pBR322

The plasmid pBR322 was constructed by Francisco Bolivar and Raymond Rodriguez in 1977 and was one of the first artificially produced plasmids. It is a derivative of a R plasmid carrying an origin of replication, ori ( origin of replication ), the genes for ampicillin resistance and tetracycline resistance in itself. In contrast to ordinary R- plasmids him the ability to independently transfer to other cells ( tra genes, oriT (origin of transfer) ) is missing. It was a much used vector, especially in the 1980's. pBR322 has recently been replaced by more advanced, simple -to-use vectors, such as pUC19 or pBluescript.

Name

The name of the plasmid pBR322 is made up as follows:

  • P stands for plasmid
  • B and R as an abbreviation for the names of those who have constructed the plasmid ( F. Bolivar & RL Rodriguez )
  • 322 is a sequential number to distinguish similar, but not identical plasmids can.

Construction

The plasmid pBR322 was constructed from the following DNA elements:

  • An ampicillin resistance gene from Tn3
  • A tetracycline resistance gene from pSC101
  • A DNA replication origin ( oriV ) from pMB1

The plasmid construct consists of 4361 base pairs. The ampicillin resistance gene is located at position 4153 ( Commencement Date) to 3293, the tetracycline resistance gene at position 88 (start ) to 1276 and the origin of replication at position 3133 ( Commencement Date) to 2519.

Use

Essential for working with the plasmid vector as are the numerous existing restriction endonuclease interfaces. The special feature is the fact that many interfaces occur only at one site in the vector. For example, BamHI, HindIII or SalI sites found in the tetracycline resistance gene, a PstI site in the ampicillin resistance gene and an EcoRI and NdeI site in the noncoding region of the vector.

PBR322 is used in Klonierungsarbeiten in E. coli. This can be, for example, incorporate foreign DNA into the vector by exploiting the restriction sites of the vector and the foreign DNA. In this case, when inserting the foreign DNA into the tetracycline resistance gene, by transforming it brings the advantages of selection of E. coli carrying the vector pBR322 including the integrated foreign DNA. Would you react sensitively and die on a Tetracyclinmedium. For selection of the desired cells so the bacterial culture is first spread on a nutrient medium containing the antibiotic ampicillin to obtain only those bacteria that have (with or without integrated foreign DNA ) was obtained by transformation of the plasmid. After an impression is taken, which is treated with tetracycline. At the locations where the crops to die, thus Cultures are obviously available, which contain a sensitivity to tetracycline, due to the insertion of foreign DNA.

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