Biology subjects

Azizoglu, A.

Publications and source records attributed to Azizoglu, A..

4 recordsLinked to original sources

bioRxiv2025

Orthogonal Inducible Transcriptional Control Systems forInvestigation of Gene-Gene Interactions

Genes do not act in isolation but through complex networks of interactions. While many double and triple gene interactions have been uncovered in Saccharomyces cerevisiae, most have been identified through overexpression or loss-of-function studies, leaving dosage-dependent interactions largely unexplored. Multi-gene control systems suffer from various drawbacks, including limited range and high cell-to-cell variation of expression across the population. To address this gap, we extend a recently developed inducible expression system that enables precise, tunable control of gene expression to additional repressor-inducer pairs: LacI/IPTG and LexA-hER/{beta}-estradiol. We demonstrate that both new systems can control endogenous genes on their own by placing various low, medium and high expressed endogenous genes under their control. Using all three WTC systems, we show simultaneous and orthogonal double and triple gene control that confirms known gene-gene interactions within the anaphase signaling network and reveals new dosage-dependent phenotypes. Given their precision, orthogonality and compatibility with all commonly used growth media, this collection of WTC systems represents a new tool suitable for precise investigation of dosage-dependent gene-gene interactions in yeast.

synthetic biology↗

bioRxiv2024

Evolverator: An engineered in cellulo yeast system to drive rapid continuous evolution of proteins

Continuous in vivo evolution of non-essential proteins requires an engineered link between target protein and host cell fitness, which is difficult to establish and vulnerable to escape mutations. We systematically designed and tested Saccharomyces cerevisiae gene circuits guided by quantitative mathematical models that predicted circuit, organism, and population behavior. We identified four generalizable design principles for escape-resistant continuous evolution and built the Evolverator, a yeast platform that couples protein binding to proliferation rate while suppressing escape mutations. We used the Evolverator to improve human estrogen receptor ligand affinity and generate stronger yeast repressors from the bacterial LacI protein. The resulting variants revealed previously undescribed hER substitutions within and outside the binding pocket, as well as LacI mutations that enhance repression in yeast despite reduced activity in Escherichia coli. Our work establishes a generalizable, model-guided framework for building yeast strains to allow continuous evolution of diverse protein functions in vivo.

synthetic biology↗

bioRxiv2022

Autorepression-based conditional gene expression system in yeast for variation-suppressed control of protein dosage

Conditional control of gene expression allows an experimenter to investigate many aspects of a genes function. In the model organism Saccharomyces cerevisiae, a number of methods to control gene expression are widely practiced, including induction by metabolites, small molecules, and even light. However, all current methods suffer from at least one of a set of drawbacks, including need for specialized growth conditions, leaky expression, or the requirement of specialized equipment. Here we describe protocols using two transformations to construct strains that carry a new controller, in which all these drawbacks are overcome. In these strains, the expression of a controlled gene (gene of interest, or GOI) is repressed by the bacterial repressor TetR, and induced by anhydrotetracycline. TetR also regulates its own expression, creating an autorepression loop. This autorepression allows tight control of gene expression/ protein dosage with low cell to cell variation in expression. A second repressor, TetR-Tup1, prevents any leaky expression. We also present a protocol showing a particular workhorse application of such strains, to generate synchronized cell populations. We turn off the expression of the cell cycle regulator CDC20 completely, arresting the cell population, and then back on so that the synchronized cells resume cell cycle progression. This control system can be applied to any endogenous or exogenous gene for precise expression. Basic Protocol 1Generating a parent WTC846 strain. Basic Protocol 2Generating a WTC846 strain with controlled expression of the targeted gene Alternate Protocol 1CRISPR-mediated promoter replacement Basic Protocol 3Cell cycle synchronization/Arrest and Release using the WTC846-K3::CDC20 strain

genetics↗

bioRxiv2019

A precisely-titratable, variation-suppressed transcriptional controller to enable genetic discovery

Methods to express genes conditionally into phenotype remain central to biological experimentation and biotechnology. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a "well-tempered" controller, WTC846, for precisely adjustable, graded and growth condition independent conditional expression of genes in Saccharomyces cerevisiae. In WTC846 strains, the controlled genes are expressed from a strong, native promoter engineered to be repressed by the prokaryotic TetR protein and induced by tetracycline and analogues. A second instance of this promoter drives TetR itself. This autorepression loop exhibits low cell-to-cell variation in gene expression and allows precise adjustment of the steady state abundance of any protein with inducer. A second, constitutively expressed zeroing repressor abolishes basal expression in the absence of inducer. WTC846-controlled, stable (Cdc42, Tpi1) and unstable (Ipl1) proteins recapitulated known knockout and overexpression phenotypes. WTC846::CDC20 strains enabled inducer regulated cell cycle synchronization. WTC846 alleles of CDC28, TOR1, PBR1 and PMA1 exhibited expected gene dosage-dependent growth rates and morphological phenotypes, and WTC846::WHI5 strains exhibited inducer controlled differences in cell volume. WTC846 controlled genes comprise a new kind of "expression clamped" allele, for which variation in expression is minimized and gene dosage can be set by the experimenter across the range of cellular protein abundances. In yeast, we expect WTC846 alleles to find use in assessment of phenotypes now incompletely penetrant due to variable dosage of the causative protein, and in genome-wide epistasis screens. Implementation in higher cells should enable experiments now impossible due to cell-to-cell variation and imprecise control.

molecular biology↗