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

Abstract

Tetracycline repressor (TetR) bears an unstructured loop region between helices α8 and α9, 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 α8–α9 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 α8-α9 loop and demonstrate the possible use of the regulator as a read-out tool for the activity of site-specific recombinases.

A neural network for intermale aggression to establish social hierarchy

“…While the use of wild-type outbred animals in neurophysiology and behavior provides an obvious benefit since it takes into consideration the genetic variability found in a natural population and therefore serves as a more faithful model for human conditions, transgenic inbred animals have become an indispensable tool in neuroscience research. The development of a powerful genetic toolbox has enabled tissue-specific transgene expression, with timing and location control [275][276][277][278][279][280] , making inbred mouse lines a dominant animal model.…”

Lox’d in translation: contradictions in the nomenclature surrounding common lox-site mutants and their implications in experiments

“…Of the 21 articles that unambiguously showed the correct loxsite annotations, 18/21 used the same lox orientation as the original paper by Albert et al [8], with the lox directionality reading from right to left [8,12,26,[30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. In contrast, five of the eight papers with mis-labelled lox sites portray them from left to right [17,[20][21][22]24].…”

Status quo of tet regulation in bacteria

“…Zeng et al (2018) constructed repression-proficient and inducible 'split' TetR variants which in their active form are not composed of two but of four polypeptide chains held together by short interacting peptides. In a study conducted in B. subtilis, tetR was activated by intragenic Cre-lox recombination resulting in a functional TetR variant with an altered loop sequence between helices a8 and a9 (Bertram et al, 2009). Natively, TetR binds [Tc-Mg] + complexes in a 1:1 stoichiometry relative to the monomer (Hinrichs et al, 1994).…”