Plasmids are a type of extrachromosomal, circular DNA molecules found in bacteria and several types of eukaryotes. They are a type of self-replicative molecules inside a cell and are independent of the genomic DNA. Hence, they can be used as carriers of foreign DNA fragments into various types of cells in genetic engineering. The molecular biology technique involved here is cloning. Genetic engineering creates organisms with novel characteristics. These novel organisms are known as genetically modified organisms (GMO). This article focuses on the process of genetic engineering, describing the use of plasmids in the creation of new organisms through altering the genomes.
Key Areas Covered
Key Terms: Cloning, DNA, Genetic Engineering, Genetically Modified Organisms (GMO), Plasmids
What are Plasmids
Plasmids are small circular DNA molecules mainly found in bacteria. They are extrachromosomal DNA elements, capable of replicating independently from the bacterial genome. The genes encoded in plasmids help bacteria to survive under stress conditions. Several to many copies of plasmids can naturally occur inside a bacterial cell. Plasmids can be used as vectors that carry foreign DNA molecules into both eukaryotic and prokaryotic cells. The features that help plasmids in order to be used as vectors are described below.
Features of Plasmids
- Plasmids can be readily isolated from bacterial cells.
- They are self-replicative inside cells.
- They are composed of unique restriction sites for one or more restriction enzymes.
- The insertion of a foreign DNA fragment may not alter the replication properties of plasmids.
- Plasmids can be sequentially transformed into different types of cells and the transformants can be selected based on the antibiotic resistance properties of the transformed plasmids.
How are Plasmids Used in Genetic Engineering
Genetic engineering is the modification of DNA in order to produce new types of organisms by inserting or deleting genes. The introduction of genes can be done by means of vectors such as plasmids. The main steps of genetic engineering are given below.
- PCR amplification of the target DNA sequence
- Digestion of DNA fragments and plasmids by the same restriction enzyme
- Ligation of plasmids and the foreign DNA fragments, producing recombinant DNA molecules.
- Transformation of the recombinant DNA molecules into a desired type of cells.
- Selection of transformed cells.
The most common vectors used in cloning are isolated from E. coli. Each plasmid contains three functional regions: origin of replication, a gene responsible for the antibiotic resistance, and restriction recognition site for the insertion of a foreign gene. A particular restriction enzyme is used to cut both plasmid and the foreign DNA fragment. During restriction digestion, the circular plasmid becomes linear and during ligation, the foreign DNA fragment can be inserted into the two ends, making the plasmid again circular. The recombinant plasmid is transformed into a receptive cell that can be bacteria, yeast, plant, or animal cell. The production of a large number of recombinant DNA molecules inside the receptive cell is known as cloning. The transformed cells can be identified by the antibiotic resistance of the plasmid. However, the transformant may contain the mutual plasmid or the recombinant plasmid. Both types of plasmids show resistance to antibiotics. Hence, another gene such as LacZ is required in order to identify the transformants with recombinant plasmids. The transformants with the recombinant plasmids are called the GMOs.
The detailed process of molecular cloning is shown in figure 2.
Plasmids are circular DNA molecules that naturally occur in bacteria. They contain genes mainly for antibiotic resistance. Plasmids are used in genetic engineering to transfer foreign genetic material into different types of cells. The foreign DNA fragment is inserted into the plasmid and the recombinant DNA molecule is transformed into the recipient cell. The transformed cells are selected by the antibiotic resistance of the used plasmid.
1. Lodish, Harvey. “DNA Cloning with Plasmid Vectors.” Molecular Cell Biology. 4th edition., U.S. National Library of Medicine, 1 Jan. 1970, www.ncbi.nlm.nih.gov/books/NBK21498/.