Easy calibration of the Light Plate Apparatus for optogenetic experiments

Sweeney, Kieran; Morales, Neydis Moreno; Burmeister, Zachary; Nimunkar, Amit J.; McClean, Megan N.

doi:10.1016/j.mex.2019.06.008

Cited by 10 publications

(10 citation statements)

References 8 publications

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“…Light intensity was measured and validated using a standard photodiode power sensor and power meter (Thorlabs #S120VC, Thorlabs #PM100D). The LPA was calibrated as described in Sweeney, Moreno Morales, Burmeister, Nimunkar, and McClean (2019) so that consistent light doses could be delivered between LPAs and between experiments.…”

Section: Blue-light Inductionmentioning

confidence: 99%

A yeast optogenetic toolkit (yOTK) for gene expression control in Saccharomyces cerevisiae

An-Adirekkun

Stewart

Geller

et al. 2019

Biotech & Bioengineering

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Optogenetic tools for controlling gene expression are ideal for tuning synthetic biological networks due to the exquisite spatiotemporal control available with light.Here we develop an optogenetic system for gene expression control integrated with an existing yeast toolkit allowing for rapid, modular assembly of light-controlled circuits in the important chassis organism Saccharomyces cerevisiae. We reconstitute activity of a split synthetic zinc-finger transcription factor (TF) using light-induced dimerization mediated by the proteins CRY2 and CIB1. We optimize function of this split TF and demonstrate the utility of the toolkit workflow by assembling cassettes expressing the TF activation domain and DNA-binding domain at different levels.Utilizing this TF and a synthetic promoter we demonstrate that light intensity and duty cycle can be used to modulate gene expression over the range currently available from natural yeast promoters. This study allows for rapid generation and prototyping of optogenetic circuits to control gene expression in S. cerevisiae. K E Y W O R D Slight-inducible promoter, MoClo, optogenetics, Saccharomyces cerevisiae, yeast Jidapas (My) An-adirekkun, Cameron J. Stewart, and Stephanie H. Geller contributed equally to this study.

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Section: Blue-light Inductionmentioning

confidence: 99%

A yeast optogenetic toolkit (yOTK) for gene expression control in Saccharomyces cerevisiae

An-Adirekkun

Stewart

Geller

et al. 2019

Biotech & Bioengineering

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“…The Light Plate Apparatus (LPA) is a published optogenetic tool which provides programmed illumination to each well of a 24-well plate. We assembled our LPA as described in 87 and calibrated as previously described 88,89 .…”

Section: Methodsmentioning

confidence: 99%

Optogenetic tools for public goods control in Saccharomyces cerevisiae

Nm¹,

Cj³

et al. 2021

Preprint

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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.

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“…Blue (Zhao et al, 2019) CRY2/CIB1 Blue (Sweeney, Moreno Morales, Burmeister, Nimunkar, & McClean, 2019) AtLOV2 Blue (Scheffer, Hasenjäger, & Taxis, 2019) AsLOV2 (LINuS plus CRISPR-dCas9) Blue (Geller, Antwi, di Ventura, & McClean, 2019) CRY2/CIB1 (yOTK) Blue (An-Adirekkun et al, 2020) CRY2/CIB1 Blue (Rivera-Tarazona, Bhat, Kim, Campbell, & Ware, 2020) AuLOV Blue (Hepp et al, 2020) EL222 Blue (Perkins, Benzinger, Arcak, & Khammash, 2020) PYP Blue (Woloschuk, Reed, McDonald, Uppalapati, & Woolley, 2020) AsLOV2 (LINX) Blue (Meriesh, Lerner, Chandrasekharan, & Strahl, 2020) CrPHOT Blue (Suzuki et al, 2020) AsLOV2 (CLASP) Blue (Chen et al, 2020) AsLOV2 (LOV2GIVe) Blue (Garcia-Marcos et al, 2020)…”

Section: Blue-light Optogenetic Systemsmentioning

confidence: 99%

“…Importantly, some of these devices have already been used to perform optogenetics in yeast, such as the Light Plate Apparatus (LPA). Using the CRY2-CIB1 switch, LPA was validated by expressing mRuby and mCherry fluorescent reporters upon blue-light exposure (Gerhardt et al, 2016;Sweeney et al, 2019 Figure 4a) (Kojadinovic et al, 2008). Importantly, bacterial phytochromes do not use PCB or PϕB and require biliverdin IX as cofactor, which is abundant in eukaryotic cells (Bhoo, Davis, Walker, Karniol, & Vierstra, 2001).…”

Section: Optogenetic Switches For Yeast Biotechnologymentioning

confidence: 99%

The rise and shine of yeast optogenetics

Figueroa

Rojas

Romero

et al. 2020

Yeast

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Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared with traditional chemically inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost, and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, and protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast. Take Away The budding yeast Saccharomyces cerevisiae has become a powerful biological platform for optogenetics, allowing the use of light to control diverse phenotypes. In this review, we summarized the main optogenetic tools implemented in yeast and their applications in metabolic engineering and biotechnology. Finally, we propose different sources of building blocks to expand the current repertoire of optogenetic systems in yeast.

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Easy calibration of the Light Plate Apparatus for optogenetic experiments

Abstract: Graphical abstract

Cited by 10 publications

References 8 publications

A yeast optogenetic toolkit (yOTK) for gene expression control in Saccharomyces cerevisiae

A yeast optogenetic toolkit (yOTK) for gene expression control in Saccharomyces cerevisiae

Optogenetic tools for public goods control in Saccharomyces cerevisiae

The rise and shine of yeast optogenetics

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