How to train your microbe: methods for dynamically characterizing gene networks

Castillo-Hair, Sebastian M.; Igoshin, Oleg A.; Tabor, Jeffrey J.

doi:10.1016/j.mib.2015.01.008

Cited by 27 publications

(26 citation statements)

References 124 publications

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“…The precipitated protein was removed by centrifugation at 15,000 ϫ g for 5 min. The supernatant was filtered through a 0.22-m-pore-size filter (MicroSolv) and analyzed by reversed-phase high-performance liquid chromatography (HPLC) as described previously (17).…”

Section: Methodsmentioning

confidence: 99%

Optogenetic Module for Dichromatic Control of c-di-GMP Signaling

et al. 2017

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Many aspects of bacterial physiology and behavior, including motility, surface attachment, and the cell cycle, are controlled by cyclic di-GMP (c-di-GMP)-dependent signaling pathways on the scale of seconds to minutes. Interrogation of such processes in real time requires tools for introducing rapid and reversible changes in intracellular c-di-GMP levels. Inducing the expression of genes encoding c-di-GMP-synthetic (diguanylate cyclases) and -degrading (c-di-GMP phosphodiesterase) enzymes by chemicals may not provide adequate temporal control. In contrast, light-controlled diguanylate cyclases and phosphodiesterases can be quickly activated and inactivated. A red/near-infrared-light-regulated diguanylate cyclase, BphS, was engineered previously, yet a complementary light-activated c-di-GMP phosphodiesterase has been lacking. In search of such a phosphodiesterase, we investigated two homologous proteins from Allochromatium vinosum and Magnetococcus marinus, designated BldP, which contain C-terminal EAL-BLUF modules, where EAL is a c-di-GMP phosphodiesterase domain and BLUF is a blue light sensory domain. Characterization of the BldP proteins in Escherichia coli and in vitro showed that they possess light-activated c-di-GMP phosphodiesterase activities. Interestingly, light activation in both enzymes was dependent on oxygen levels. The truncated EAL-BLUF fragment from A. vinosum BldP lacked phosphodiesterase activity, whereas a similar fragment from M. marinus BldP, designated EB1, possessed such activity that was highly (Ͼ30-fold) upregulated by light. Following light withdrawal, EB1 reverted to the inactive ground state with a half-life of ϳ6 min. Therefore, the bluelight-activated phosphodiesterase EB1 can be used in combination with the red/nearinfrared-light-regulated diguanylate cyclase BphS for the bidirectional regulation of c-di-GMP-dependent processes in E. coli as well as other bacterial and nonbacterial cells.IMPORTANCE Regulation of motility, attachment to surfaces, the cell cycle, and other bacterial processes controlled by the c-di-GMP signaling pathways occur at a fast (seconds-to-minutes) pace. Interrogation of these processes at high temporal and spatial resolution using chemicals is difficult or impossible, while optogenetic approaches may prove useful. We identified and characterized a robust, blue-lightactivated c-di-GMP phosphodiesterase (hydrolase) that complements a previously engineered red/near-infrared-light-regulated diguanylate cyclase (c-di-GMP synthase). These two enzymes form a dichromatic module for manipulating intracellular c-di-GMP levels in bacterial and nonbacterial cells.KEYWORDS biofilms, c-di-GMP, diguanylate cyclase, gene expression, motility, optogenetics, phosphodiesterase, photoreceptors, photosensory reception, signal transduction S ignaling via second messengers occurs at various time scales, from seconds to hours. To control relatively slow processes, intracellular concentrations of a second messenger can be modulated by the constitutive or inducible (over)e...

