Plasmid
In 1952, Joshua Lederberg coined the term plasmid, in reference to any extrachromosomal heritable
determinant. Plasmids are fragments of double-stranded DNA that typically carry genes and can replicate
independently from chromosomal DNA. Although they can be found in archaea and eukaryotes, they play the
most significant biological role in bacteria where they can be passed from one bacterium to another by a type
of horizontal gene transfer (conjugation), usually providing a benefit to the host, such as antibiotic resistance.
This benefit can be context-dependent, and thus the plasmid exists in a symbiotic relationship with the host
cell. Like the bacterial chromosomal DNA, plasmid DNA is replicated upon cell division, and each daughter
cell receives at least one copy of the plasmid.
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) for physical
isolation (purification) and manipulation. 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 easily make an endless number of
copies of the plasmid using bacteria, which can uptake plasmids and amplify them during cell division.
Because bacteria are easy to grow in a lab, divide relatively quickly, and exhibit exponential growth
rates, plasmids can be replicated easily and efficiently in a laboratory setting.
Stable - Plasmids are stable long-term either as purified DNA or within bacterial cells that have been
preserved 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 were originally used to understand gene function, they are now used for a
variety of studies used to investigate promoters, small RNAs, or other genetic elements
Plasmid 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 (ORI), 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.
Common plasmid elements:
Plasmid
Element Description
DNA sequence which directs initiation of plasmid replication (by bacteria) by recruiting
Origin of bacterial transcriptional machinery. The ORI is critical for the ability of the plasmid to be
Replication copied (amplified) by bacteria, which is an important characteristic of why plasmids are
(ORI) convenient and easy to use.
Allows for selection of plasmid-containing bacteria by providing a survival advantage to the
bacterial host. Each bacterium can contain multiple copies of an individual plasmid, and
ideally would replicate these plasmids upon cell division in addition to their own genomic
DNA. Because of this additional replication burden, the rate of bacterial cell division is
reduced (i.e., it takes more time to copy this extra DNA). Because of this reduced fitness,
bacteria without plasmids can replicate faster and out-populate bacteria with plasmids, thus
selecting against the propagation of these plasmids through cell division. To ensure the
retention of plasmid DNA in bacterial populations, an antibiotic resistance gene (i.e., a gene
whose product confers resistance to ampicillin) is included in the plasmid. These bacteria are
then grown in the presence of ampicillin. Under these conditions, there is a selective pressure
to retain the plasmid DNA, despite the added replication burden, as bacteria without the
plasmid DNA would not survive antibiotic treatment. It is important to distinguish that the
antibiotic resistance gene is under the control of a bacterial promoter, and is thus expressed
Antibiotic in the bacteria by bacterial transcriptional machinery.
Resistance
Gene
Multiple Short segment of DNA which contains several restriction enzyme sites, enabling easy
Cloning insertion of DNA by restriction enzyme
digestion and ligation. In expression plasmids, the MCS is often located downstream from a
Site (MCS) promoter, such that when a gene
is inserted within the MCS, its expression will be driven by the promoter. As a general rule,
the restriction sites in the MCS are
unique and not located elsewhere in the plasmid backbone, which is why they can be used
for cloning by restriction enzyme
digestion.
The insert is the gene, promoter, or other DNA fragment cloned into the MCS. The insert is
Insert typically the genetic element one
wishes to study using a particular plasmid.
Promoter Drives transcription of the insert. The promoter is designed to recruit transcriptional
Region machinery from a particular organism or
group of organisms. Meaning, if a plasmid in intended for use in human cells, the promoter
will be a human or mammalian
promoter sequence. The promoter can also direct cell-specific expression, which can be
achieved by a tissue-specific
promoter (e.g., a liver-specific promoter). The strength of the promoter is also important
for controlling the level of insert
expression (i.e., a strong promoter directs high expression, whereas weaker promoters can
direct low/endogenous expression
levels).
Selectable The selectable marker is used to select for cells that have successfully taken up the plasmid
Marker for the purpose of expressing the
insert. This is different than selecting for bacterial cells that have taken up the plasmid for
the purpose of replication. The
selectable marker enables selection of a population of cells that have taken up the plasmid
and that can be used to study the
insert. The selectable marker is typically in the form of another antibiotic resistance gene
(this time, under the control of a non-
bacterial promoter) or a fluorescent protein (that can be used to select or sort the cells by
visualization or FACS).
Primer A short single-stranded DNA sequence used as an initiation point for PCR amplification or
Binding Site DNA sequencing of the plasmid.
Primers can be utlized to verify the sequence of the insert or other regions of the plasmid.