AbstractOne way to create new components for synthetic transcription circuits is to re-purpose naturally occurring transcription factor proteins and their cognate DNA operators. For the proteins, re-engineering can be accomplished via domain recombination (to create chimeric regulators) and/or amino acid substitutions. The resulting activities of new protein regulators are often assessed in vitro using a representative operator. However, when functioning in vivo, transcription factors can

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... ct with multiple operators. We compared in vivo and in vitro results for two LacI-based transcription repressor proteins, their mutational variants, and four operator sequences. The two sets of repressor variants differed in their overall in vivo repression, even though their in vitro binding affinities for the primary operator spanned the same range. Here, we show that the offset can be explained by different abilities to simultaneously bind and "loop" two DNA operators. Further in vitro studies of the looping-competent repressors were carried out to measure binding to a secondary operator sequence. Surprisingly, binding to this operator was largely insensitive to amino acid changes in the repressor protein. In vitro experiments with additional operators and analyses of published data indicates that amino acid changes in these repressor proteins leads to complicated changes in ligand specificity. These results raise new considerations for engineering components of synthetic transcription circuits and – more broadly – illustrate difficulties encountered when trying to extrapolate information about specificity determinant positions among protein homologs.