Title: The Importance of Plasmids
Course: BTEC-1100-301-302-303-F21 > Section: Module 2 - Molecular Biology

Genes and the Central Dogma

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.

Plasmids and Recombinant DNA Technology

Techniques in chemistry enable isolation and purification of cellular components, such as DNA, but practically this isolation is only feasible for relatively short DNA molecules. In order to isolate a particular gene from human chromosomal DNA, it would be necessary to isolate a sequence of a few hundred or few thousand basepairs from the other six billion basepairs of the human genome. Digesting the human genome with restriction enzymes would yield about two million DNA fragments, which is far too many to separate from each other for the purposes of isolating one specific DNA sequence. This obstacle has been overcome by the field of recombinant DNA technology, which enables the preparation of more managable (i.e., smaller) DNA fragments.

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.

By the 1970s the combined discoveries of restriction enzymes, DNA ligase, and gel electrophoresis allowed for the ability to move specific fragments of DNA from one context to another, such as from a chromosome to a plasmid. These tools are essential to the field of recombinant DNA, in which many identical DNA fragments can be generated. The combination of a DNA fragment with a plasmid or vector DNA backbone generates a recombinant DNA molecule, which can be used to study DNA fragments of interest, such as genes.

Molecular Cloning

Plasmids that are used most commonly in the field of recombinant DNA technology have been optimized for their use of studying and manipulating genes. For instance, most plasmids are replicated in E. coli and are relatively small (∼3000 - 6000 basepairs) to enable easy manipulation. Typically plasmids contain the minimum essential DNA sequences for this purpose, which includes a DNA replication origin, an antibiotic-resistance gene, and a region in which exogenous DNA fragments can be inserted. When a plasmid exists extrachromosomally in E. coli, it is replicated upon cell division and segregated to the resulting daughter cells. These daughter cells contain the same genetic information as the parental cell, and are thus termed clonesof the original cell. The plasmid DNA is similarly referred to as cloned DNA, and this process of generating multiple identical copies of a recombinant DNA molecule is known as DNA or molecular cloning. The process of molecular cloning enabled scientists to break chromosomes down to study their genes, 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 basepairs) 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.

Below are some common plasmid elements:

Plasmid Element | Description
Origin of Replication (ORI) | DNA sequence which directs initiation of plasmid replication (by bacteria) by recruiting bacterial transcriptional machinery. The ORI is critical for the ability of the plasmid to be copied (amplified) by bacteria, which is an important characteristic of why plasmids are convenient and easy to use.
Antibiotic Resistance Gene | 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 in the bacteria by bacterial transcriptional machinery.
Multiple Cloning Site (MCS) | Short segment of DNA which contains several restriction enzyme sites, enabling easy insertion of DNA by restriction enzyme digestion and ligation. In expression plasmids, the MCS is often located downstream from a 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.
Insert | The insert is the gene, promoter, or other DNA fragment cloned into the MCS. The insert is typically the genetic element one wishes to study using a particular plasmid.
Promoter Region | Drives transcription of the insert. The promoter is designed to recruit transcriptional 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 Marker | The selectable marker is used to select for cells that have successfully taken up the plasmid 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 Binding Site | A short single-stranded DNA sequence used as an initiation point for PCR amplification or DNA sequencing of the plasmid. Primers can be utlized to verify the sequence of the insert or other regions of the plasmid.

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 of replication, 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 (for the purposes of gene study). Expression vectors must contain a promoter sequence, 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 are modified viral genomes that are used to efficiently deliver 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.