pBR322

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Schematic plasmid map of pBR322. The genes for ampicillin resistance, tetracycline resistance and the origin of replication are shown. The base pair position of exemplary restriction sites is highlighted in blue.

The plasmid pBR322 was constructed in 1977 by Francisco Bolivar and Raymond Rodriguez in the laboratory of Herbert Boyer at the University of San Francisco (UCSF) and was one of the first man-made plasmids. It is a derivative of a R-plasmid containing an origin of replication ori ( origin of replication ), genes for ampicillin - resistant and tetracycline carries -resistance up. In contrast to common R plasmids, it lacks the ability to transfer itself to other cells ( tra genes, oriT ( origin of transfer )). It was a widely used vector , especially in the 1980s. pBR322 has recently been replaced by more advanced, easier-to-use vectors, such as pUC19 or pBluescriptII.

Surname

The name of the plasmid pBR322 is composed as follows:

  • p stands for plasmid,
  • B and R are abbreviations for the names of those who constructed the plasmid (F. Bolivar & RL Rodriguez),
  • 322 is a consecutive number in order to be able to differentiate between similar but not identical plasmids.

construction

Detailed plasmid map of pBR322

The plasmid pBR322 was constructed from the following DNA elements:

  • an ampicillin resistance gene from Tn3
  • a tetracycline resistance gene from pSC101
  • an origin of DNA replication (oriV) from pMB1

The constructed plasmid consists of 4361 base pairs . The ampicillin resistance gene extends from 4153 (start) to 3293, the tetracycline resistance gene from position 86 (start) to 1276 and the origin of replication comprises the sequence element between position 3133 and 2519.

use

The large number of restriction endonuclease cleavage sites are essential for working with the plasmid as a vector. What is also special is the fact that many interfaces only appear at one point in the vector. For example, BamHI , HindIII or SalI cleavage sites are found in the tetracycline resistance gene, a PstI cleavage site in the ampicillin resistance gene and an EcoRI and an NdeI cleavage site in the non-coding region of the vector.

pBR322 is used in E. coli cloning work. Here, for example, foreign DNA can be built into the vector by using the restriction cleavage sites of the vector and the foreign DNA. If you insert the foreign DNA into the tetracycline resistance gene, this brings advantages after transformation in the selection of E. coli bacteria that carry the pBR322 vector including built-in foreign DNA. They would be sensitive to a tetracycline medium and die. To select the desired cells, the bacterial culture is therefore first spread on a nutrient medium that contains the antibiotic ampicillin in order to obtain only those bacteria that have received the plasmid (with or without built-in foreign DNA) through transformation. An impression is then made, which is treated with tetracycline (replica plating). In the places where the cultures die, there are obviously cultures that contain a sensitivity to tetracycline, which is due to the introduction of foreign DNA.

The development of many modern cloning vectors, such as the pUC vectors constructed in the laboratory of Joachim Messing , e.g. B. pUC19 , is based on the plasmid pBR322.

Individual evidence

  1. ↑ F. Bolivar, RL Rodriguez, PJ Greene, MC Betlach, HL Heyneker, HW Boyer (1977): Construction and characterization of new cloning vehicles . In: Genes . Vol. 2, No. 2, pp. 95-113. PMID 344137
  2. ↑ N. Watson (1988): A new revision of the sequence of plasmid pBR322 . In: Genes . Vol. 70, No. 2, pp. 399-403, PMID 3063608
  3. ↑ pBR322 Sequence and Map. In: snapgene.com. GSL Biotech LLC, accessed April 24, 2017 .

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