Plasmids have been essential to the development of molecular biotechnology. Any novice scientist entering a molecular biology lab can expect to be assigned to create, modify and construct plasmids. Thanks to advancements in plasmid DNA construction, medical researchers have been able to create a wide variety of medicines and treatments.
What is plasmid in DNA?
A plasmid is a circular-shaped extrachromosomal molecule within a cell. Plasmid DNA in a cell is in close proximity to chromosomal DNA and both have a similar chemical composition. However, plasmid DNA is able to exist and replicate separately from chromosomal DNA.
What is plasmid DNA made of?
Just like the DNA in a cell, plasmids are made of nucleic acids. Plasmids are polynucleotides, with nucleic acids composed of nitrogen-based aromatic bases attached to a pentose sugar, which in turn connects to a phosphate group. Most plasmid DNA is double-stranded, a feature that gives it higher stability.
DNA sequences are constituted out of identical building blocks known as nucleotides. These can contain four out of five possible nitrogen-containing bases, commonly recognized by a letter:
- Adenine (A)
- Guanine (G)
- Cytosine (C)
- Thymine (T)
- Uracil (U)
Nucleotides A and G are cataloged as purines, which means that they have a two-ringed composition made of carbon and nitrogen atoms. In turn, C, T, and U are understood to be pyrimidines, as they have a six-ringed structure of five carbon atoms together with a single nitrogen atom.
DNA (Dextroribonucleic acid) is built out of A, G, C, and T nucleotides. In RNA (ribonucleic acid), a single-stranded molecule similar to DNA, T is replaced by U nucleotides. While most plasmids have a double-stranded DNA structure, RNA-based single-stranded plasmids also exist.
Do humans have plasmid DNA?
The nuclei of human cells do not contain any foreign DNA, therefore human cells do not contain plasmid DNA. Nonetheless, the field of gene therapy uses plasmids for the insertion of therapeutic genes in the human body. These plasmids can positively affect diseased or infected cells by triggering therapeutic genes in them.
What is the main function of plasmid DNA?
The main function of plasmids is to carry genetic material and spread its influence over the body of a host. A plasmid DNA gene can contain information that improves the body’s antibiotic resistance and can also provide other benefits to the survivability of its host.
Functions of plasmids include:
- Carrying and spreading antibiotic-resistant genes (this is how many diseases are treated)
- Carrying genes involved in metabolic activities, which help the body digest environmental pollutants
- Producing antibacterial proteins
- Carrying genes that can cause an increase in the pathogenicity of bacteria
How does a plasmid work?
The genetic material contained in plasmid DNA is advantageous to its host. The circular DNA molecule resides in close proximity to chromosomal DNA. Within a cell, plasmids can influence its genetics, enhancing the resilience and constitution of organisms.
Plasmids possess a double-stranded DNA structure shaped in the form of a circle. This circular structure is created by both ends of a plasmid’s DNA being connected through covalent bonds. It is important to know that not all plasmids share this form, as plasmids with a linear DNA structure have also been discovered.
Plasmids replicate independently within a cell. To do so, smaller plasmids make use of the host cell’s replication enzymes. In turn, larger plasmids may have genes responsible for their replication. As self-replicating units, plasmids use replicons as their origin of replication. Also known as Ori, the origin of replication is a particular sequence in a genome where it can begin to be copied.
Plasmid copy number
It is important for plasmids to be able to regulate the rate at which they self-replicate. High-copy plasmids run the risk of overwhelming the host cell due to an overuse of cellular machinery and energy. On the other hand, plasmids with a low-copy number may not transfer their genes to the cell’s progeny.
A plasmid’s DNA sequence and regulation of its origin of replication are the main factors that influence copy number. Other factors that influence a plasmid’s copy number include the culture inoculum, the bacterial strain being used to propagate the plasmids, and growth conditions such as temperature, aeration, and bacterial culture volume.
Plasmids in nature
In nature, plasmids are commonly used by bacterial cells to regulate their stress-related responses. For instance, plasmid DNA in a bacteria can help it generate antibiotics when it has been affected by a foreign toxin or help cells survive in otherwise lethal environments. In the same vein, plasmids can give bacterial cells “weapons” to hunt or defend themselves, such as the ability to secrete poison.
Although most plasmids are composed of double-stranded DNA sequences, single-stranded DNA and double-stranded RNA plasmids also exist. RNA plasmids have been found in fungi and various species of plants. Even so, it is considerably hard to distinguish RNA plasmids from other strands of RNA.
Types of plasmids
There are 5 types of plasmids:
- Resistance plasmids: These plasmids usually carry the genetic material that grants poison or antibiotic resistance. Resistance plasmids are often involved in bacterial conjugation and carry the genes necessary for the construction of conjugation pili.
