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Tetracycline repressor (TetR) bears an unstructured loop region between helices alpha8 and alpha9, which is moderately permissive to amino acid exchanges and length variations. Recognition sites for the site-specific recombinases Flp (FRT) or Cre (lox) were inserted in-frame into tetR, substituting some of this loop's codons. A number of the deduced TetR variants displayed efficient regulation in vivo, thus allowing the establishment of a new mode of TetR activation on the genetic level. Chromosomally encoded tetR in Bacillus subtilis was disrupted and inactivated by insertion of a lox66-aphAIII-lox71 kanamycin resistance cassette. Marker excision by Cre recombinase led to the assembly of a novel tetR allele. The encoded regulator, termed TetR(lox72/1), is distinguished from wt-TetR by a slightly elongated and altered alpha8-alpha9 loop only, harboring an amino acid stretch encoded by lox72. Despite decreased intracellular protein amounts, TetR(lox72/1) displayed efficient in vivo activity in B. subtilis and E. coli, indistinguishable from that of wt-TetR. These results underline the sequence flexibility of TetR in the alpha8-alpha9 loop and demonstrate the possible use of the regulator as a read-out tool for the activity of site-specific recombinases.

In vivo Activation of Tetracycline Rep ressor by Cre/<i>lox</i>-Mediated Gene Assembly