lose it. Wouldn't it be great if the bacteria did all the work maintaining the plasmid for us? Actually, that's one of the main reasons scientists put a gene into a bacterial plasmid. As we learned previously, the origin of replication is the place where the process of DNA replication begins. Therefore, if our plasmid possesses an origin of replication, the bacteria will automatically make a new copy of the plasmid during the replication process.
Recall that DNA replication creates an exact copy of DNA in the cell. This means that the two daughter cells both receive a copy of the DNA found in the mother cell when the mother cell divides. If our genetically-engineered plasmid has an origin of replication, it is also replicated. This means that each daughter cell also receives a copy of our human insulin gene whenever the bacterial cell undergoes replication. In this way, our bacterial host has become a living storage vessel for the genetically-engineered plasmid. As long as this genetically-engineered bacteria is alive, it will maintain our insulin plasmid.
Okay. So, we have a means of getting the insulin gene into a DNA plasmid using the multiple cloning site. And, we will have a continual supply of our plasmid because the origin of replication will ensure that a new copy of the plasmid is created each time the host bacterial cell divides. That's great. With those plasmid characteristics alone, we can create genetically-engineered bacteria that will make insulin for us.
But, isn't bacteria all around us? How are we going to differentiate between the genetically-engineered bacteria and 'regular' bacteria from the environment? Right now, there's no way we'd be able to distinguish between the two. Since bacteria is everywhere and mass-producing insulin is going to entail use of optimal bacterial growth conditions, what's to prevent contaminating bacteria from interfering with industrial production or laboratory experiments?
The answer is a selectable marker. Consider the proverbial needle in a haystack. How do you propose finding the needle amongst all that hay? Obviously, we could sift through the haystack by hand 'til we find the needle, but that seems like a lot of work. Adding a magnet as a tool would be a more sophisticated version of this search method, but, at its heart, this is still a search as well. We're still actively doing something, albeit less work because of the magnet.
Wouldn't it be great if we could just sit on a porch drinking lemonade while the needle finds itself? How's this for an idea? Let's toss a match into the haystack and get that lemonade while we consider the consequences of our fiery plan. The fire is going to burn the hay to the ground, allowing us to just waltz up and pick up our needle once we've finished our lemonade. Note that we didn't need to do any work once we set the haystack ablaze.
This is what a selectable marker provides. A selectable marker is a gene that enables identification of genetically modified organisms of interest by removing or inhibiting unwanted organisms. It is the third major characteristic of DNA plasmids. Note the difference between 'selection,' which requires no work on our part and 'screening,' which requires work on the part of the scientist to identify the bacteria of interest.
What is the biological equivalent of fire from this haystack analogy? For bacteria, the answer is antibiotics. Recall that an antibiotic is a substance that specifically kills or inhibits the growth of bacteria without harming other organisms. Also recall that bacteria can acquire resistance to antibiotics. In the medical field, that's a bad thing. In the research field, that is simply another trick to add to our laboratory tool belt.
What would happen if our insulin plasmid included an antibiotic-resistance gene as a selectable marker? The antibiotic-resistance gene produces a protein that allows the bacteria to survive in the presence of an antibiotic that would normally kill the bacteria. Do you see where this is going? If the plasmid contains both the antibiotic-resistance gene and our insulin gene, we can ensure we are always dealing with the genetically-engineered bacteria if we grow our bacteria in the presence of the antibiotic. Random bacterial contaminants will be killed by the antibiotic, leaving only the resistant genetically-engineered ones that we want.
In summary, a plasmid is a circular piece of DNA, which can be used as a vector for DNA of interest in molecular biology experiments. A multiple cloning site is the location in a plasmid where a sequence of DNA can be inserted.
An origin of replication is the place where the process of DNA replication begins. It is a critical component of a DNA plasmid because it ensures the plasmid is passed from mother to daughter cells during cell division.
A selectable marker is a gene that enables identification of a genetically modified organism of interest by removing or inhibiting unwanted organisms. When dealing with a bacterial host, the selectable marker is usually an antibiotic-resistance gene.
You will be able to describe the three characteristics of plasmids - multiple cloning site, origin of replication and selectable marker - that make plasmids important to genetic engineering at the conclusion of this video.
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Back To CourseMCAT Test: Practice and Study Guide
88 chapters | 863 lessons