- Degradative plasmids: This type of plasmid can help a cell degrade or digest organic matter. By consuming dead organic matter from plants and animals, cells can generate energy and recycle the matter. Organic matter is also used for biosynthesis, a process crucial for cell metabolism where enzymatic reactions create natural products.
- Fertility plasmids: These plasmids are responsible for carrying genes related to bacterial conjugation. Their main function is to carry tra-genes to aid in the transferring of genetic material between bacterial cells.
- Col plasmids: By supplying the genes to produce an antibiotic called colicin, these plasmids help cells generate proteins that protect them while killing other bacteria. Col plasmids are the reason for E. coli’s harmfulness, for example.
- Virulence plasmids: As the name suggests, these plasmids carry genes that cause diseases and are responsible for turning bacteria into pathogens.
What is the difference between DNA and plasmid DNA?
While chromosomal DNA is crucial for the cell’s existence and development, plasmid DNA is unrelated to the cell’s genetic structure. However, plasmid DNA can supply genes that enhance the cell’s genetics.
The differences between chromosomal and plasmid DNA include the following:
- Size: Plasmid DNA is smaller than chromosomal DNA and contains fewer genes.
- Shape: Plasmid DNA is commonly circular-shaped, while chromosomal DNA can be either circular or linear, depending on the organism.
- Host cell: While mostly prokaryotes carry plasmids, chromosomal DNA can be found in both prokaryotic and eukaryotic cells.
- Number: The number of chromosomal DNA strands depends on the species. In contrast, the number of plasmids within a cell varies widely. Some cells may contain only one plasmid, while others can have hundreds of them.
- Replication: Chromosomal DNA replicates with the genome, while plasmid DNA is self-replicating.
- Reading frame: Chromosomal DNA in eukaryotes contains exons and introns, which facilitate the encoding of proteins. Plasmid DNA and the chromosomal DNA of prokaryotes, however, have an open reading frame.
- Transference: Cell division processes like mitosis and meiosis transfer chromosomal DNA. On the other hand, transferring of plasmid DNA requires a horizontal gene transfer.
Why are plasmids useful?
Plasmids are incredibly useful to scientists and researchers due to their ability to transform cells. Artificially-engineered plasmid vectors are important tools in laboratories, where they can be used to clone, amplify, or express genes.
How is plasmid DNA constructed in the lab?
Plasmid DNA is artificially modified and reconstituted in a lab from bacterial cultures, using various methods to break down and rebuild DNA into its intended form. The construction of plasmids is a crucial process in molecular biology.
Plasmid DNA is commonly constructed in special environments known as restriction sites, where restriction enzymes are used to cut DNA fragments for later reconstitution. Another plasmid construction method is based on gap-repair cloning (GRC). This second method allows for further flexibility during construction, as no restriction sites are needed. It is common to insert the plasmids into bacteria such as E. coli cells to observe their properties.
How do scientists use plasmid DNA?
Plasmid DNA encoding is performed by scientists to harness the benefits bacterial cells receive from plasmids. Properties that enhance the cell’s growth or give selective advantages to bacteria can be isolated, reconstructed, and given scientific or medical use.
Scientists can use plasmid DNA to manipulate a target cell’s gene expressions. Plasmids can offer flexibility, versatility, and safety, giving them an expansive range of applications. It is common for scientists to produce large quantities of plasmids, as they can be reserved for later work.
Scientists can use plasmids for the following operations:
- Producing large protein amounts so it can be purified and studied in a controlled environment.
- Producing bioluminescent proteins, so scientists can better track their location and quantity inside of a cell.
- Detecting how prevalent a chemical is in a particular environment.
- Producing synthetic viruses for research or therapeutic purposes
- Genome engineering. This process consists in producing enzymes that will influence specific, controlled changes in an organism’s genome structure.
Isolation of plasmid DNA
Plasmid DNA purification is an essential step for plasmid DNA to be useful. Molecular biologists need to have plasmid DNA in an isolated form to perform several procedures, including DNA sequencing, cloning, transfection, and gene therapy. The preferred plasmid purification method among scientists is alkaline lysis.
Plasmids and gene therapy
Plasmids are incredibly important in the field of gene therapy. Plasmids have the ability to target infected cells and trigger a therapeutic response in them; plus they’re easy to manipulate. The replication of plasmids on the target cell is also not complicated. Thereby, plasmids are used to insert therapeutic genes into the human body to help it fight diseases.
Plasmids and recombinant DNA technology
There are many uses of plasmids in recombinant DNA technology. For example, drug delivery technology makes use of plasmids to insert drugs into the body. Plasmid DNA can also be used to create antibiotic resistance and kill harmful bacteria. Common recombinant DNA technology applications of plasmids include the manufacturing of insulin and the insertion of human growth hormones in mammalian animal cells.