bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2022.11.17.516977

Exploration of the autonomous replication region and its utilization for expression vectors in cyanobacteria.

Sakamaki, Y.·Maeda, K.·Nimura-Matsune, K.·Chibazakura, T.·Watanabe, S.
View original source ↗Export metadata ↓

Abstract

Due to their photosynthetic capabilities, cyanobacteria is expected to be an ecologically friendly host for the production of biomaterials. However, compared to other bacteria, there is little information of autonomous replication sequences, and tools for genetic engineering, especially expression vector systems, are limited. In this study, we established an effective screening method, namely AR-seq (Autonomous Replication sequencing), for finding autonomous replication regions in cyanobacteria and utilized the region for constructing expression vector. AR-seq using the genomic library of Synechocystis sp. PCC 6803 revealed that a certain region containing Rep-related protein (here named as Cyanobacterial Rep protein A2: CyRepA2) exhibits high autonomous replication activity in a heterologous host cyanobacterium, Synechococcus elongatus PCC 7942. The reporter assay using GFP showed that the expression vector pYS carrying CyRepA2 can be maintained in a wide range of multiple cyanobacterial species, not only S. 6803 and S. 7942, but also Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120. In S. 7942, the GFP expression in pYS-based system can be tightly regulated by IPTG, achieving 10-fold higher levels than that of chromosome-based system. Furthermore, pYS can be used together with conventional vector pEX, which was constructed from an endogenous plasmid in 5. 7942. The combination of pYS with other vectors is useful for genetic engineering, such as modifying metabolic pathways, and is expected to improve the performance of cyanobacteria as bioproduction chassis.

Explore related subjects

synthetic biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Sakamaki, Y., Maeda, K., Nimura-Matsune, K., Chibazakura, T., Watanabe, S.. 2022-11-18. Exploration of the autonomous replication region and its utilization for expression vectors in cyanobacteria.. https://doi.org/10.1101/2022.11.17.516977

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

KEEP EXPLORING

Related preprints

bioRxiv2026

Programmable interphyla communication for Staphylococcus aureus by transferability of homoserine lactone sensors between Bacillales bacteria

Staphylococcus aureus is a Gram-positive opportunistic pathogen of the order Bacillales and a frequent member of human-associated microbiomes. Despite its clinical relevance, engineering S. aureus and the communities it inhabits remains limited by a shortage of well-characterized genetic tools for precise control of gene expression and intercellular signaling. Biosensors for homoserine lactones (HSLs) are widely used in Gram-negative bacteria, as HSLs are small, passively diffusible signaling molecules for and their sensors are tightly regulated. Here, we show that synthetically designed HSL sensors using LuxR-family regulators are highly functional in S. aureus. Notably, synthetic designs previously built for the Gram-positive model Bacillus subtilis, a distantly related Bacillales, transferred functionally and establish repurposing the genetic toolbox as an effective strategy for importing these functions into S. aureus. Sensor performance correlated strongly between the two Bacillales, and activation in S. aureus reached 55-fold, exceeding the dynamic range of reported inducible systems. We further demonstrate tightly regulated, programmable interphyla communication between Gram-negative Escherichia coli and S. aureus via HSL signaling. These results provide a new toolset for engineering interbacterial communication and open a route to extending programmable HSL signaling to other tough-to-engineer bacteria.

synthetic biology↗
bioRxiv2026

DIALECT: DNA-Programmable Synthetic Cells Communicate with Mammalian Cells through Synthetic Receptors

