Conditional expression of genes and observation of phenotype remain central to biological discovery. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a 'well-tempered' controller, WTC846, for precisely adjustable, graded, growth condition independent expression of genes in Saccharomyces cerevisiae. Controlled genes are expressed from a strong semisynthetic promoter repressed by the prokaryotic TetR, which also represses its own synthesis; with basal expression abolished by a second, 'zeroing' repressor. The autorepression loop lowers cell-to-cell variation while enabling precise adjustment of protein expression by a chemical inducer. WTC846 allelic strains in which the controller replaced the native promoters recapitulated known null phenotypes (CDC42, TPI1), exhibited novel overexpression phenotypes (IPL1), showed protein dosage-dependent growth rates and morphological phenotypes (CDC28, TOR2, PMA1 and the hitherto uncharacterized PBR1), and enabled cell cycle synchronization (CDC20). WTC846 defines an 'expression clamp' allowing protein dosage to be adjusted by the experimenter across the range of cellular protein abundances, with limited variation around the setpoint.

For more than 65 years, means to express genes conditionally into phenotype have remained central to biological experimentation and discovery. Current experimental methods typically enable either on/o↵ gene expression or growth-condition-specific, imprecisely controlled graded expression in response to exogenous inducers. Here we describe a "well-tempered" controller, one that allows precise and graded conditional expression of genes in Saccharomyces cerevisiae. This system, WTC 846 , appropriates genetic and design elements from bacterial, eukaryotic, and engineered systems. Its main autorepressing circuitry relies on two identical instances of a strong, growth rate regulated promoter P TDH3 engineered to be repressible by the TetR protein. This allows for titration of inducer concentration to drive variation-reduced expression of regulated genes across the entire range of the proteome. To reduce basal expression to zero, a TetR-Tup1 fusion protein is expressed at a low, constitutive level. We showed that strains carrying conditional, WTC 846 allelic forms of genes encoding stable low abundance proteins (eg. Cdc42), unstable low abundance proteins (eg. Ipl1), and highly expressed proteins including the glycolytic enzyme Tpi1 recapitulated known knockout and overexpression phenotypes. Strains bearing WTC 846:Cdc20 alleles allowed inducer controlled cell cycle synchronization of batch cultures and release. Chemical titration of WTC 846 allelic strains of CDC28, TOR1, PBR1 and PMA1 brought about distinct, gene dosage dependent controlled growth rates, and highly penetrant morphological phenotypes expected for the di↵erent gene doses. The ability to generate WTC 846 controlled, "expression-clamped" genes may operationally define a new kind of conditional allele whose cell-to-cell variation in expression is minimized and whose e↵ective dosage is under the experimenter's control. We expect Well-tempered Controller 846 (WTC 846 ) strains to find use in assessment of phenotypes now incompletely penetrant due to variable dosage of the causative gene product, in targeted cell biological experimentation, and in genome-wide studies such as gene by gene epistasis screens. In higher cells, we hope that implementation of titratable, expression clamped control logic via mammalian specific transcription elements will enable experiments now impossible due to cell-to-cell variation and imprecise control.

Background The continued spread of SARS-CoV-2 and emergence of new variants with higher transmission rates and/or partial resistance to vaccines has further highlighted the need for large-scale testing and genomic surveillance. However, current diagnostic testing (e.g., PCR) and genomic surveillance methods (e.g., whole genome sequencing) are performed separately, thus limiting the detection and tracing of SARS-CoV-2 and emerging variants. Results Here, we developed DeepSARS, a high-throughput platform for simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2 by the integration of molecular barcoding, targeted deep sequencing, and computational phylogenetics. DeepSARS enables highly sensitive viral detection, while also capturing genomic diversity and viral evolution. We show that DeepSARS can be rapidly adapted for identification of emerging variants, such as alpha, beta, gamma, and delta strains, and profile mutational changes at the population level. Conclusions DeepSARS sets the foundation for quantitative diagnostics that capture viral evolution and diversity. Graphical abstract DeepSARS uses molecular barcodes (BCs) and multiplexed targeted deep sequencing (NGS) to enable simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2. Image was created using Biorender.com.

Cell-to-cell variability originating, for example, from the intrinsic stochasticity of gene expression, presents challenges for designing synthetic gene circuits that perform robustly. Conversely, synthetic biology approaches are instrumental in uncovering mechanisms underlying variability in natural systems. With a focus on reducing noise in individual genes, the field has established a broad synthetic toolset. This includes noise control by engineering of transcription and translation mechanisms either individually, or in combination to achieve independent regulation of mean expression and its variability. Synthetic feedback circuits use these components to establish more robust operation in closed-loop, either by drawing on, but also by extending traditional engineering concepts. In this perspective, we argue that major conceptual advances will require new theory of control adapted to biology, extensions from single genes to networks, more systematic considerations of origins of variability other than intrinsic noise, and an exploration of how noise shaping, instead of noise reduction, could establish new synthetic functions or help understanding natural functions.