PAC (P1-derived artificial chromosome) is commonly used in modern research.
The vectors of this series contain genes of the moderate bacteriophage P1, which ensure replication of the phage chromosome in infected bacterial cells. Recombinant DNAs based on them (insert size 150-200 kbp) are introduced into bacterial cells using electroporation.
However, unlike the bacteriophage, which, during a latent (lysogenic) state, integrates its chromosome into the chromosome of the host bacteriophage, the P1 phage maintains the chromosome in the cytoplasm of bacterial cells in the form of a circular covalently closed molecule resembling a plasmid, the size of which is 100 kbp. The size of the replicon, which is able to provide replication of the P1 chromosome in the lysogenic state, is only 1.5 kb.
To overcome the difficulties arising when using artificial yeast chromosomes, alternative vector systems have been designed, among which the most popular at present are systems based on artificial chromosomes of bacteria – BAC.
A bacterial artificial chromosome (BAC) is a vector system based on the E. coli F-plasmid, cos sites of the lambda phage and loxP of the P1 phage, used for cloning long (150-350 kbp) DNA sequences. The F-plasmid encodes genes that regulate replication and control copy number (1-2 molecules per cell). At the loxP site, plasmid DNA can be cleaved by the Cre protein of the P1 phage, at the cos site by the corresponding enzyme of the lambda phage. A similar vector system called PAC (p1-derived artificial chromosome) was made on the basis of a bacterial P1 plasmid from the DNA of the P1 phage.
Artificial bacterial chromosomes are often used to sequence the genomes of organisms in various projects, such as the Human Genome project. A short piece of DNA from the organism under study is inserted into the chromosome and then amplified and sequenced. After that, the read sequences are aligned in silico, resulting in the complete sequence of the organism’s genome. This approach has now been superseded by faster and less labor-intensive sequencing methods, such as the shotgun method or next-generation sequencing methods.