Plasmid


Plasmids are molecules of extra-chromosomal DNA, found most often in bacteria but occasionally in eukaryotic organisms such as yeast. Plasmids are double-stranded, usually circular, and capable of autonomous replication (i.e. they are replicons). They range in size from 1 to 1,000kbp (or alternatively 1,000 - 1,000,000 base pairs) and usually encode traits that are often selectively advantageous to their host cell, such as antibiotic resistance, tolerance to environmental toxins, proteins required for the destruction of other bacteria, pathogenicity factors, or the ability to fix nitrogen in times of nutrient depletion. Plasmids, like viruses, are mobile genetic elements without their own capacity for growth, environmental response or metabolism and thus are not generally recognised as 'living' entities. Unlike viruses, plasmids exist as naked DNA without any protein or lipid encasing.

Plasmids are a key agent of horizontal gene transfer, since they can be transmitted between individuals within a population. Some plasmids, notably fertility (or F) plasmids, ensure their own transfer by encoding a sex pilus that links two bacteria and enables the plasmid to be transferred from a donor to a recipient via a mechanism called conjugation. In other circumstances, host-encoded proteins are necessary to enable the uptake of plasmids from the environment - this is called transformation. Either way, plasmid transfer between bacteria is a key mechanism in the spread of antibiotic resistance among a bacterial population.

Plasmids generally come in two categories:
  • Conjugative plasmids, which are contain the tra genes necessary to direct their transfer by conjugation
  • Non-conjugative plasmids, which are incapable of directing conjugation and thus can only be transferred with conjugative plasmids 'by accident'

There is an intermediate group of mobilisable plasmids which contain some of the genes necessary for conjugation, but generally 'parasitise' conjugative plasmids and thus are transferred at a higher frequency in their presence.

Plasmids have various mechanisms to ensure they will remain present in the next generation of bacterial cells. One such system, the post-segregational killing system (PSK), acts by creating a selective disadvantage for any of the host's daughter cells who do not carry a copy of the plasmid. In this system, the plasmid encodes a long-term poison and a short-term antidote. Daughter cells that do not inherit a copy of the plasmid will die from the presence of the poison after binary fission due to not carrying the antidote gene.

Plasmids are often used in genetic engineering for the purpose of gene cloning. In this capacity, they act as vectors. Plasmids with genes for antibiotic resistance are usually selected, as this offers a reliable genetic marker, and the plasmid will also contain a multiple cloning site (or polylinker) with several restriction sites to make splicing a foreign gene into the plasmid easier. This is vital in the mass-production of important proteins, such as insulin for diabetic individuals, because the engineered plasmid replicates at a high rate, resulting in many gene copies and thus lots of protein product. Plasmids have a limited capacity for foreign genes however - generally inserts cannot be larger than 10-20kbp. When cloning longer fragments of DNA, typically it is more common to use one of the options below.

  • Bacteriophages with the genes for lysogeny removed. The lack of genes for lysogeny means that transformed bacteria are lysed by the recombinant phage, acting as in intrinsic genetic marker

  • Bacterial artificial chromosomes (BACs) to clone fragments typically between 150 and 350kb, but sometimes closer to 700kb. BACs are based on functional fertility (F) plasmids. F-plasmids are important because they contain partition genes that ensure their even distribution between daughter cells during binary fission. BACs are often used to carry DNA fragments used in genome sequencing by shotgun sequencing.

  • Yeast artificial chromosomes (YACs) to clone fragments between 100 and 3000kb in length. These are constructed by breaking a circular plasmid into two linear fragments using restriction enzymes and then using the linearised plasmid as a template to which a gene of interest can be hybridised (using DNA ligase). YACs are constructed to contain telomeres, centromeres and origins of replication that are necessary for their preservation, and then they are transfected into yeast cells. YACs are preferable to BACs when expressing eukaryotic proteins that require post-translational modifications, although YACs have reduced stability to compared to BACs.

  • Cosmids, which are hybrid plasmids that have been artificially combined with the cohesive (cos) sites typical of bacteriophages. Because of the presence of these cos sites, cosmids can be packaged into phage heads in vitro ready to transduce a recipient bacterium.

It has been hypothesised that plasmids could also act as vectors in gene therapy. Plasmids would enable site-specific insertion of healthy genes into the human genome without causing unwanted mutagenesis. This could be done with a plasmid that encodes zinc finger nucleases, a family of enzymes that facilitate homologous recombination.

Plasmids can be purified from bacterial strains either by mini-prep or maxi-prep. The former extracts impure plasmid DNA, suitable for restriction digest analysis, while the latter extracts purer plasmid DNA as a result of subsequent purification steps that are not present in the mini-prep procedure.