By Megan Knight, BSc
Bacterial cells often possess molecules of closed, circular DNA, otherwise known as ‘plasmids’. They can also be present at much lower frequencies in certain eukaryotic cell types, such as yeast. They are non-essential, self-replicating DNA molecules which are important for the prokaryotic mobile gene pool.
Plasmids can only exist and replicate within a cell, where it uses host cell machinery. They consist of small circular double-stranded DNA and have a huge diversity in size i.e. from 2kb-200kb.
Plasmids are vertically distributed to daughter cells after host cell division. They can also be transferred horizontally between different strains of prokaryotic cells, during a process called bacterial conjugation. Their copy number in bacterial cells varies greatly. The plasmid type determines copy number; high copy number plasmids can be more than 100 copies per cell, whereas others are limited to very few copies per cell.
A bacterial plasmid is a small DNA molecule that are commonly used bacterial cloning. The cloning plasmids contain a site for a DNA insert. Image Credit: Soleil Nordic / Shutterstock
Types of Plasmids
Plasmids are important for bacterial evolution and adaptation to the changing environment, as they carry genes which carry beneficial traits for the bacterial cell.
Different types of plasmids can coexist in one bacterial cell. For example, strains such as Escherichia coli can have three different small plasmids in many copies, and one large single copy plasmid. Copy number depends on how actively transcribed the plasmids origin of replication is. If it is extremely active, plasmids can undergo several replication cycles during a single cell cycle.
Individual plasmids carry very few genes, but they can carry huge selective advantages in certain environments. For example, plasmids can contain antibiotic resistance genes, posing a risk to public health. Plasmids carrying resistance genes are known as R plasmids. Increased use of antibiotics globally has caused bacterial strains carrying R plasmids to multiply in size; this means resistant pathogens are becoming more common, making treatment of certain bacterial infections much more difficult. Another complication is that certain R plasmids can carry up to 10 resistance genes to different antibiotics, and these plasmids often contain genes which allow bacterial conjugation to occur.
Other naturally occurring plasmids include those carrying the sex factor (F), which allows the cell to perform bacterial conjugation, replication origins and maintenance functions.
Certain types of plasmids can insert part of their DNA into the host cell genome, by a process known as integration. These plasmids can exist in two forms, which are extrachromosomal replicons or integrated plasmids. These plasmids are known as ‘episomes’.
The mitochondria within human cells also contains a closed circular DNA molecule; this is known as mtDNA. It encodes 37 genes and 16500bp. The endosymbiont theory states that mitochondria were once an independent prokaryotic species, which formed a symbiotic relationship with a larger eukaryotic cell. mtDNA can only be inherited from the mother, and mutations in it lead to a diverse range of diseases due to varying metabolic demands in organs of the human body.
The Use of Plasmids in Basic DNA Cloning
Most DNA cloning techniques uses modified bacterial cells as host cells e.g. removed virulence factors. This is due to short cell cycle times and rapid multiplication.
Plasmids which replicate in E. coli are one of the most common choices of vector in DNA cloning. A vector is a molecule which is used to carry target DNA as a passenger into cells, which allows the target DNA sequence to be replicated. Plasmids are a popular choice of vector as they are one of the most efficient ways to propagate DNA, they have an independent replication cycle and possess their own origin of replication.
Natural plasmids must be genetically modified before being used as a vector for cloning. They are designed so target restriction fragments, cut by specific restriction enzymes, have a unique location in that plasmid. These sites flank the cloning site in the plasmid. Many modified plasmids have a variety of unique restriction fragment sites, formed by unique combinations of restriction enzymes. This makes the plasmid a more flexible cloning vector.
Modified plasmids also carry a marker gene. The most common form is a gene conferring resistance to commonly used antibiotics. For cloning to be effective, the bacterial host cells must be sensitive to the chosen antibiotic; this means that surviving colonies have been transformed by plasmid vector, allowing them to grow in that environment.
A standard cloning procedure begins with the ligation of a plasmid vector, with a marker gene, and the target DNA fragment. This requires a ‘ligase’ enzyme and forms a recombinant plasmid. The plasmid is then introduced to bacterial host cells by a heat shock. This step multiples the recombinant vector’s copy number. The bacterial cells are then cultured on agar plates containing the antibiotic which the vector confers resistance too. This selects for transformed colonies. The recombinant plasmids can then be extracted, purified and sequenced from the transformed colonies and used in a variety of studies i.e. expression studies.
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