Plasmids

Introduction to Plasmids

A plasmid is a small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell.

Learning Objectives

Outline the utility of plasmids

Key Takeaways

Key Points

  • Plasmids can be found in all three major domains: Archaea, Bacteria, and Eukarya. Similar to viruses, plasmids are not considered by some to be a form of life.
  • Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state.
  • Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or the proteins produced may act as toxins under similar circumstances.

Key Terms

  • plasmid: A circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa.
  • mobilome: The entirety of the mobile (transposable) elements of a genome.
  • replicons: a region of DNA or RNA, that replicates from a single origin of replication.

In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of the chromosomal DNA.They are double-stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in archaea, and even in eukaryotic organisms (e.g., the 2-micrometre ring in Saccharomyces cerevisiae).

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Step by step of cloning a gene using a plasmid: This image shows a line drawing that compares the activity of non-integrating plasmids, on the top, with episomes, on the bottom, during cell division. The upper half of the image shows a bacterium with its chromosomal DNA and plasmids dividing into two identical bacteria, each with their chromosomal DNA and plasmids. The lower half of the image shows a bacterium with its chromosomal DNA, but with an episome. Next to this bacterium, we see the same bacterium, but after the episome has integrated into the chromosomal DNA and has become a part of it. This second bacterium now divides into two bacteria identical to it, each with an episome integrated into it.

Plasmid sizes vary from 1 to over 1,000 kbp. The number of identical plasmids in a single cell can range anywhere from one to thousands under some circumstances. Plasmids can be considered part of the mobilome because they are often associated with conjugation, a mechanism of horizontal gene transfer.

The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952.

Plasmids are considered replicons.They can be found in all three major domains: Archaea, Bacteria, and Eukarya. Similar to viruses, plasmids are not considered by some to be a form of life. Unlike viruses, they are naked DNA and do not encode genes necessary to encase the genetic material for transfer to a new host, though some classes of plasmids encode the sex pilus necessary for their own transfer. Plasmid host-to-host transfer requires direct mechanical transfer by conjugation, or changes in incipient host gene expression allowing the intentional uptake of the genetic element by transformation. Microbial transformation with plasmid DNA is neither parasitic nor symbiotic in nature, because each implies the presence of an independent species living in a commensal or detrimental state with the host organism. Rather, plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or the proteins produced may act as toxins under similar circumstances. Plasmids can also provide bacteria with the ability to fix elemental nitrogen or to degrade recalcitrant organic compounds that provide an advantage when nutrients are scarce.

Types of Plasmids and Their Biological Significance

Plasmids are commonly used to multiply (make many copies of) or express particular genes.

Learning Objectives

Recognize the characteristics of, and thus the functions, of plasmids

Key Takeaways

Key Points

  • The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics, and into a multiple cloning site (MCS, or polylinker), allowing the easy insertion of DNA fragments.
  • A major use of plasmids is to make large amounts of proteins. Bacterium can be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for; for example, insulin or even antibiotics.
  • It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli. However, related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions.

Key Terms

  • Col plasmids: These plasmids contain genes that code for bacteriocins, proteins that can kill other bacteria.
  • F-plasmid: Fertility F-plasmids contain tra genes and are capable of conjugation resulting in the expression of sex pilli.
  • Resistance plasmids: These plasmids contain genes that provide resistance against antibiotics or poisons.

Types of Plasmids

Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics. The gene is also inserted into a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments.

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A Plasmid Map of pUC19: pUC19 is one of a series of plasmid cloning vectors created by Messing and co-workers in the University of California. The p in its name stands for plasmid and UC represents the University in which it was created. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the most widely used vector molecules as the recombinants, or the cells into which foreign DNA has been introduced, can be easily distinguished from the non-recombinants based on color differences of colonies on growth media. pUC18 is similar to pUC19, but the multiple cloning site region is reversed.

Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria that take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way, the antibiotics act as a filter, selecting only the modified bacteria. Finally, these bacteria can be grown in large amounts, harvested, and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.

Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for; for example, insulin or even antibiotics.

One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain tra genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium. Non-conjugative plasmids are incapable of initiating conjugation, hence they can be transferred only with the assistance of conjugative plasmids. An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can parasitize a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA, and may possibly be a tool for curing many diseases.

It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli. However, related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Thus, plasmids can be assigned into incompatibility groups.

Another way to classify plasmids is by function. There are five main classes:

  • Fertility F-plasmids, which contain tra genes. They are capable of conjugation and result in the expression of sex pilli.
  • Resistance plasmids, which contain genes that provide resistance against antibiotics or poisons. They were historically known as R-factors, before the nature of plasmids was understood.
  • Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other bacteria.
  • Degradative plasmids, which enable the digestion of unusual substances, e.g. toluene and salicylic acid.
  • Virulence plasmids, which turn the bacterium into a pathogen.