A novel strategy is proposed for positive selection of recombinant plasmids, based on the use of a conditionally lethal gene. Expression in Escherichia coli of the gene for a bacterial ribonuclease, barnase, kills the host cell. Incorporation of foreign DNA into the barnase gene results in synthesis of a defective catalytically inactive protein that does not affect the cell viability. Thus, colonies are formed only by the cells that carry plasmids with cloned DNA inserts, whereas any "background" that can arise from cells that have acquired the vector not cleaved by the restriction enzyme or with the ribonuclease gene restored by ligation or other events is obviated because such cells die. This approach was implemented using plasmids that carried the native barnase gene as well as variants with in-frame recognition sites for commonly used restriction enzymes. Computer modeling of the spatial structure of the corresponding barnase derivatives enabled prediction of their catalytic properties in order to choose the optimal version. The pMT440 vector construct contained the entire pUC19 polylinker instead of the Val36 codon of the barnase gene. The cytotoxity of barnase was not affected by this 19-aa insert, which validated our structural-functional premises.
|Number of pages||6|
|Publication status||Published - Jan 2000|
- Molecular cloning
ASJC Scopus subject areas
- Structural Biology