An optogenetic toolkit for light-inducible antibiotic resistance

Sheets, Michael B.; Tague, Nathan; Dunlop, Mary J.

doi:10.1038/s41467-023-36670-2

Cited by 21 publications

(16 citation statements)

References 82 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Optical signals can also be multiplexed through spectral [19,20] or amplitude modulation [21]. A growing number of optogenetics tools have been recently applied in prokaryotes to control different aspects of bacterial physiology [22], such as growth [23][24][25], antibiotic resistance [26], motility [27,28] and adhesion [29]. A widely used optogenetic system is the light-switchable two-component system, CcaS-CcaR from Synechocystis PCC 6803 [30].…”

Section: Introductionmentioning

confidence: 99%

Light-driven synchronization of optogenetic clocks

Cannarsa,

Liguori,

Pellicciotta

et al. 2023

Preprint

View full text Add to dashboard Cite

Synthetic genetic oscillators can serve as internal clocks within engineered cells to program periodic expression. However, cell-to-cell variability introduces a dispersion in the characteristics of these clocks that drives the population to complete desynchronization. Here we introduce the optorepressilator, an optically controllable genetic clock that combines the repressilator, a three-node synthetic network in E. coli, with an optogenetic module enabling to reset, delay, or advance its phase using optical inputs. We demonstrate that a population of optorepressilators can be synchronized by transient green light exposure or entrained to oscillate indefinitely by a train of short pulses, through a mechanism reminiscent of natural circadian clocks. Furthermore, we investigate the system's response to detuned external stimuli observing multiple regimes of global synchronization. Integrating experiments and mathematical modeling, we show that the entrainment mechanism is robust and can be understood quantitatively from single cell to population level.

show abstract

Section: Introductionmentioning

confidence: 99%

Light-driven synchronization of optogenetic clocks

Cannarsa,

Liguori,

Pellicciotta

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…LOV domains make up the core of most non-ion-channel optogenetic systems [39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] , presumably because they are small, reliable, and do not need exogenous chromophores. Interest in LOV domains is reflected in multiple diversification campaigns 34,[54][55][56] .…”

Section: )mentioning

confidence: 99%

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Gligorovski,

Labagnara,

Rahi

2024

Preprint

View full text Add to dashboard Cite

Directed evolution is a powerful method in biological engineering. Current approaches were devised for evolving steady-state properties such as enzymatic activity or fluorescence intensity. A fundamental problem remains how to evolve dynamic, multi-state, or computational functionalities, e.g., folding times, on-off kinetics, state-specific activity, stimulus-responsiveness, or switching and logic capabilities. These require applying selection pressure on all of the states of a protein of interest (POI) and the transitions between them. We realized that optogenetics and cell cycle oscillations could be leveraged for a novel directed evolution paradigm (‘optovolution’) that is germane for this need: We designed a signaling cascade in budding yeast where optogenetic input switches the POI between off (0) and on (1) states. In turn, the POI controls a Cdk1 cyclin, which in the re-engineered cell cycle system is essential for one cell cycle stage but poisonous for another. Thus, the cyclin must oscillate (1-0-1-0…) for cell proliferation. In this system, evolution can act efficiently on the dynamics, transient states, and input-output relations of the POI in every cell cycle. Further, controlling the pacemaker, light, directs and tunes selection pressures. Optovolution isin vivo, continuous, self-selecting, and genetically robust. We first evolved two optogenetic systems, which relay 0/1 input to 0/1 output: We obtained 25 new variants of the widely used LOV transcription factor El222. These mutants were stronger, less leaky, or green- and red-responsive. The latter was conjectured to be impossible for LOV domains but is needed for multiplexing and lowering phototoxicity. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss ofYOR1makes supplementing the expensive and unstable chromophore phycocyanobilin (PCB) unnecessary. Finally, we demonstrate the generality of the method by creating and evolving a destabilized rtTA transcription factor, which performs an AND operation between transcriptional and doxycycline input. Optovolution makes coveted, difficult-to-change protein functionalities evolvable.

show abstract

“…In a recent study, Opto-Cre-Vvd was used to induce antibiotic resistance upon light exposure and also for metabolic engineering. 97 Split-Cre fused to chemical-induced dimerization domains (CIDs) can be subject to optical control by photolabile small molecule ligands (Figure 6D). 98 In one such study, the authors used a UV-cleavable rapamycin dimer (dRap) to activate split Cre fused to the FKBP12-FRB domains.…”

Section: Optical Control Ofmentioning

confidence: 99%

“…Opto-Cre-VVd also shows low sensitivity to ambient light and a light-dose-dependent increase in activity and can be completely activated by prolonged exposure (4 h) to very low-intensity blue light (−0.005 to 0.010 mW/cm 2 ). In a recent study, Opto-Cre-Vvd was used to induce antibiotic resistance upon light exposure and also for metabolic engineering …”

Section: Optical Control Of Dna Manipulation Using Cre Recombinasementioning

confidence: 99%

Optically Controlled CRISPR-Cas9 and Cre Recombinase for Spatiotemporal Gene Editing: A Review

Devarajan

2023

ACS Synth. Biol.

View full text Add to dashboard Cite

CRISPR-Cas9 and Cre recombinase, two tools extensively used for genome interrogation, have catalyzed key breakthroughs in our understanding of complex biological processes and diseases. However, the immense complexity of biological systems and off-target effects hinder clinical applications, necessitating the development of platforms to control gene editing over spatial and temporal dimensions. Among the strategies developed for inducible control, light is particularly attractive as it is noninvasive and affords high spatiotemporal resolution. The principles for optical control of Cas9 and Cre recombinase are broadly similar and involve photocaged enzymes and small molecules, engineered split-and single-chain constructs, light-induced expression, and delivery by light-responsive nanocarriers. Few systems enable spatiotemporal control with a high dynamic range without loss of wild-type editing efficiencies. Such systems posit the promise of light-activatable systems in the clinic. While the prospect of clinical applications is palpably exciting, optimization and extensive preclinical validation are warranted. Judicious integration of optically activated CRISPR and Cre, tailored for the desired application, may help to bridge the "bench-to-bedside" gap in therapeutic gene editing.

show abstract

An optogenetic toolkit for light-inducible antibiotic resistance

Cited by 21 publications

References 82 publications

Light-driven synchronization of optogenetic clocks

Light-driven synchronization of optogenetic clocks

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Optically Controlled CRISPR-Cas9 and Cre Recombinase for Spatiotemporal Gene Editing: A Review

Contact Info

Product

Resources

About