Biology subjects

Mishin, A. S.

Publications and source records attributed to Mishin, A. S..

8 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

Developing a versatile technology for agroinfiltration in multiple plants

Agrobacterium-mediated transient transformation methods are widely used in plant science and molecular pharming. However, plant immune response and post-transcriptional gene silencing (PTGS) can compromise gene expression, limiting the adaptability of these methods across plant species. The lack of a robust reporter system further hinders advancements in agroinfiltration technology. To address these challenges, we developed a fungal bioluminescence pathway (FBP)-based reporter system that utilizes caffeic acid to generate self-sustained luminescence. By incorporating NahG and P19, our FBP assay enhances transient transformation efficiency in over 20 plant families, including major crops, vegetables, ornamental plants, and trees. Furthermore, we demonstrate its utility for assessing protein localization, interactions, and transcriptional regulation in multiple plant species, thereby broadening our understanding of gene function throughout the plant kingdom.

plant biology↗

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↗

bioRxiv2023

The two key substitutions in the chromophore environment of mKate2 to produce an enhanced FusionRed-like red fluorescent protein

Red fluorescent proteins (RFPs) are often probes of choice for living tissue microscopy and whole-body imaging. When choosing a specific RFP variant, the priority may be focused on fluorescence brightness, maturation rate, monomericity, excitation/emission wavelengths, and low toxicity, which are rarely combined in optimal way in a single protein. If the additional requirements such as prolonged fluorescence lifetime and/or blinking ability are applied, the available probes repertoire could become surprisingly narrow. Since the whole diversity of the conventional single-component RFPs belongs to just a few phylogenetic lines (with DsRed-, eqFP578- and eqFP611-derived being the major ones), it is not unexpected that their advantageous properties are split between close homologs. In such cases, a systematic mutagenetic analysis focused on variant-specific amino acid residues can shed light on the origins of sibling RFPs distinctness and might be beneficial for consolidation of their strengths in new RFP variants. For instance, the protein FusionRed, although being efficient in the fluorescence labeling due its good monomericity and low cytotoxicity, has undergone a considerable loss in fluorescence brightness/lifetime compared to the parental mKate2. In this contribution, we describe a fast-maturing monomeric RFP designed semi-rationally based on the mKate2 and FusionRed templates, outperforming both its parents in molecular brightness, having extended fluorescence lifetime, and showing spontaneous blinking pattern promising for nanoscopy use.

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↗

bioRxiv2020

NanoFAST: Structure-based design of a small fluorogen-activating protein with only 98 amino acids

One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We shown that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.

biochemistry↗

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↗