What is a plasmid? A plasmid is a genetic unit capable of autonomous replication outside the chromosome, including organelles of eukaryotes and deoxyribonucleic acid (DNA) molecules except for chromosomes in bacterial cells. It is now customary to refer to DNA molecules in bacteria, yeasts, and actinomycetes other than chromosomes. In genetic engineering, plasmids are often used as vectors for genes. Many bacteria have a large number of small circular DNA molecules in addition to chromosomes. This is a plasmid (some plasmids are RNA). The plasmid often has antibiotic resistance genes, such as tetracycline resistance gene or kanamycin resistance gene. Some plasmids are called episomes, and these plasmids can be integrated into the chromosome of the bacteria. From the integration position, it becomes a DNA molecule that is free from extrachromosomes.
At present, hundreds of bacteria have been found to have plasmids. Most of the known bacterial plasmids are closed circular DNA molecules (cccDNA). The relative molecular mass of bacterial plasmids is generally small, about 0.5%~3% of bacterial chromosomes. According to the relative molecular mass, plasmid can be roughly divided into two types: the relative larger one is 40×106 or more, and the relative smaller one is 10×106 or less. (A few molecular masses of plasmids are somewhere in between.) The number of plasmids in each cell is mainly determined by the replication characteristics of the plasmid itself. According to the nature of replication, the plasmids can be divided into two categories: one is a tight-type plasmid. When the cell chromosome is replicated once, the plasmid is also replicated once, and there are only 1 or 2 plasmids in each cell; the other is a relaxed plasmid, which can continue to replicate when the chromosome replication is stopped. There are generally about 20 plasmids in each cell. The replication of these plasmids is under the control of the relaxation of the host cells, containing 10–200 copies per cell. If a certain drug treatment inhibits the synthesis of the host protein, the plasmid copy number is increased to several thousand. The earlier plasmid pBR322 belongs to the relaxation plasmid, which can be treated with chloramphenicol to reach higher copy number. Generally, the plasmid with larger molecular weight is tight-type. The plasmid with smaller molecular weight is relaxed-type. The replication of plasmids sometimes is related to their Host cell.
In genetic engineering, artificially constructed plasmids are commonly used as vectors. Artificially constructed plasmids can integrate a variety of useful features, such as a variety of single enzyme cleavage sites, antibiotic resistance, etc. Commonly used artificial plasmid carriers have pBR322 and pSC101. pBR322 contains anti-tetracycline gene (Tcr) and ampicillin resistance gene (Apr), and contains a single point of 5 endonucleases. If the DNA fragment is inserted into the EcoRI cut point, it will not affect the two antibiotic genes expression. However, if the DNA fragment is inserted into the Hind III, Bam HI or Sal I point, the anti-tetracycline gene will be inactivated. At this time, the pBR322 containing the DNA insert will make the host bacterium resistant to ampicillin but sensitive to tetracycline. pBR322 without a DNA insert would make the host bacterium resistant to both ampicillin and tetracycline, whereas bacteria without the pBR322 plasmid would be sensitive to ampicillin and tetracycline. pSC101 is similar to pBR322 except that it has no anti-ampicillin gene and PstI nicking. The largest insert of the plasmid vector is approximately 10 kb (kb is expressed as kilobase pairs).
Before starting a plasmid sequencing project, one should consider:
The DNA quality and purity
Is there host DNA contamination?
Sole plasmid or genome and plasmid?
Plasmid sequencing Applications
Ÿ Mutation studies
Ÿ Vector verifications
Ÿ Characterization of production strains