Engineering the Ultrasensitive Transcription Factors by Fusing a Modular Oligomerization Domain

Abstract

The dimerization and high-order oligomerization of transcription factors has endowed them with cooperative regulatory capabilities that play important roles in many cellular functions. However, such advanced regulatory capabilities have not been fully exploited in synthetic biology and genetic engineering. Here, we engineered a C-terminally fused oligomerization domain to improve the cooperativity of transcription factors. First, we found that two of three designed oligomerization domains significantly increased the cooperativity and ultrasensitivity of a transcription factor for the regulated promoter. Then, seven additional transcription factors were used to assess the modularity of the oligomerization domains, and their ultrasensitivity was generally improved, as assessed by their Hill coefficients. Moreover, we also demonstrated that the allosteric capability of the ligand-responsive domain remained intact when fusing with the designed oligomerization domain. As an example application, we showed that the engineered ultrasensitive transcription factor could be used to significantly improve the performance of a "stripe-forming" gene circuit. We envision that the oligomerization modules engineered in this study could act as a powerful tool to rapidly tune the underlying response profiles of synthetic gene circuits and metabolic pathway controllers.

A Novel Design of Transcriptional Factor-mediated Dynamic Control of DNA Recombination

“…We suspected that the small difference between zero, one, and two sites in the preliminary experiment may be due to the relatively weak repression ability of lacI. Thus, we replaced lacI with PhlF TF fused with oligomerization domains from another TF protein cI434 (PhlF*) to increase repression level 10 . The new TF PhlF* plasmid also implements an inducible Ptac promoter to enable convenient control of TF expression.…”

“…Previous researches have proposed the model of cooperation between TFs with oligomerization domains 10 . According to this theory, the presence of at least two operators enables them to oligomerize and bend the DNA region between them to disable actions to the region.…”

Scaling up genetic circuit design for cellular computing: advances and prospects

“…These include changing the strength of the promoter sequence, hybrid combinations of promoter sequences (Chen et al 2018 ), operator site modification (Ang et al 2013 ), ribosome binding site (RBS) modification (Salis et al 2009 ), altering plasmid copy number (Guido et al 2006 ), using decoy DNA operators (Lee and Maheshri 2012 ), RNA interference (RNAi), degradation tags (Bonger et al 2011 ), or co-expression with sequestering proteins or molecules (Wang et al 2014 ). Cooperativity has been improved using oligomerization domains (Hou et al 2018 ). Positive feedback loops and signal cascades have improved the ON/OFF ratio in digital-like circuits which is often poor due to an inherent basal level of ‘leaky’ gene expression even without the presence of activators or in the presence of repressors (Bradley et al 2016a ).…”

FRETting about the affinity of bimolecular protein–protein interactions

“…To address this question, we expressed FP‐tagged proteins in E. coli from a single plasmid. This approach enabled efficient live‐cell measurements of E with advantages of (a) the rapid growth of E. coli , shortening the measurement‐time, (b) minimal anticipated interference of eukaryotic proteins (used here) from host proteins, (c) factors influencing protein expression in E. coli cells have been well studied (SI pp S7), and, therefore, can be manipulated for altering protein expression, and (d) the convenience of cell‐storage for repeating measurements. We created a set consisting of nine pairs of interacting proteins (Table ), referred to as the different ‐fold set (Supplementary Material, p. S5).…”

“…(B), top]. We had previously determined affinity of these complexes (e.g., KDM5B/D) using (ITC) . As these complexes are absent in the set used for generating the linear equation [Fig.…”