Plasmids were first discovered in Escherichia coli in the early 1950s. They are closed circular double-stranded DNA molecules independent of chromosomes (or nucleoids) found in organisms such as bacteria, yeasts, and actinomycetes. These molecules have the ability to replicate autonomously, maintain a constant copy number in progeny cells, and express the genetic information they carry.
Currently, plasmids have become a commonly used, simple vector in genetic engineering, characterized by autonomous replication, amplifiability, transferability, and incompatibility. Besides their widespread application in research fields, plasmids are also extensively used in the development and production of various biopharmaceutical products, such as recombinant protein drugs, vaccines, gene therapy drugs, and CAR-T drugs.
Nowadays, the plasmid vectors used in molecular biology are no longer the naturally occurring plasmids found in bacteria or cells but have been artificially modified. Different types of plasmid vectors have been designed to cater to various experimental objectives.
The "p" in construction of pBR322 stands for plasmid; "BR" represents the initials of the researchers Bolivar and Rodriguez, and "322" is the experiment number.
The following are the characteristics of construction of pBR322:
It has a relatively small molecular weight, with a vector length of 4361bp.
It contains a replication origin that ensures the plasmid functions only within Escherichia coli cells.
It has two antibiotic resistance genes, which can be used for the selection of transformants.
It has a high copy number. After chloramphenicol amplification, each cell can accumulate 1000-3000 copies, greatly facilitating the preparation of recombinant DNA.
To insert a gene into a vector, scientists can use various cloning methods (enzyme digestion and ligation, Gateway, Golden Gate, Gibson, etc.). The choice of cloning method depends on the plasmid backbone used. Once the cloning step is complete, the vector containing the newly inserted gene will be transformed into bacterial cells, and the transformed bacterial culture will be plated on antibiotic-containing plates for selection.
Traditionally, plasmid recombination is based on the principle of complementary base pairing. The general procedure is:
Introduce restriction sites at both ends of the target fragment through primers;
Use the same restriction enzymes to digest both the target fragment and the vector;
Ligate the digested products of the target fragment and the vector in vitro;
Transform the ligated product into Escherichia coli;
Screen for recombinants.
Artificially constructed plasmids can integrate multiple useful features, such as multiple unique restriction sites and antibiotic resistance. Commonly used artificial plasmid vectors include pBR322 and pSC101. pBR322 contains tetracycline resistance genes (Tcr) and ampicillin resistance genes (Apr) and includes five unique restriction sites. Inserting a DNA fragment at the EcoRI site does not affect the expression of the two antibiotic genes.
However, inserting a DNA fragment at the Hind III, BamH I, or Sal I sites will inactivate the tetracycline resistance gene. In this case, pBR322 containing the DNA insert will render the host bacteria resistant to ampicillin but sensitive to tetracycline. pBR322 without the DNA insert will render the host bacteria resistant to both ampicillin and tetracycline, while bacteria without the pBR322 plasmid will be sensitive to both antibiotics.
