Biology subjects

McClean, M. N.

Publications and source records attributed to McClean, M. N..

8 recordsLinked to original sources

bioRxiv2023

Dynamic Multiplexed Control and Modeling of Optogenetic Systems Using the High-Throughput Optogenetic Platform, Lustro

The ability to control cellular processes using optogenetics is inducer-limited, with most optogenetic systems responding to blue light. To address this limitation we leverage an integrated framework combining Lustro, a powerful high-throughput optogenetics platform, and machine learning tools to enable multiplexed control over blue light-sensitive optogenetic systems. Specifically, we identify light induction conditions for sequential activation as well as preferential activation and switching between pairs of light-sensitive spit transcription factors in the budding yeast, Saccharomyces cerevisiae. We use the high-throughput data generated from Lustro to build a Bayesian optimization framework that incorporates data-driven learning, uncertainty quantification, and experimental design to enable the prediction of system behavior and the identification of optimal conditions for multiplexed control. This work lays the foundation for designing more advanced synthetic biological circuits incorporating optogenetics, where multiple circuit components can be controlled using designer light induction programs, with broad implications for biotechnology and bioengineering. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/572411v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1d560ecorg.highwire.dtl.DTLVardef@1ce5614org.highwire.dtl.DTLVardef@1756e3corg.highwire.dtl.DTLVardef@986683_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Tuning Interdomain Conjugation Toward in situ Population Modification in Yeast

The ability to modify and control natural and engineered microbiomes is essential for biotechnology and biomedicine. Fungi are critical members of most microbiomes, yet technology for modifying the fungal members of a microbiome has lagged far behind that for bacteria. Interdomain conjugation (IDC) is a promising approach, as DNA transfer from bacterial cells to yeast enables in situ modification. While such genetic transfers have been known to naturally occur in a wide range of eukaryotes, and are thought to contribute to their evolution, IDC has been understudied as a technique to control fungal or fungal-bacterial consortia. One major obstacle to widespread use of IDC is its limited efficiency. In this work, we utilize interactions between genetically tractable Escherichia coli and Saccharomyces cerevisiae to control the incidence of IDC. We test the landscape of population interactions between the bacterial donors and yeast recipients to find that bacterial commensalism leads to maximized IDC, both in culture and in mixed colonies. We demonstrate the capacity of cell-to-cell binding via mannoproteins to assist both IDC incidence and bacterial commensalism in culture, and model how these tunable controls can predictably yield a range of IDC outcomes. Further, we demonstrate that these lessons can be utilized to lastingly alter a recipient yeast population, by both "rescuing" a poor-growing recipient population and collapsing a stable population via a novel IDC-mediated CRISPR/Cas9 system.

bioengineering↗

bioRxiv2023

Lustro: High-throughput optogeneticexperiments enabled by automation anda yeast optogenetic toolkit

Optogenetic systems use genetically-encoded light-sensitive proteins to control cellular processes. This provides the potential to orthogonally control cells with light, however these systems require many design-build-test cycles to achieve a functional design and multiple illumination variables need to be laboriously tuned for optimal stimulation. We combine laboratory automation and a modular cloning scheme to enable high-throughput construction and characterization of optogenetic split transcription factors in Saccharomyces cerevisiae. We expand the yeast optogenetic toolkit to include variants of the cryptochromes and Enhanced Magnets, incorporate these light-sensitive dimerizers into split transcription factors, and automate illumination and measurement of cultures in a 96-well microplate format for high-throughput characterization. We use this approach to rationally design and test an optimized Enhanced Magnet transcription factor with improved light-sensitive gene expression. This approach is generalizable to high-throughput characterization of optogenetic systems across a range of biological systems and applications.

synthetic biology↗

bioRxiv2022

Transcription Factor Localization Dynamics and DNA Binding Drive Distinct Promoter Interpretations

Environmental information may be encoded in the temporal dynamics of transcription factor (TF) activation and subsequently decoded by gene promoters to enact stimulus-specific gene expression programs. Previous studies of this behavior focused on the encoding and decoding of information in TF nuclear localization dynamics, yet cells control the activity of TFs in myriad ways, including by regulating their ability to bind DNA. Here, we use light-controlled mutants of the yeast TF Msn2 as a model system to investigate how promoter decoding of TF localization dynamics is affected by changes in the ability of the TF to bind DNA. We find that yeast promoters directly decode the light-controlled localization dynamics of Msn2 and that the effects of changing Msn2 affinity on that decoding behavior are highly promoter dependent, illustrating how cells could potentially regulate TF localization dynamics and DNA binding in concert for improved control of gene expression.

