What are Plasmids?

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Plasmids.

Most cells contain at least one chromosome but some cells also contain an additional DNA element or elements called plasmids. Plasmids are DNA molecules, generally circular, which can replicate in Bacterial, Archaeal and Eukaryotic cells. They take advantage of the cellular environment of the cell but can also carry a rich diversity of genes which can be beneficial for the cell. Some plasmids confer the ability to degrade organic compounds and to fix nitrogen. Other plasmids carry antibiotic resistance genes and their spread in pathogenic bacteria is of great medical significance. Plasmids are used in molecular studies of various organisms and are important in many branches of biology, medicine, ecology and evolution as well as basic research in microbiology, molecular biology and structural biology.

 

Plasmids are also used for gene cloning of the bacteria for research purpose. In some bacteria, many plasmids are present at one time in one single bacterium. Plasmid sizes vary from 1 to over 1,000 kilobase pairs (kbp). The number of identical plasmids in a single cell can range anywhere from one to even thousands under some circumstances. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Plasmids also can provide bacteria with an ability to fix elemental nitrogen or to degrade recalcitrant organic compounds which provide an advantage when nutrients are scarce.

 

What are the various types of Plasmids?

One way of grouping plasmids is by their ability to transfer to other bacteria.

  • Conjugative plasmids contain ‘tra’ genes (genes necessary for non-sexual transfer of genetic material), 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 only be transferred with the assistance of conjugative plasmids.
  • Intermediate classes 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. Therefore, plasmids can be assigned into compatibility groups.

 

How are Plasmids classified by their function?

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

  • Fertility F-plasmids, which contain ‘tra’ genes. They are capable of conjugation.
  • Resistance (R) plasmids, which contain genes that can build a resistance against antibiotics or poisons and help bacteria produce pili (a hair like appendage found on the surface of many bacteria). Traditionally 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. salicylic acid.
  • Virulence plasmids, which turn the bacterium into a pathogen.

 

What are Yeast Plasmids?

Some types of plasmids are often related to yeast cloning vectors that include:

  • Yeast integrative plasmid (YIp), yeast vectors that rely on integration into the host chromosome for survival and replication, and are usually used when studying the functionality of a solo gene or when the gene is toxic.
  • Yeast Replicative Plasmid (YRp), which transport a sequence of chromosomal DNA that includes an origin of replication. These plasmids are less stable, as they can "get lost" during the budding.

 

What are the Conformations of plasmids?

Plasmid DNA may appear in one of five conformations. Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:

  • Nicked Open-Circular: DNA has one strand cut.
  • Relaxed Circular: DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed).
  • Linear: DNA has free ends, either because both strands have been cut, or because the DNA was linear.
  • Supercoiled: (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form.
  • Supercoiled Denatured: DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.

 

What is the role of Plasmids in genetic engineering?

  • Vectors are the plasmids which are used in genetic engineering . Plasmids serve as a essential 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 and a multiple cloning site (MCS), which is a short region containing several commonly used restriction sites (locations on a DNA molecule) allowing the easy insertion of DNA fragments at this location.
  • Next, the plasmids are inserted into bacteria by a process called “Transformation”.
  • Then, the bacteria are exposed to the particular antibiotics. Only bacteria which 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 to select only the modified bacteria.
  • Now these bacteria can be grown in large amounts, harvested and lysed to isolate the plasmid of interest. Breaking down of a You do not have access to view this node, often by viral, enzymic, or osmotic mechanisms is known as Lysis.
  • One more key 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 bacteria produce 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. Though, a plasmid can only contain inserts of about 1–10 kbp. To clone longer lengths of DNA, cosmids ( type of hybrid plasmid), bacterial artificial chromosomes or yeast artificial chromosomes could be used.

 

What is Plasmid DNA extraction?

Plasmids are often used to purify a specific sequence, since they can easily be purified away from the rest of the genome. For their use as vectors, and for molecular cloning (process of making multiple molecules), plasmids often need to be isolated. There are several methods to isolate plasmid DNA (plasmid preparation is a method used to extract and purify plasmid DNA) from bacteria, the archetypes of which are the miniprep and the maxiprep/bulkprep.

  • Miniprep: This can be used to quickly find out whether the plasmid is correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and for some cloning techniques.
  • Maxiprep/bulkprep: In this, much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. Essentially this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several micrograms) of very pure plasmid DNA.
  • In recent times many commercial kits have been created to perform plasmid extraction at various scales, purity and levels of automation. Commercial services can prepare plasmid DNA at quoted prices below $300/mg in milligram quantities and $15/mg in gram quantities (early 2007).

 

What are the Applications of plasmids?

  •  Disease models: Plasmids were historically used to genetically engineer the embryonic stem cells of rats in order to create rat genetic disease models. The limited efficiency of plasmid based techniques prohibited their use in the creation of more accurate human cell models. Providentially, developments in Adeno-associated virus (a small virus which infects humans and some other primate species) recombination techniques, and Zinc finger nucleases (artificial restriction enzymes), have enabled the creation of a new generation of iso genic human disease models ( family of cells that are selected or engineered to accurately model the genetics of a specific patient population).
  • Gene therapy: The success of some strategies of gene therapy rely on the efficient insertion of therapeutic genes at the appropriate chromosomal target sites within the human genome, without causing cell injury, cancer or an immune response. Plasmid vectors are one of many approaches that could be used for this purpose. Zinc finger nucleases (ZFNs) offer a way to cause a site-specific double strand break to the DNA genome and cause homologous recombination. This makes targeted gene correction a possibility in human cells. Plasmids encoding ZFN could be used to deliver a therapeutic gene to a pre-selected chromosomal site with a frequency higher than that of random integration. Although the practicality of this approach to gene therapy has yet to be proven, some aspects of it could be less problematic than the alternative viral-based delivery of therapeutic genes.
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