Expanding the toolkit of LacI/GalR chimeras

Abstract

When creating synthetic transcription circuits, multi-input regulation is desirable. However, the size and complexity of prokaryotic circuits are constrained by the number of transcription factors that can simultaneously bind a promoter region. This limitation has been circumvented by leveraging the conserved architecture of LacI/GalR transcription repressors: The DNA binding domain of one repressor can be fused to paralogous ligand binding domains that bind different allosteric ligands; function can be optimized by mutating domain interfaces. When such chimeras were used in prior studies to co-regulate transcription, their set of allosteric ligands conveyed Boolean “AND”, “NOT”, and “NOR” logic from a single DNA operator. Here, we report construction and characterization of additional chimeras that can be used to expand the LacI/GalR toolkit. For both novel and previously reported chimeras, we assessed (and in most cases ruled out) cross reactivity among their ligands. As such, we propose that three of the novel chimeras, along with a previously uncharacterized fourth chimera, could be co-expressed in engineered systems to expand the options available for Boolean “AND” logic. Gratuitous inducers were identified for another prior chimera that would allow “OR” logic using a single transcription factor. Surprisingly, another novel chimera was anti-induced by the ligand that induces its parent protein. This allosteric switch illustrates what may be a general feature of the LacI/GalR proteins: they appear poised to switch between induction and anti-induction via changes in ligands or amino acid mutations. Practically speaking, this anti-induced chimera could be co-expressed with a previous anti-induced chimera to perform “NOR” logic.