A modular toolbox for the optogenetic deactivation of transcription DOI Creative Commons

Philipp Muench,

Matteo Fiumara,

Nicholas Southern

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Ноя. 6, 2023

Abstract Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited light-triggered induction expression. Here, we inverted this mode action and created two complementary systems capable efficiently terminating in response blue light. First, designed highly compact regulators, by photo-controlling VP16 transactivation peptide exposure. Then, applying two-hybrid strategy, engineered LOOMINA ( l ight o ff- perated m odular ductor ctivation), versatile control platform for mammalian cells adaptable compatible various effector proteins. Leveraging the flexibility CRISPR systems, integrated Cas9 as DNA-binding domain transcription from endogenous promoters exceptionally dynamic ranges multiple cell lines, including neuron-like cells. Both functionally mechanistically, represents valuable addition repertoire regulation.

Язык: Английский

Integrating bioelectronics with cell-based synthetic biology DOI
Jonathan Rivnay, Ritu Raman, Jacob T. Robinson

и другие.

Nature Reviews Bioengineering, Год журнала: 2025, Номер unknown

Опубликована: Янв. 7, 2025

Язык: Английский

Процитировано

3
Lighting the way: recent developments and applications in molecular optogenetics DOI Creative Commons

Anja Armbruster,

Asim ME Mohamed, Hoang Trong Phan

и другие.

Current Opinion in Biotechnology, Год журнала: 2024, Номер 87, С. 103126 - 103126

Опубликована: Март 29, 2024

Molecular optogenetics utilizes genetically encoded, light-responsive protein switches to control the function of molecular processes. Over last two years, there have been notable advances in development novel optogenetic switches, their utilization elucidating intricate signaling pathways, and progress toward practical applications biotechnological processes, material sciences, therapeutic applications. In this review, we discuss these areas, offer insights into recent developments, contemplate future directions.

Язык: Английский

Процитировано

9
Light-inducible protein degradation in E. coli with the LOVdeg tag DOI Creative Commons
Nathan Tague,

Cristian Coriano-Ortiz,

Michael B. Sheets

и другие.

eLife, Год журнала: 2024, Номер 12

Опубликована: Янв. 25, 2024

Molecular tools for optogenetic control allow spatial and temporal regulation of cell behavior. In particular, light-controlled protein degradation is a valuable mechanism because it can be highly modular, used in tandem with other mechanisms, maintain functionality throughout growth phases. Here, we engineered LOVdeg, tag that appended to interest inducible Escherichia coli using blue light. We demonstrate the modularity LOVdeg by range proteins, including LacI repressor, CRISPRa activator, AcrB efflux pump. Additionally, utility pairing existing enhance performance developing combined EL222 system. Finally, use metabolic engineering application post-translational metabolism. Together, our results highlight system introduce powerful new tool bacterial optogenetics.

Язык: Английский

Процитировано

4
Engineering transcriptional regulation for cell-based therapies DOI Creative Commons
Matthias Recktenwald,

Evan Hutt,

Leah Davis

и другие.

SLAS TECHNOLOGY, Год журнала: 2024, Номер 29(2), С. 100121 - 100121

Опубликована: Фев. 8, 2024

A major aim in the field of synthetic biology is developing tools capable responding to user-defined inputs by activating therapeutically relevant cellular functions. Gene transcription and regulation response external stimuli are some most powerful versatile these functions being explored. Motivated success chimeric antigen receptor (CAR) T-cell therapies, transmembrane receptor-based platforms have been embraced for their ability sense extracellular ligands subsequently activate intracellular signal transduction. The integration receptors with transcriptional activation has not yet achieved its full potential. Transient expression plasmid DNA often used explore gene vitro. However, applications targeting endogenous or stably integrated genes more clinically relevant. may allow engineered cells traffic into tissues interest secrete functional proteins space differentiate cells. Transmembrane that regulate potential revolutionize cell therapies a myriad applications, including cancer treatment regenerative medicine. In this review, we will examine current engineering approaches control mammalian an emphasis on systems can be selectively activated signals. We also speculate therapeutic technologies promising expand capabilities tighten therapies.

Язык: Английский

Процитировано

3
Light-inducible protein degradation in E. coli with the LOVdeg tag DOI Creative Commons
Nathan Tague,

Cristian Coriano-Ortiz,

Michael B. Sheets

и другие.

eLife, Год журнала: 2023, Номер 12

Опубликована: Май 30, 2023

Molecular tools for optogenetic control allow spatial and temporal regulation of cell behavior. In particular, light-controlled protein degradation is a valuable mechanism because it can be highly modular, used in tandem with other mechanisms, maintain functionality throughout growth phases. Here, we engineered LOVdeg, tag that appended to interest inducible Escherichia coli using blue light. We demonstrate the modularity LOVdeg by range proteins, including LacI repressor, CRISPRa activator, AcrB efflux pump. Additionally, utility pairing existing enhance performance developing combined EL222 system. Finally, use metabolic engineering application post-translational metabolism. Together, our results highlight system introduce powerful new tool bacterial optogenetics.