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Section: Methodsmentioning

confidence: 99%

Optogenetic Module for Dichromatic Control of c-di-GMP Signaling

et al. 2017

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“…Light is a powerful actuator as it is inexpensive, easily controlled in time and space, and S. cerevisiae contains no known native photoreceptors (Salinas, Rojas, Delgado, Agosin, & Larrondo, ). The ability to rapidly add and remove light from cell culture or spatially target specific cells makes it particularly advantageous for applications that require spatiotemporal precision such as dynamic stimulation or real‐time feedback control of cellular processes (Benzinger & Khammash, ; Castillo‐Hair, Igoshin, & Tabor, ; Harrigan, Madani, & El‐Samad, ; Lugagne & Dunlop, ; Milias‐Argeitis, Rullan, Aoki, Buchmann, & Khammash, ; Ng et al, ; Rullan, Benzinger, Schmidt, Milias‐Argeitis, & Khammash, ; Toettcher, Gong, Lim, & Weiner, ). To continue expanding the utility of optogenetics in S. cerevisiae , here we report the construction and optimization of a light‐activated transcription factor and associated components for use with an existing toolkit of yeast parts (Lee, Deloache, Cervantes, & Dueber, ).…”

Section: Introductionmentioning

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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“…Such an approach could accelerate the gene expression signals that we have generated in this and our previous study 11 , enabling characterization of gene circuit dynamics on faster timescales. Finally, multiplexed biological function generation could be used to evaluate how the timing of expression of two genes impacts cellular decision making [30][31][32] . For example, in B. subtilis, the gene circuits that regulate sporulation and competence compete via a 'molecular race' in the levels of the corresponding master regulators 30 .…”

Section: Discussionmentioning

confidence: 99%

A two-state photoconversion model predicts the spectral response dynamics of optogenetic systems

Olson

Tzouanas

Tabor

2016

Preprint

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In optogenetics, light signals are used to control genetically engineered photoreceptors, and in turn manipulate biological pathways with unmatched precision. Recently, evolved photoreceptors with diverse in vitro-measured wavelength and intensity-dependent photoswitching properties have been repurposed for synthetic control of gene expression, proteolysis, and numerous other cellular processes. However, the relationship between the input light spectrum and in vivo photoreceptor response dynamics is poorly understood, restricting the utility of these optogenetic tools. Here, we advance a classic in vitro two-state photoreceptor model to reflect the in vivo environment, and combine it with simplified mathematical descriptions of signal transduction and output gene expression through our previously engineered green/red and red/far red photoreversible bacterial two-component systems (TCSs). Additionally, we leverage our recent open-source optical instrument to develop a workflow of spectral and dynamical characterization experiments to parameterize the model for both TCSs. To validate our approach, we challenge the model to predict experimental responses to a series of complex light signals very different from those used during parameterization. We find that the model generalizes remarkably well, predicting the results of all categories of experiments with high quantitative accuracy for both systems. Finally, we exploit this predictive power to program two simultaneous and independent dynamical gene expression signals in bacteria expressing both TCSs. This multiplexed gene expression programming approach will enable entirely new studies of how metabolic, signaling, and decision-making pathways integrate multiple gene expression signals. Additionally, our approach should be compatible with a wide range of optogenetic tools and model organisms. Significance statementLight-switchable signaling pathways (optogenetic tools) enable precision studies of how biochemical networks underlie cellular behaviors. We have developed a versatile mathematical model based on a two-state photoconversion mechanism that we have applied to the E. coli CcaSR and Cph8-OmpR optogenetic tools. This model enables accurate prediction of the gene expression response to virtually any light source or mixture of light sources. We express both optogenetic tools in the same cell and apply our model to program two simultaneous and independent gene expression signals in the same cell. This method can be used to study how biological pathways integrate multiple inputs and should be extensible to other optogenetic tools and host organisms.

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How to train your microbe: methods for dynamically characterizing gene networks

Cited by 27 publications

References 124 publications

Optogenetic Module for Dichromatic Control of c-di-GMP Signaling

Optogenetic Module for Dichromatic Control of c-di-GMP Signaling

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

A two-state photoconversion model predicts the spectral response dynamics of optogenetic systems

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