Synthetic cells can regulate mammalian signalling through the controlled release of diffusible protein ligands, but coupling programmable synthetic-cell senders to orthogonal, engineered mammalian receptors remains challenging. Here we present DIALECT (DNA-Instructed Assembly of Ligands for Engineered Cellular Transduction), a platform in which proteinosome-based synthetic cells translate sequence-defined DNA inputs into coiled-coil signalling ligand outputs for engineered mammalian receptors. DNA-ligand conjugates are localized within semipermeable proteinosomes through DNA hybridization and released by toehold-mediated DNA strand displacement (TMSD). The released ligands diffuse to neighbouring mammalian cells and activate cognate CC-GEMS receptors, inducing JAK/STAT3 signalling and reporter activation, with a higher response in cognate than non-cognate receiver cells under activation conditions. We further implement distributed Boolean AND logic using two independently programmed proteinosome populations that respond to distinct DNA inputs and release complementary ligand components. Simultaneous addition of both inputs produces substantially higher reporter activity than the respective single-input conditions, consistent with extracellular assembly of an active receptor agonist. DIALECT thus establishes a modular interface linking DNA-based molecular computation in synthetic cells to orthogonal signalling pathways in mammalian cells, providing a foundation for hybrid artificial-biological communication networks.

synthetic biology↗
bioRxiv2026

Intracellular bio-synthesizing Gold Nanoparticles using bacterial systems and their applications in photothermal therapy (A green approach)

Background: Conventional cancer therapies are heavily constrained by systemic toxicity, off-target side effects, and poor deep-tissue penetration due to the highly heterogeneous tumor microenvironment (TME). Although gold nanoparticles (AuNPs) possess exceptional localized surface plasmon resonance (LSPR) properties ideal for photothermal therapy (PTT), their clinical translation is bottlenecked by inconsistent passive accumulation and chemical toxicity arising from conventional synthetic surfactants. Objective: This study developed a sustainable, biocompatible bio-hybrid platform utilizing the probiotic Escherichia coli Nissle 1917 (EcN) as an autonomous, tumor-homing living carrier integrated with AuNPs synthesized via an eco-friendly intracellular green chemistry approach. Methods: EcN cultures were prepared in Luria-Bertani (LB) medium and subsequently exposed to a 1 mM chloroauric acid (HAuCl4) metallic precursor. A facile, room-temperature green synthesis protocol was established by utilizing ascorbic acid (Vitamin C) as a non-toxic co-reductant to drive a seed-mediated crystallization onto intra-bacterial metallic seeds. The bio-hybrid system (EcN::AuNPs) was characterized via visual inspection, UV-Vis spectroscopy, and in vitro PTT assays under continuous-wave near-infrared (NIR) laser irradiation. Results: Visual confirmation showed an irreversible color shift from pale yellow to a deep dark-brownish hue, serving as a direct diagnostic indicator of successful Au3+ to Au0 reduction. UV-Vis spectroscopic analysis revealed a distinct, broadened surface plasmon resonance (SPR) band centered at 560nm, indicating the formation of stabilized, isotropic gold nanostructures (30-60nm) subjected to near-field electromagnetic coupling within the complex intracellular compartments (periplasm and cytoplasm). Biocompatibility assays demonstrated a significant collapse in cell viability from 10^8CFU/mL down to 10^3CFU/mL, driven by a combination of trivalent gold heavy-metal toxicity, mechanical disruption of the peptidoglycan wall from spatial confinement, and severe metabolic/oxidative stress. Nevertheless, the remaining surviving population maintained required active chemotactic capabilities, while the lysed bacterial matrix provided a natural protein capping layer that prevented nanoparticle aggregation. Under 5 minutes of continuous NIR laser irradiation, the EcN::AuNPs platform exhibited exceptional concentration-dependent hyperthermia kinetics, triggering a drastic thermal escalation from 20 C to an absolute peak of 75.4 C, vastly outperforming all baseline control groups (pure EcN, bare HAuCl4, and standalone ascorbic acid). Conclusion: The engineered EcN::AuNPs bio-hybrid platform represents a highly efficient and biosafe "bacteriobot" paradigm. By shifting the SPR footprint and leveraging the native bacterial cell envelope for colloidal stability, this system achieves an optimal equilibrium between active biological tumor targeting and rapid, localized photothermal ablation, offering a promising alternative for targeted oncological interventions.

synthetic biology↗