Modular, Multi-Input Transcriptional Logic Gating with Orthogonal LacI/GalR Family Chimeras

Abstract

In prokaryotes, the construction of synthetic, multi-input promoters is constrained by the number of transcription factors that can simultaneously regulate a single promoter. This fundamental engineering constraint is an obstacle to synthetic biologists because it limits the computational capacity of engineered gene circuits. Here, we demonstrate that complex multi-input transcriptional logic gating can be achieved through the use of ligand-inducible chimeric transcription factors assembled from the LacI/GalR family. These modular chimeras each contain a ligand-binding domain and a DNA-binding domain, both of which are chosen from a library of possibilities. When two or more chimeras have the same DNA-binding domain, they independently and simultaneously regulate any promoter containing the appropriate operator site. In this manner, simple transcriptional AND gating is possible through the combination of two chimeras, and multiple-input AND gating is possible with the simultaneous use of three or even four chimeras. Furthermore, we demonstrate that orthogonal DNA-binding domains and their cognate operators allow the coexpression of multiple, orthogonal AND gates. Altogether, this work provides synthetic biologists with novel, ligand-inducible logic gates and greatly expands the possibilities for engineering complex synthetic gene circuits.

AlloRep: A Repository of Sequence, Structural and Mutagenesis Data for the LacI/GalR Transcription Regulators

“…LacI is widely used in biotechnology, and synthetic LacI/GalR homologs are being developed to carry out novel, complex, biosynthetic circuitry [40]. …”

“…Primers used were 5′-CGGACAGCGCGTCGACGGGGTCCTCCTGC and 5′-CGTCGACGCGCTGTCCGGCCAGGTAGCCG. Repression and induction of the LLhE-3mut variants were assayed as described in Shis et al [40]. …”

Microbiome therapeutics — Advances and challenges

“…Directed evolution has been successfully applied to change substrate specificity of enzymes [155] and promoter specificity of RNA polymerases [156] and similar strategies could be applied for the development of novel biosensors. Moreover, hybrid transcription factors composed of distinct DNA-binding and ligand-binding domains has expanded the collection of available sensors [157–159]. A generalized approach for de novo discovery of sensors of clinically relevant molecules is greatly needed, as well as fluid dialogue between synthetic biologist and clinicians to identify relevant candidate disease biomarkers.…”

Applications and advances of metabolite biosensors for metabolic engineering

“…In one example, an isopentenyl diphosphate (IPP) biosensor was created by fusing the AraC DNA binding domain and the linker with IPP isomerase Idi (Chou and Keasling, 2013). These general approaches are useful to develop sensors for a variety of compounds, as demonstrated for triacetic acid lactone (Tang et al, 2013), D-arabinose (Tang et al, 2008), fructose, ribose (Meinhardt et al, 2012;Shis et al, 2014), etc.…”