bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.05.18.725961

A Spectrum of Possibilities: A Systematic Evaluation of Fluorescent Proteins in Cyanobacteria

Hasenklever, D.·Boecker, J.·Grankin, A.·Sener, F.·Axmann, I. M.·Behle, A.
View original source ↗Export metadata ↓

Abstract

Fluorescent reporters cover a wide range of applications in both basic and applied research. Whether a study involves microscopic imaging to study (co)-localization of proteins, FRET, biosensing, or quantifying gene expression, fluorophores are attractive reporter candidates due to their relatively straightforward in vivo readout. For microbiological applications, a wide variety of fluorescent proteins with varying excitation and emission wavelengths, brightness levels, and maturation times are available. Careful consideration is required when selecting from this large suite of proteins, especially when choosing multiple fluorophores. This is further complicated in phototrophic organisms, which exhibit strong autofluorescence, especially towards the red part of the spectrum, effectively eliminating common candidates such as mCherry. In this study, the specific properties and performance of a selection of fluorescent proteins are systematically evaluated against the background of photosynthetic pigment-derived autofluorescence in the cyanobacterium Synechocystis sp. PCC 6803. Specific readouts of different combinations of fluorescent proteins are also analyzed using high-throughput methods, namely plate reader fluorescent scans and single-cell flow cytometry to quantify fluorescence. The ultimate goal is to assess each fluorescent protein with regard to: 1.) Its ability to be discerned from cyanobacterial autofluorescence. 2.) Its compatibility with other fluorophores in this context. 3.) Its overall suitability in cyanobacterial research. Several highly suitable fluorescent proteins for use in cyanobacteria are identified, including mTagBFP2, mNeonGreen and mScarlet-I and suitable combinations, covering nearly the whole spectrum of visible light. This study expands the knowledge and toolset for current and future researchers and uncovers a whole spectrum of possibilities for fluorescent protein selection in cyanobacterial cell biology.

Explore related subjects

synthetic biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Hasenklever, D., Boecker, J., Grankin, A., Sener, F., Axmann, I. M., Behle, A.. 2026-05-19. A Spectrum of Possibilities: A Systematic Evaluation of Fluorescent Proteins in Cyanobacteria. https://doi.org/10.64898/2026.05.18.725961

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Associative Learning and Memory Erasure in a Synthetic Multicellular System

Learning is a basic form of adaptation that organisms display from neural systems to single cells. In associative learning, the pairing of two stimuli lets one of them evoke the response to the other. Engineering this behavior in a living cell is difficult, because coincidence detection, memory, and conditional readout must be wired into a single regulatory network. Here we build a consortium of five E. coli strains that distributes the operations of associative learning and communicates through one diffusible quorum-sensing signal. The same signal acts as the memory trace. One strain detects the coincidence of the two inputs, a second maintains the memory through positive feedback in the shared medium, a third reads it out on the conditioned stimulus, a fourth reports the unconditioned response, and a fifth erases the memory on command. We show experimentally that the assembled consortium learns the association, since a stimulus that is silent before training evokes a strong response after pairing. Memory is retained through a medium exchange in which both stimuli are removed, and then the conditioned stimulus alone evokes the response. The reporter is not produced without its input. An erase command returns the response to the background. A population model of the consortium accounts for the sequence of responses and gives design rules for writing, retention, and erasure, and a trade-off in storing memory in a shared extracellular signal. Distributing the operations over strains makes each of them separately tunable and testable and provides a route to microbial communities that learn from their history.

synthetic biology↗
bioRxiv2026

Self-organized criticality in a mammalian RNA-interference circuit

Living systems have been argued to operate near critical points, but their relevance at the cellular level is poorly understood. Critical states allow for a high sensitivity to external inputs and maximal information transfer and display fluctuations at all scales. Critical points are very special, and require the fine tuning of a control parameter, which is hard to reconcile with the robustness of living cells. Self-organized criticality (SOC) proposes that feedback, rather than tuning, brings a system to its critical point. Here we show that RNA interference, in which messenger RNAs compete for shared silencing machinery and so queue for service, together with negative feedback gives rise to SOC in mammalian cells. In a synthetic circuit, a protein repressor whose mRNA competes with the mRNA of a fluorescent reporter that it represses senses the congestion of the queue, closing the feedback loop. Theory and stochastic simulations show that the loop holds the queue near its critical point despite changes in its parameters, and that expression becomes intermittent, with scale-free bursts. Measurements in human cell lines show the predicted power-law distributions of single-cell fluorescence. RNA interference could thus support engineered SOC in animal cells and may also contribute to SOC in natural systems.

synthetic biology↗
bioRxiv2026

An anaerobic cell-free protein synthesis formulation for the in vitro study of oxygen sensitive proteins

Anaerobic microbes harbor unique enzymes and metabolic pathways with potential biotechnological applications, yet these microbes remain difficult to study and engineer. Research is limited by the labor-intensive workflows required to maintain anoxic environments and the lack of genetic tools available for non-model anaerobes. Here, we present an anaerobic cell-free protein synthesis (CFPS) platform for the in vitro study of oxygen-sensitive enzymes. This study establishes two methods for conducting anaerobic CFPS, achieving an anoxic environment by (1) sparging with inert gas or (2) lyophilizing to remove oxygenated water. We show the functionality of our anaerobic CFPS formulation by producing oxygen-independent fluorescent protein reporters and a [4Fe-4S] cluster pyruvate:ferredoxin oxidoreductase (PFOR) with a specific activity of 5.2 {+/-} 0.3 x 10-4 U/{micro}L cell-free extract. This highly modular platform will allow study of diverse oxygen-sensitive proteins and requires only minor modifications to commonly used CFPS workflows.

synthetic biology↗