What is a Plasmid?
Origins of Molecular Genetics
The concept of genes as carriers of phenotypic information was introduced in the early 19th century by Gregor Mendel, who later demonstrated the properties of genetic inheritance in peas. Over the next 100 years, many significant discoveries lead to the conclusions that genes encode proteins and reside on chromosomes, which are composed of DNA. These findings culminated in the central dogma of molecular biology, that proteins are translated from RNA, which is transcribed from DNA.
In 1952, Joshua Lederberg coined the term plasmid, in reference to any extrachromosomal heritable determinant. Plasmids are fragments of double-stranded DNA that can replicate independently of chromosomal DNA, and usually carry genes. Although they can be found in Bacteria, Archaea and Eukaryotes, they play the most significant biological role in bacteria where they can be passed from one bacterium to another by horizontal gene transfer, usually providing a context-dependent selective advantage, such as antibiotic resistance.
By the 1970s the combined discoveries of restriction enzymes, DNA ligase and gel electrophoresis together allowed for the ability to move specific fragments of DNA from one context to another, such as from a chromosome to a plasmid. This process of molecular cloning allowed scientists to break chromosomes down to study their genes individually, marking the birth of molecular genetics.
Today, scientists can easily study and manipulate genes and other genetic elements using specifically engineered plasmids, commonly referred to as vectors, which have become possibly the most ubiquitous tools in the molecular biologist’s toolbox.
Importance of Plasmids
Plasmids have become an essential tool in molecular biology for a variety of reasons, including that they are:
- Easy to work with - Plasmids are a convenient size (generally 1,000-20,000 base pairs). With current cloning technology, it is easy to create and modify plasmids containing the genetic element that you are interested in.
- Self-replicating - Once you have constructed a plasmid, you can make an endless number of copies of the plasmid by growing the plasmid in bacteria.
- Stable - Plasmids are stable long-term either as purified DNA or within bacteria (as glycerol stocks).
- Functional in many species and can useful for a diverse set of applications - Plasmids can drive gene expression in a wide variety of organisms, including plants, worms, mice and even cultured human cells. Although plasmids are commonly used to understand gene function, they can also be used to investigate promoters, small RNAs, or other genetic elements.
Plasmids used by scientists today come in many sizes and vary broadly in their functionality. In their simplest form, plasmids require a bacterial origin of replication, an antibiotic resistance gene, and at least one unique restriction enzyme recognition site. These elements allow for the propagation of the plasmid within bacteria, while allowing for selection against any bacteria not carrying the plasmid. Additionally, the restriction enzyme site(s) allow for the cloning of a fragment of DNA to be studied into the plasmid.
Below are some common plasmid elements:
|Origin of Replication (ORI)||DNA sequence which allows initiation of replication within a plasmid by recruiting transcriptional machinery proteins|
|Antibiotic Resistance Gene||Allows for selection of plasmid-containing bacteria. Learn more in the Antibiotic reference.|
|Multiple Cloning Site (MCS)||Short segment of DNA which contains several restriction sites allowing for the easy insertion of DNA. In expression plasmids, the MCS is often downstream from a promoter.|
|Insert||Gene, promoter or other DNA fragment cloned into the MCS for further study.|
|Promoter Region||Drives transcription of the target gene. Vital component for expression vectors: determines which cell types the gene is expressed in and amount of recombinant protein obtained.|
|Selectable Marker||The antibiotic resistance gene allows for selection in bacteria. However, many plasmids also have selectable markers for use in other cell types.|
|Primer Binding Site||A short single-stranded DNA sequence used as an initiation point for PCR amplification or sequencing. Primers can be exploited for sequence verification of plasmids. See Addgene's sequencing primers.|
Types of Plasmids
The combination of elements often determines the type of plasmid. Below are some common plasmid types:
- Cloning Plasmids - Used to facilitate the cloning of DNA fragments. Cloning vectors tend to be very simple, often containing only a bacterial resistance gene, origin and MCS. They are small and optimized to help in the initial cloning of a DNA fragment. Commonly used cloning vectors include Gateway entry vectors and TOPO cloning vectors.
- Expression Plasmids - Used for gene expression. Expression vectors must contain a promoter sequence as well as a transcription terminator sequence, and the inserted gene. The promoter region is required for the generation of RNA from the insert DNA via transcription. The terminator sequence on the newly synthesized RNA signals for the transcription process to stop. An expression vector can also include an enhancer sequence which increases the amount of protein or RNA produced. Expression vectors can drive expression in various cell types (mammalian, yeast, bacterial, etc), depending largely on which promoter is used to initiate transcription.
- Gene knock-down Plasmids - Used for reducing the expression of an endogenous gene. This is frequently accomplished through expression of an shRNA targeting the mRNA of the gene of interest. These plasmids have promoters that can drive expression of short RNAs.
- Reporter Plasmids - Used for studying the function of genetic elements. These plasmids contain a reporter gene (for example, luciferase or GFP) that offers a read-out of the activity of the genetic element. For instance, a promoter of interest could be inserted upstream of the luciferase gene to determine the level of transcription driven by that promoter.
- Viral Plasmids - These plasmids use technology evolved by viruses to aid in delivery of genetic material into target cells . You can use these plasmids to create viral particles, such as lentiviral, retroviral, AAV, or adenoviral particles, that can infect your target cells at a high efficiency.
If you are looking for an empty plasmid backbone for your experiment, see Addgene's empty backbone page for more information.