What Are Plasmids?
Plasmids are small, circular, extrachromosomal, double-stranded DNA molecules, naturally found in bacterial species. However, their presence has also been found in some archaea and eukaryotes.
Plasmids are physically different from chromosomal DNA. All bacterial plasmids replicate on their own and contain an origin of replication (which controls the plasmid’s host range and copy number) and genes that contribute to the survival of bacteria, such as antibiotic resistance genes.
On the other hand, bacterial chromosomes contain genes required for the development and life of the microbes, under normal conditions.
Figure: An illustrative diagram of bacteria showing bacterial chromosomal DNA and Plasmids.
Plasmids are present in variant lengths in different microbes, ranging from around one thousand base pairs to hundreds and thousands of base pairs. During cell division, the plasmid in bacterial cells is copied and one plasmid is transferred to each daughter cell through a process called bacterial conjugation. This exchange of bacterial genomes can also occur between two different bacterial strains.
Furthermore, plasmids are also regarded as mobile genetic elements and the process of their transmission of genetic material, through conjugation, is a type of mechanism of horizontal gene transfer.
Plasmids are artificially altered and constructed in labs to use as a tool for transferring foreign DNA in specific cell types. They are widely used as a cloning vector to drive recombinant DNA replication in host cells. And, because of their ability to replicate on their own within hosts, plasmids are also known as replicons.
In this article, we will review the types of plasmids, how they are artificially designed in labs for laboratory assays, and their applications.
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Types of Plasmid
Plasmids can be classified in many ways. For example, based on their ability to transfer genes through conjugation, they are grouped as conjugative and non-conjugative plasmids. Whereas, based on their functions they are classified into:
- Fertility F-plasmids
- Resistance (R) plasmids
- Virulence plasmids
- Degradative plasmids
- Col plasmids
Also known as F-plasmid, fertility plasmids contain TRA genes that help in the transfer of genes to other bacterial species through conjugation. They fall in the category of conjugative plasmids.
Because of their ease and ability to be inserted into chromosomal DNA, they are also referred to as episomes in the context of prokaryotes.
In the process of transferring a plasmid, the bacteria that receive the plasmid are known as F+ bacteria and the bacteria that do not are known as F- bacteria. However, the fusion between these two bacteria results in the production of two F+ plasmids.
Resistance plasmids are also known as R-plasmids or R-factors. They contain the genes that help bacteria to protect themselves in unfavorable environments, such as in the presence of antibiotics or poison.
Often, they can be transferred to other bacterial species through conjugation, providing that species is resistant to specific antibiotics.
These plasmids, when present in a bacterial strain, turn it into a pathogen. They can easily spread and multiply among affected organisms.
For example, certain Escherichia coli (or E.coli) strains present in the human gut and other animals when containing virulence plasmids, cause vomiting and diarrhea. Other organisms having virulence plasmid include Agrobacterium tumefaciens (contain Ti plasmid) and Salmonella enterica.
Degradative plasmids belong to the group of conjugative plasmids. They contain genes encoding proteins that help the bacteria to digest those compounds that are not commonly present in nature, such as xylene, camphor, toluene, and salicylic acid.
Col plasmids contain genes that produce bacteriocin, helping one bacterial species to kill other bacterial species.
How Are Plasmids Constructed?
Plasmids are one of the essential tools in research and medicinal labs to understand, design, and develop a range of therapeutics for deadly diseases. A plasmid can transfer the desired piece of nucleic acid to the target location to cure mutation-driven diseases, such as cancer.
The common components present in all man-designed plasmids are the origin of replication, selection marker, antibiotic resistance gene, promoter region, primer binding sites, and cloning site. Currently, plasmids are constructed by first digesting DNA sequences using restriction enzymes and then ligating the ends of the DNA fragments using the enzyme DNA ligase.
Figure: An illustration of a plasmid construct with its labeled elements.
Another technique to build plasmids is known as gap-repair cloning (GRC) which involves using the homologous recombination activity of budding yeast cells.
The artificially created plasmids are generally referred to as vectors. And, other than the above-mentioned techniques, a variety of other cloning techniques, such as Gibson, Gateway, and ligation independent methods, are used to insert a desired nucleotide sequence in the plasmid.
After the desired DNA fragment is inserted in the vector, it’s transformed into bacterial cells that are grown on culture plates containing selective antibiotics. This only allows the growth of the bacterial species that contain the plasmid.
Since plasmids are isolated from bacteria, it makes them an efficient medium to increase the copy number of plasmids in the amount they want.
What Are Plasmids Used For?
Plasmids have extensive use in molecular biology, biotechnology, and microbiology labs. They are artificially constructed in labs and designed to carry and introduce foreign DNA or nucleotide sequences into cells. They are an essential tool to clone, transfer, and manipulate genes.
Based on the purpose of the study, different types are created, such as expression vectors, cloning plasmids, RNA plasmids, reporter plasmids, gene knock-down plasmid vectors, and genome engineering plasmids.
The artificially designed recombinant plasmids have a range of applications including transcriptional analysis of organisms and altering specific gene expression in the target cell. Below is a brief on some common uses of plasmids.
Genetic engineering involves the use of recombinant technologies to insert or delete genes, altering the complete genetic makeup and producing a new type of organism. The process involves the following basic steps:
- The target DNA sequence is amplified using the PCR technique.
- The target DNA and plasmids are digested using the same restriction enzyme.
- The resulting DNA fragments due to digestion are ligated using DNA ligase.
- The resulting recombinant nucleic acid is transformed into a specific cell type.
- The transformed cells are selected based on a selection marker inserted in the plasmid.
The plasmid is one of the effective, easy, and affordable tools in use for the mass production of proteins. A gene of interest is inserted in the plasmid, and the bacteria can induce the production of proteins through the inserted genes.
One example of proteins produced using a plasmid vector is insulin, which is used to treat diabetic patients.
Figure: An illustration of the process of producing therapeutic proteins using artificially constructed plasmids.
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Plasmids are small and circular, double-stranded DNA that replicate autonomously and are naturally found in bacterial species. They contain genes that help microbes to survive in unfavorable conditions.
In labs, plasmids are one of the extensively used tools to integrate foreign DNA into specific cell types. And, because of their versatility, safety, flexibility, and cost-effectiveness, they are utilized by biotechnologists, molecular biologists, and microbiologists in a wide range of applications.
Executing these applications in labs becomes much easier when they are combined with automated, high-throughput equipment with advanced software, which helps researchers to generate reliable and validated data in a large amount.
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