Язык: Английский

Процитировано

8
Light inducible protein degradation inE. coliwith the LOVdeg tag DOI Open Access
Nathan Tague,

Cristian Coriano-Ortiz,

Michael B. Sheets

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Фев. 26, 2023

Abstract Molecular tools for optogenetic control allow spatial and temporal regulation of cell behavior. In particular, light controlled protein degradation is a valuable mechanism because it can be highly modular, used in tandem with other mechanisms, maintain functionality throughout growth phases. Here, we engineered LOVdeg, tag that appended to interest inducible Escherichia coli using blue light. We demonstrate the modularity LOVdeg by range proteins, including LacI repressor, CRISPRa activator, AcrB efflux pump. Additionally, utility pairing existing enhance performance developing combined EL222 system. Finally, use metabolic engineering application post-translational metabolism. Together, our results highlight system, introduce powerful new tool bacterial optogenetics.

Язык: Английский

Процитировано

5
Comprehensive Screening of a Light-Inducible Split Cre Recombinase with Domain Insertion Profiling DOI
Nathan Tague, Virgile Andreani, Yunfan Fan

и другие.

ACS Synthetic Biology, Год журнала: 2023, Номер 12(10), С. 2834 - 2842

Опубликована: Окт. 3, 2023

Splitting proteins with light- or chemically inducible dimers provides a mechanism for post-translational control of protein function. However, current methods engineering stimulus-responsive split often require significant expertise and the laborious screening individual constructs. To address this challenge, we use pooled library approach that enables rapid generation nearly all possible constructs in parallel, where results can be read out by using sequencing. We perform our method on Cre recombinase optogenetic as proof concept, resulting comprehensive data sites throughout protein. improve accuracy predicting behavior, develop Bayesian computational to contextualize errors inherent experimental procedures. Overall, streamlined achieving interest.

Язык: Английский

Процитировано

4
Light inducible protein degradation in E. coli with the LOVdeg tag DOI Open Access
Nathan Tague,

Cristian Coriano-Ortiz,

Michael B. Sheets

и другие.

Опубликована: Янв. 3, 2024

Molecular tools for optogenetic control allow spatial and temporal regulation of cell behavior. In particular, light controlled protein degradation is a valuable mechanism because it can be highly modular, used in tandem with other mechanisms, maintain functionality throughout growth phases. Here, we engineered LOVdeg, tag that appended to interest inducible Escherichia coli using blue light. We demonstrate the modularity LOVdeg by range proteins, including LacI repressor, CRISPRa activator, AcrB efflux pump. Additionally, utility pairing existing enhance performance developing combined EL222 system. Finally, use metabolic engineering application post-translational metabolism. Together, our results highlight system, introduce powerful new tool bacterial optogenetics.

Язык: Английский

Процитировано

1
Opticool: Cutting-edge transgenic optical tools DOI Creative Commons
Kelli D. Fenelon, Julia Krause, Theodora Koromila

и другие.

PLoS Genetics, Год журнала: 2024, Номер 20(3), С. e1011208 - e1011208

Опубликована: Март 22, 2024

Only a few short decades have passed since the sequencing of GFP, yet modern repertoire transgenically encoded optical tools implies an exponential proliferation ever improving constructions to interrogate subcellular environment. A myriad tags for labeling proteins, RNA, or DNA arisen in last decades, facilitating unprecedented visualization components and processes. Development broad array genetically sensors allows real-time, vivo detection molecule levels, pH, forces, enzyme activity, other extracellular phenomena expanding contexts. Optogenetic, optically controlled manipulation systems gained traction biological research community facilitate single-cell, real-time modulation protein function broadening, novel applications. While this field continues explosively expand, references are needed assist scientists seeking use improve these transgenic devices new exciting ways development disease. In review, we endeavor highlight state trajectory tools.

Язык: Английский

Процитировано

0
Local disorder is associated with enhanced catalysis in an engineered photoswitch DOI Creative Commons
James W. McCormick, Jerry C. Dinan, Marielle AX Russo

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Ноя. 28, 2024

Abstract The A. sativa LOV2 domain is commonly harnessed as a source of light-based regulation in engineered optogenetic switches. In prior work, we used to create light-regulated Dihydrofolate Reductase (DHFR) enzyme and showed that structurally disperse mutations DHFR were able tune the allosteric response light. However, it remained unclear how light allosterically activates DHFR, modulate effect. A mechanistic understanding these phenomena would improve our ability rationally design new enzymes. We combination Eyring analysis CD spectroscopy quantify relationship between allostery, catalytic activity, global thermal stability. found DHFR/LOV2 fusion was marginally stable at physiological temperatures. photoactivation simultaneously: (1) thermally destabilized (2) lowered transition free energy lit state relative dark state. energetic effect activation on composed two opposing forces: favorable reduction enthalpic barrier offset by an entropic penalty. Allostery-tuning acted through this tradeoff, either accentuating benefit or minimizing penalty but never improving both. Many allostery tuning negative correlation induced change stability suggesting activity-stability tradeoff.

Язык: Английский

Процитировано

0