pBR322 is one of the earliest and most widely used cloning vectors in Escherichia coli. Created in 1977 at the University of California, San Francisco, in the laboratory of Herbert Boyer, it is named after postdoctoral researchers Francisco Bolivar Zapata and Raymond L. Rodriguez. The "p" stands for "plasmid", and "BR" represents "Bolivar" and "Rodriguez". Plasmids were first discovered in Escherichia coli in the early 1950s. They are closed circular double-stranded DNA molecules that exist independently of the chromosome (or nucleoid) in organisms such as bacteria, yeast, and actinomycetes. Plasmids have the ability to replicate autonomously, maintain a constant copy number in progeny cells, and express the genetic information they carry.
Today, plasmids have become a common and simple vector in genetic engineering, characterized by autonomous replication, amplifiability, transferability, and incompatibility. In addition to their widespread application in research, plasmids are also extensively used in the development and production of various biomedical products, such as recombinant protein drugs, vaccines, gene therapy drugs, and CAR-T drugs. The plasmid vectors used in molecular biology today are no longer the natural plasmids found in bacteria or cells but have been artificially modified. Various types of plasmid vectors have been designed for different experimental purposes.
pBR322 DNA is one of the most extensively studied, earliest used, and most widely applied plasmid vectors in Escherichia coli. The length of the vector is 4361 base pairs. pBR322 is resistant to ampicillin and tetracycline and can be used for gene cloning. It is also sometimes used for preparing DNA molecular weight standards.
The simplified construction of pBR322 (using restriction enzyme digestion and ligation as an example) involves several steps: extracting the plasmid, amplifying the target gene fragment, treating the plasmid and the target fragment with restriction enzymes, ligating, transforming, culturing, and identifying.
The construction of pBR322 is a complex yet clear process. The construction of pBR322 first uses the pMB1 plasmid as a basis, introducing the Tn3 transposon from the Rldrd19 plasmid to form the pMB3 plasmid. Next, most of the unnecessary fragments of pMB3 are removed by EcoRI digestion, forming sticky ends that are ligated to obtain the pMB8 plasmid. Then, under EcoRI active conditions, another pSC101 plasmid is digested to produce a DNA fragment containing tetracycline resistance, which integrates with pMB8 to form the pMB9 plasmid. To perfect the vector functionality, the Tn3 transposon from pSF2124 is introduced, forming the pBR313 plasmid, and ensuring the stability of Tn3 by cutting out its gene for transposase expression. Subsequently, the PstI and EcoRII sites are removed from pBR313, forming two complementary function plasmids, pBR318 and pBR320. Finally, hybridizing these two plasmids yields the highly versatile vector plasmid pBR322. This process demonstrates the complete steps of constructing the pBR322 plasmid vector, providing an important tool for genetic engineering and molecular biology research.