systems biology↗

bioRxiv2022

Strain dependent differences in coordination of yeast signaling networks

The yeast mitogen activated protein kinase pathways serve as a model system for understanding how network interactions affect the way in which cells coordinate the response to multiple signals. We have quantitatively compared two yeast strain backgrounds YPH499 and {Sigma}1278b (both of which have previously been used to study these pathways) and found several important differences in how they coordinate the interaction between the high osmolarity glycerol (HOG) and mating pathways. In the {Sigma}1278b background, in response to simultaneous stimulus, mating pathway activation is dampened and delayed in a dose dependent manner. In the YPH499 background, only dampening is dose dependent. Further, leakage from the HOG pathway into the mating pathway (crosstalk) occurs during osmostress alone in the {Sigma}1278b background only. The mitogen activated protein kinase Hog1p suppresses crosstalk late in an induction time course in both strains but does not affect the early crosstalk seen in the {Sigma}1278b background. Finally, the kinase Rck2p plays a greater role suppressing late crosstalk in the {Sigma}1278b background than in the YPH499 background. Our results demonstrate that comparisons between laboratory yeast strains provide an important resource for understanding how signaling network interactions are tuned by genetic variation without significant alteration to network structure.

molecular biology↗

bioRxiv2021

Integrating multiple single-cell phenotypes links stress acclimation to prior life history in yeast

Stress defense and cell growth are inversely related in bulk culture analyses; however, these studies miss substantial cell-to-cell heterogeneity, thus obscuring true phenotypic relationships. Here, we devised a microfluidics system to characterize multiple phenotypes in single yeast cells over time before, during, and after salt stress. The system measured cell and colony size, growth rate, and cell-cycle phase along with nuclear trans-localization of two transcription factors: stress-activated Msn2 that regulates defense genes and Dot6 that represses ribosome biogenesis genes during an active stress response. By tracking cells dynamically, we discovered unexpected discordance between Msn2 and Dot6 behavior that revealed subpopulations of cells with distinct growth properties. Surprisingly, post-stress growth recovery was positively corelated with activation of the Dot6 repressor. In contrast, cells lacking Dot6 displayed slower growth acclimation, even though they grow normally in the absence of stress. We show that wild-type cells with a larger Dot6 response display faster production of Msn2-regulated Ctt1 protein, separable from the contribution of Msn2. These results are consistent with the model that transcriptional repression during acute stress in yeast provides a protective response, likely by redirecting translational capacity to induced transcripts.

molecular biology↗

bioRxiv2021

Optogenetic tools for public goods control in Saccharomyces cerevisiae

Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light-control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in Saccharomyces cerevisiae to perturb both intercellular and interspecies interactions. ImportanceRecent advances in microbial ecology have highlighted the importance of intercellular interactions in controlling the development, composition and resilience of microbial communities. In order to better understand the role of these interactions in governing community development it is critical to be able to alter them in a controlled manner. Optogenetically-controlled interactions offer advantages over static perturbations or chemically-controlled interactions as light can be manipulated in space and time and doesnt require the addition of nutrients or antibiotics. Here we report a system for rapidly achieving light-control of a gene of interest in the important model organism Saccharomyces cerevisiae and demonstrate that by controlling expression of the enzyme invertase we can control cooperative interactions. This approach will be useful for understanding intercellular and interspecies interactions in natural and synthetic microbial consortia containing Saccharomyces cerevisiae and serves as a proof-of-principle for implementing this approach in other consortia.

bioengineering↗

bioRxiv2017

Single-cell RNA-seq reveals intrinsic and extrinsic regulatory heterogeneity in yeast responding to stress

From bacteria to humans, individual cells within isogenic populations can show significant variation in stress tolerance, but the nature of this heterogeneity is not clear. To investigate this, we used single-cell RNA sequencing to quantify transcript heterogeneity in single S. cerevisiae cells treated with and without salt stress, to explore population variation and identify cellular covariates that influence the stress-responsive transcriptome. Leveraging the extensive knowledge of yeast transcriptional regulation, we uncovered significant regulatory variation in individual yeast cells, both before and after stress. We also discovered that a subset of cells decouple expression of ribosomal protein genes from the environmental stress response, in a manner partly correlated with the cell cycle but unrelated to the yeast ultradian metabolic cycle. Live-cell imaging of cells expressing pairs of fluorescent regulators, including the transcription factor Msn2 with Dot6, Sfp1, or MAP kinase Hog1, revealed both coordinated and decoupled nucleocytoplasmic shuttling. Together with transcriptomic analysis, our results reveal that cells maintain a cellular filter against decoupled bursts of transcription-factor activation but mount a stress response upon coordinated regulation, even in a subset of unstressed cells.

systems biology↗