Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.02.24.707249

CyanOperon: an operon building expansion for the CyanoGate MoClo toolkit

Astbury, M. J.·Schiavon Osorio, A. A.·Victoria, A. J.·McCormick, A. J.
View original source ↗Export metadata ↓

Abstract

Operons are gene clusters controlled by a single promoter that enable coordinated translation from a single messenger RNA. Here we describe an expansion of the CyanoGate MoClo toolkit to assemble synthetic operons. The versatile CyanOperon system includes two Level 0 acceptor vectors for building interchangeable promoter-ribosome bind site (RBS) combinations and 15 Level 1 acceptor vectors for the hierarchical assembly and expression of up to six genes within a single operon. The system also allows for operon assembly into a self-replicating vector or for chromosomal integration by homologous recombination. To showcase CyanOperon, we assembled the violacein biosynthesis pathway as an operon and demonstrated violacein production in Escherichia coli. We then constructed a 20-part RBS library to examine how spacer length between the Shine-Dalgarno sequence and start codon affects translation in E. coli and the model cyanobacterium Synechocystis sp. PCC 6803. Lastly, we compared the expression of up to three operonic fluorescent markers following chromosomal integration or from a self-replicating vector in E. coli and Synechocystis sp. PCC 6803. The CyanOperon system is publicly available and can be readily integrated with other MoClo systems to accelerate the development of standardized operon assemblies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/707249v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@c9e14corg.highwire.dtl.DTLVardef@1e6566borg.highwire.dtl.DTLVardef@133c63forg.highwire.dtl.DTLVardef@1c5dff_HPS_FORMAT_FIGEXP M_FIG C_FIG

Source connections

Explore related subjects

synthetic biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Astbury, M. J., Schiavon Osorio, A. A., Victoria, A. J., McCormick, A. J.. 2026-02-24. CyanOperon: an operon building expansion for the CyanoGate MoClo toolkit. https://doi.org/10.64898/2026.02.24.707249

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

KEEP EXPLORING

Related preprints

bioRxiv2026

Gene expression noise is reduced in communicating synthetic cell populations

A major goal in bottom-up synthetic biology is the construction of multicellular synthetic systems capable of coordinated and robust collective behaviours. However, robustness is often limited by noise and variability arising from increased molecular complexity. Whilst communication has been implemented in synthetic multi-cellular systems, the ability for communication to suppress cell free gene expression variability in populations of synthetic cells remain unexplored. To address this, we encapsulated the Lux and Las quorum sensing gene circuits in lipid vesicles under cell-free conditions to test the effect of communication on reducing cell-free gene expression variability across the population. Our results show that communication, limiting expression resources, and membrane surface effects can reduce gene expression variability. Resource limited Gillespie simulations for transcription and translation show that communication-mediated coupling reduces population-level expression noise under constrained and excess resource conditions. Together, our work provides simple strategies to reduce gene expression variability and thereby improve robustness in synthetic multicellular systems, an important criteria for the future applications of synthetic cells.

synthetic biology↗
bioRxiv2026

Boolean Logic-responsive FRET Biosensors via Genetically Encoded Autonomous Compilation

Forster resonance energy transfer (FRET) is commonly used to monitor protein-protein interactions in situ. The high spatiotemporal resolution and facile implementation inside complex molecular environments have spearheaded FRET's widespread adoption in biosensing. Despite these advantages, current FRET biosensors are largely restricted to the detection of the presence/absence of individual inputs and are thus unable to sense several multiplexable inputs simultaneously within complex milieu of biological environments. In this work, we introduce a generalizable strategy to construct genetically encoded protein-based FRET biosensors capable of recognizing multiple inputs following Boolean logic-type (YES/OR/AND) operations. These topologically specified FRET sensors powerfully expand the input capacity in sensing protein-protein interactions while providing a user-programmable platform for monitoring heterogeneous biological activities both in vitro and in living cells.

synthetic biology↗
bioRxiv2026

AI-Guided Multi-Objective Engineering of Glucoamylase Enables Acidification-Free Starch Saccharification

Glucoamylase is essential for industrial starch saccharification, but the limited thermostability and near-neutral pH tolerance of fungal glucoamylases necessitate cooling and acidification of liquefied starch. Here, we developed an artificial intelligence-guided strategy to simultaneously improve the thermostability, pH tolerance, and catalytic activity of glucoamylase from Penicillium oxalicum (PoGA). Two property-specific machine-learning models, CASPE-T and CASPE-A, identified substitutions associated with thermostability and pH tolerance, respectively. Experimental screening identified beneficial substitutions in 11 of 21 CASPE-T and 12 of 22 CASPE-A candidates. Folding-energy-guided recombination integrated the two traits while maintaining structural compatibility. The optimal variant, PoGA T513E/Q305N, exhibited 2.21-fold higher specific activity than the wild type, with half-life extended from 22.3 to 57.9 min at 60 degrees C and from 16.6 to 64.7 min at pH 8.0. Molecular dynamics simulations attributed these improvements to reinforcement of high-occupancy hydrogen-bonding networks, suppression of conformational fluctuations in the linker and carbohydrate-binding module, enhanced long-range dynamic coordination, and preservation of a compact catalytic architecture. At 60 degrees C and pH 6.5 without acidification, PoGA T513E/Q305N produced 219.9 g/L glucose and achieved 89.1% starch conversion, 31.4% higher than the wild type. This work provides an efficient framework for multi-objective enzyme engineering and sustainable starch biorefining.

synthetic biology↗