Plasmid Vectors for Marker-Free Chromosomal Insertion of Genetic Material in Escherichia coli

Amethod to achieve the insertion of genetic material into the chromosome of Escherichia coli is described. The method is based on the use of integration vectors from the pBRINTs-rAnb^R family. These vectors offer the choice of using the antibiotics chloramphenicol, gentamycin, or kanamycin to select for chromosomal integration events. In addition, it is possible to eliminate these chromosomal antibiotic resistance markers, after integration has taken place. The overall insertion strategy is as follows: a fragment containing the gene(s) to be integrated in the chromosome is inserted into the multiple cloning site of a pBRINTs-rAnb^R vector and the resulting plasmid is used to transform E. coli cells. The plasmid is first allowed to replicate in the cell at the permissive temperature of 30�C. Next, the temperature of the culture is raised to 44�C to inhibit plasmid replication and to select for the integrants in the presence of the appropriate antibiotic. Chromosomal excision of the Anb^R gene can then be catalyzed by the Cre recombinase that is transiently expressed in the cell from the temperature-sensitive pJW168 plasmid. This plasmid is finally eliminated from the cells by increasing the temperature of the culture to 44�C.