Sm. Deyev et al., RIBONUCLEASE-CHARGED VECTOR FOR FACILE DIRECT CLONING WITH POSITIVE SELECTION, MGG. Molecular & general genetics, 259(4), 1998, pp. 379-382
Plasmid vectors for positive selection of cloned inserts in Escherichi
a coli were devised, based on an expression plasmid (pMT416) for the b
acterial ribonuclease barnase. In addition to the barnase gene under c
ontrol of a synthetic tac promoter, these plasmids carry the gene for
the barnase inhibitor, barstar, the constitutive expression of which p
rotects the bacterium from the detrimental effects of moderate barnase
production. Full expression of the barnase gene overcomes protection
by barstar and becomes lethal. Having a unique SmaI/XmaI site in the b
arnase structural gene, pMT416 itself can be used as a selective vecto
r: uncut or religated pMT416 will preclude growth while plasmids with
inserts in the barnase gene will allow the cells to survive. The entir
e pUC polylinker was inserted into the barnase gene in place of the Va
l-36 codon. This insert of nineteen largely hydrophilic amino acids do
es not prevent the lethal effect of full expression of the gene. The r
esulting plasmid, pMT440, is a generally useful selective cloning vect
or representing the ''kill-the-rest'' approach.