Biology subjects

Sarkisyan, K. S.

Publications and source records attributed to Sarkisyan, K. S..

9 recordsLinked to original sources

bioRxiv2025

Metabolically robust autoluminescent reporters

Self-sustained luminescence can be engineered in non-luminous organisms by integrating luciferin biosynthesis pathways with the hosts metabolism. When conditionally expressed, such pathways enable non-invasive monitoring of virtually any transcriptional event, providing a wide dynamic range and high resolution. However, because light emission depends on substrate availability within the cell, autoluminescence reporters are inherently non-quantitative: signal intensity reflects both gene expression and cellular metabolism. Here, we present an approach that disentangles the contributions of gene expression and metabolism, rendering autoluminescence signals robust against metabolic perturbations. By co-expressing an engineered reference luciferase variant that uses the same substrate but emits light at a different wavelength, and monitoring the resulting colour ratio, we dramatically reduce signal variation, enabling quantitative physiological imaging amid changing metabolic activity. Our strategy is applicable across known autoluminescence pathways from bacteria and fungi.

plant biology↗

bioRxiv2025

Bioluminescent sentinel plants enable autonomous diagnostics of viral infections

Plants engineered with synthetic genetic programs can transform how we monitor and manage the extension of crop pests and diseases. Here, we establish a bioluminescent platform in Nicotiana benthamiana for autonomous viral sensing based on the fungal bioluminescence pathway (FBP). We first demonstrate that recombinant viruses can deliver missing pathway components, enabling spatially resolved tracking of infection dynamics. Leveraging this starting point, we developed a dual-output sentinel circuit that uses a protease-responsive Bioluminescence Resonance Energy Transfer (BRET) module to report infection through a virus-triggered spectral shift in luminescence. In the absence of infection, plants emit a stable yellow glow indicating system integrity. Upon infection with potyviruses, cleavage of the BRET fusion by the virus-encoded NIa-Pro protease activates a distinct colour change detectable with low-cost imaging. This modular design is compatible with other pathogens carrying specific proteases and supports future multiplexing strategies. Our results highlight the potential of synthetic sentinel gene circuits as autonomous biosensors for precision crop protection.

synthetic biology↗

bioRxiv2025

Screening conditions and constructs for attempted genetic transformation of C. elegans by Agrobacterium

Manipulating gene expression within a model organism is important for reverse genetic experimentation, and while techniques to generate transgenic C. elegans are available, they are optimised for creating individual lines. The ability to create libraries of genetically modified animals using C. elegans as a model would make new types of experiments possible and would speed up studies of animal physiology. Here, we describe a range of constructs designed to establish a high-throughput method of C. elegans transformation mediated by gene transfer from Agrobacterium. We demonstrate that C. elegans are able to survive on Agrobacterium as a sole food source, and screen conditions for Agrobacterium-mediated transformation in this organism. While we do not achieve routine gene transfer from Agrobacterium to C. elegans, we suggest that this technique has potential following further optimization. The success of the approach would enable rapid and high-throughput transformation of C. elegans, providing an improvement on currently available methods. Here we provide details of optimization conditions tested, and a useful resource of T-binary constructs for use by the scientific community.

bioengineering↗

bioRxiv2025

Non-invasive imaging of salicylic and jasmonic acid activities in planta

Jasmonic and salicylic acids are the major hormones involved in plant response to pests and pathogens. Here, we engineered autoluminescent plants that report activity of these hormones with up to 53-fold contrast. Using consumer-grade cameras, we imaged reporter Arabidopsis thaliana and Nicotiana benthamiana plants throughout normal development, and in response to attacks of pests and pathogens.

plant biology↗

bioRxiv2024

A platform for lab management, note-keeping and automation

We report a lab management concept and its no-code implementation based on general-purpose database services, such as Airtable. The solution we describe allows for integrated management of samples, lab procedures, experimental notes and data within a single browser-based application, and supports custom automations. We believe that this system can benefit a wide scientific audience by offering communication-less retrieval of information, collaborative editing, unified sample labelling and data keeping style. A template database is available at airtable.com/universe/expPcKlB7VCHE6wVK/lab-management.

molecular biology↗

bioRxiv2021

Heterogeneity of the GFP fitness landscape and data-driven protein design

Studies of protein fitness landscapes reveal biophysical constraints guiding protein evolution and empower prediction of functional proteins. However, generalisation of these findings is limited due to scarceness of systematic data on fitness landscapes of proteins with a defined evolutionary relationship. We characterized the fitness peaks of four orthologous fluorescent proteins with a broad range of sequence divergence. While two of the four studied fitness peaks were sharp, the other two were considerably flatter, being almost entirely free of epistatic interactions. Counterintuitively, mutationally robust proteins, characterized by a flat fitness peak, were not optimal templates for machine-learning-driven protein design - instead, predictions were more accurate for fragile proteins with epistatic landscapes. Our work paves insights for practical application of fitness landscape heterogeneity in protein engineering.

synthetic biology↗

bioRxiv2019

A mutant of the phototoxic protein KillerRed that does not form DsRed-like chromophore

We report a mutant of the phototoxic fluorescent protein KillerRed protein with fluorescence in the green part of the spectrum. The mutant variant carries mutations I64L, D114G, and T115S and does not form a DsRed-like chromophore. The protein can be used as a template to create new genetically encodable photosensitizers that are spectrally different from KillerRed.

bioengineering↗

bioRxiv2019

Plants with self-sustained luminescence

In contrast to fluorescent proteins, light emission from luciferase reporters requires exogenous addition of a luciferin substrate. Bacterial bioluminescence has been the single exception, where an operon of five genes is sufficient to produce light autonomously. Although commonly used in prokaryotic hosts, toxicity of the aldehyde substrate has limited its use in eukaryotes1. Here we demonstrate autonomous luminescence in a multicellular eukaryotic organism by incorporating a recently discovered fungal bioluminescent system2 into tobacco plants. We monitored these light-emitting plants from germination to flowering, observing temporal and spatial patterns of luminescence across time scales from seconds to months. The dynamic patterns of luminescence reflected progression through developmental stages, circadian oscillations, transport, and response to injuries. As with other fluorescent and luminescent reporters, we anticipate that this system will be further engineered for varied purposes, especially where exogenous addition of substrate is undesirable.

plant biology↗

bioRxiv2019

Measuring C. elegans spatial foraging and food intake using bioluminescent bacteria

For most animals, feeding includes two behaviours: foraging to find a food patch and food intake once a patch is found. The nematode Caenorhabditis elegans is a useful model for studying the genetics of both behaviours. However, most methods of measuring feeding in worms quantify either foraging behaviour or food intake but not both. Imaging the depletion of fluorescently labelled bacteria provides information on both the distribution and amount of consumption, but even after patch exhaustion a prominent background signal remains, which complicates quantification. Here, we used a bioluminescent Escherichia coli strain to quantify C. elegans feeding. With light emission tightly coupled to active metabolism, only living bacteria are capable of bioluminescence so the signal is lost upon ingestion. We quantified the loss of bioluminescence using N2 reference worms and eat-2 mutants, and found a nearly 100-fold increase in signal-to-background ratio and lower background compared to loss of fluorescence. We also quantified feeding using aggregating npr-1 mutant worms. We found that groups of npr-1 mutants first clear bacteria from each other before foraging collectively for more food; similarly, during high density swarming, only worms at the migrating front are in contact with bacteria. These results demonstrate the usefulness of bioluminescent bacteria for quantifying feeding and suggest a hygiene hypothesis for the function of C. elegans aggregation and swarming.

animal behavior and cognition↗