RT Journal Article
SR Electronic
T1 Cooperative assembly confers regulatory specificity and long-term genetic circuit stability
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.05.22.492993
DO 10.1101/2022.05.22.492993
A1 Meghan D. J. Bragdon
A1 Nikit Patel
A1 James Chuang
A1 Ethan Levien
A1 Caleb J. Bashor
A1 Ahmad S. Khalil
YR 2022
UL http://biorxiv.org/content/early/2022/05/23/2022.05.22.492993.abstract
AB In eukaryotes, links in gene regulatory networks are often maintained through cooperative self-assembly between transcriptional regulators (TRs) and DNA cis-regulatory motifs, a strategy widely thought to enable highly specific regulatory connections to be formed between otherwise weakly-interacting, low-specificity molecular components. Here, we directly test whether this regulatory strategy can be used to engineer regulatory specificity in synthetic gene circuits constructed in yeast. We show that circuits composed of artificial zinc-finger TRs can be effectively insulated from aberrant misregulation of the host cell genome by using cooperative multivalent TR assemblies to program circuit connections. As we demonstrate in experiments and mathematical models, assembly-mediated regulatory connections enable mitigation of circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturally-inspired approach offers a simple, generalizable means for building evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.Competing Interest StatementN.P., C.J.B, and A.S.K. are co-inventors on a patent related to engineered cooperativity and control of gene expression. A.S.K. is a scientific advisor for and holds equity in Senti Biosciences and Chroma Medicine, and is a co-founder of Fynch Biosciences and K2 Biotechnologies.