Engineering a New Class of Anti-LacI Transcription Factors with Alternate DNA Recognition

Abstract

The lactose repressor, LacI (I + YQR ), is an archetypal transcription factor that has been a workhorse in many synthetic genetic networks. LacI represses gene expression (apo ligand) and is induced upon binding of the ligand isopropyl β-D-1-thiogalactopyranoside (IPTG). Recently, laboratory evolution was used to confer inverted function in the native LacI topology resulting in anti-LacI (antilac) function (I A YQR ), where IPTG binding results in gene suppression. Here we engineered 46 antilacs with alternate DNA binding function (I A ADR ). Phenotypically, I A ADR transcription factors are the inverse of wild-type I + YQR function and possess alternate DNA recognition (ADR). This collection of bespoke I A ADR bind orthogonally to disparate nonnatural operator DNA sequences and suppress gene expression in the presence of IPTG. This new class of I A ADR gene regulators were designed modularly via the systematic pairing of nine alternate allosteric regulatory cores with six alternate DNA binding domains that interact with complementary synthetic operator DNA sequences. The 46 I A ADR identified in this study are also orthogonal to the naturally occurring operator O 1 . Finally, a demonstration of full orthogonality was achieved via the construction of synthetic genetic toggle switches composed of two nonsynonymous unit pair operations that control two distinct fluorescent outputs. This new class of I A ADR transcription factors will facilitate the expansion of the computational capacity of engineered gene circuits, via the scalable increase in the control over the number of gene outputs by way of the expansion of the number of unique transcription factors (or systems of transcription factors) that can simultaneously regulate one or more promoter(s).

Engineered systems of inducible anti-repressors for the next generation of biological programming

“…Here we established a two part workflow to engineer non-natural anti-repressor transcription factors, combining two separate strategies that we established (in part) previously 25 , 40 , 44 (Fig. 2 ).…”

“…2 ). In this study our overarching design goal was to engineer a set of non-natural anti-repressors that complement and communally function with our current collection of non-natural repressor transcription factors 25 , 44 . In stage one of the workflow, we outlined the process for conferring anti-repression (Fig.…”

“…Results shown are from n = 12 independent biological replicates with similar results, dots shown for each replicate. a NORp gates were constructed from two NOTp operations in SE-PA. R A(1) HQN and I A(5) HQN (from Rondon and Wilson 44 ) both regulated GFP via a proximal O ttg operator. In the presence of either, or both, ligands, IPTG and D-ribose, transcription was anti-induced, causing diminished gene expression.…”

“…Discretely, each operator can be objectively described as independent NOT unit operations-i.e., core (NOTc) and proximal (NOTp). Using an architecture enabling discrete unit operations for information processing allows for a NORp gates were constructed from two NOTp operations in SE-PA. R A (1) HQN and I A (5) HQN (from Rondon and Wilson 44 ) both regulated GFP via a proximal O ttg operator. In the presence of either, or both, ligands, IPTG and D-ribose, transcription was anti-induced, causing diminished gene expression.…”

“…In addition, Chan et al have used an analogous modular design strategy to engineer TF repressors for other biotechnological applications (e.g., Deadman and passcode kill switches) 38 . Recent work has verified engineered transcription factor applicability in not just the repressor phenotype, but also a library of anti-repressor TFs in the form of a collection of anti-lacs 40 , 44 .…”