Plasmids are fragments of double-stranded DNA that can replicate independently of chromosomal DNA, and usually carry genes. What does that mean? All information must be carried or conveyed via some medium. Digital information is carried electronically, a collection of billions or trillions of tiny, organized switches that can be in of two distinct positions (on or off / 1 or 0). Written information is conveyed through language and syntax, the distinct arrangement of universal character set intended to convey a specific thought or concept.
Like other forms of information, genetic information also must be expressed through a medium, and that medium is the gene. Genes are the smallest functional unit of inheritance, and are composed of linear double-stranded DNA sequences that carry the blueprint for every distinct heritable phenotypic trait.
Plasmids: A short definition
By their simplest definition, plasmids are stretches of DNA that contain at least one gene and are:
- Small (with respect to nearly any whole genome)
- Extrachromsomal (not packaged inside a chromosome)
- Circular (No distinct 5’ or 3’beginning or end)
- Capable of autonomous replication (i.e. they containing their own origin of replication)
Plasmids occur naturally in many prokaryotes, and often contain critical genes necessary for antibiotic resistance, but they are also commonly adopted for co-opted for use in the life sciences as vectors.
A) Cloning Vectors:
In molecular biology, plasmids are used as vectors, ferrying genetic material from one cell to another, for the purposes of replication or expression.
One of the most frequent uses for a plasmid is as a “cloning vector”. Cloning vectors are used to replicate, modify, and temporarily store a specific desired gene sequence.
A plasmid used as a cloning vector will typically contain:
- A multiple cloning site (MCS), containing sequences recognized by common restriction enzymes, and designed to allow simple insertion of a desired gene sequence.
- An origin of replication (ORI) , allowing the plasmid to be simply and rapidly duplicated by the host organisms replication machinery.
- A gene that encodes an end product conveying some form of antibiotic resistance to the host organism (this allows the researcher to distinguish between bacteria that contain the plasmid, and those that don’t).
- A selection mechanism designed to confirm that a desired sequence has been inserted into the multiple cloning site.
Once all of the necessary cloning has been completed, a cloning vector is transferred into a robust host organism (most commonly a laboratory strain of E. coli) through a technique known as transformation.
Transformed E. coli will replicate the cloning vector, with each individual bacterial cell generating hundreds or even thousands of copies in a very short period of time. Upon lysis the high-copy cloning vector can be isolated, further modified, or transferred to an expression vector.
B) Expression Vectors:
Expression vectors, like cloning vectors, are used for horizontal gene transmission. There is a difference however. Cloning vectors are primarily used for replication – generating a large number of copies of the same plasmid.
By contrast, the purpose of an expression vector is to co-opt the natural transcriptional and translational machinery in the host organism, and express the final engineered gene product.
If the cloning vector is the reporter’s notebook, the expression vector is the end product. A final, edited story submitted for print.
Expression vectors will contain all of the same basic elements present in a cloning vector, but they’ll also have something else: a promoter sequence that recruits transcriptional machinery, and directs the host organism to transcribe an RNA template from the cloned gene product.
The specific promoter sequence that a researcher chooses for an expression vector is dependent upon gene cloned into the expression vector, the organism and cell type that the researcher wants the gene to be expressed in.
Many promoters sequences are cell-type specific, and while most or all of the cells in a transgenic organism will contain copies of the expression vector, only specific cells or tissues will generate RNA transcripts of the transgene.
Expression level also plays a role in choosing a promoter sequence for an expression vector. “Strong” promoters will prompt production of many copies a given RNA transcript, while other “weaker” promoters will result in only transient expression, with fewer copies produced.
Working with expression vectors
Cloning vectors can be transformed into E. coli or other prokyaryotic host cells with relative ease and simplicity. Prokaryotes lack a nucleus, and plasmid DNA need not be present in any particular subcellular location to be replicated or expressed. Application of heat or an electric field can stress E. coli enough to permeabilize its outer membrane and permit the passage of plasmid DNA into the cell.
Working with eukaryotic cells is not quite as simple. The presence of a nucleus as a central organizational structure containing genetic material, the inability to withstand many of the different stringent treatments that make E. coli transformable, and the ready and frequent destruction of foreign cytosolic extrachromosomal DNA by many eukaryotes makes transforming them more complex.
For many eukaryotic cell lines (particularly those of mammalian origin) the preference is to use a viral expression vector. Many viral organisms have evolved elegant methods for infecting mammalian cells, evading host-cell defense mechanisms, and coopting cellular machinery to produce copious quantities of their own genetic material.
By utilizing components of viral DNA in the expression vector, and employing a carefully chosen virus itself as a delivery mechanism, a researcher can relatively simply insert a desired transgene into a eukaryotic organism or cell line.
Limitations of Plasmids
Plasmids are powerful tools, but they are limited. The most frequently encountered limitation is one of size.
Plasmids are ideal for carrying small amounts of genetic material, but their delivery and function is severely hindered when larger sequences (above ~15kb) are used.
In these instances, alternative methods like cosmids or bacterial artificial chromosomes (BACs) are better options because they contain sequences that facilitate efficient DNA packaging.
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