Biology subjects

Capin, J.

Publications and source records attributed to Capin, J..

7 recordsLinked to original sources

bioRxiv2025

A scalable architecture for tuning multistate differentiation ratios in synthetic microbial consortia

Establishing synthetic microbial consortia in competitive environments is often compromised by stochastic colonization bottlenecks, where founder effects lead to the unpredictable dominance of a single strain. Here, we overcome this challenge by engineering a differentiation abacus, a scalable, single-layer recombinase architecture that enables a single progenitor cell to differentiate into up to twelve distinct subpopulations. By arranging competitive excision sites in a linear array, we demonstrate that differentiation ratios can be programmed through rationally tuning recombination-site kinetics and inter-site spacing. This architecture allows the generation of strictly mutually exclusive phenotypes with tunable composition, scaling from simple two-state systems to complex multi-state ensembles without the need for multilayered regulation. Finally, we validate the systems utility in a mouse tumor model, showing that in situ differentiation establishes robust, homogeneous consortia that overcome the colonization variability associated with pre-assembled mixtures. This work provides a versatile and scalable framework for reliably controlling consortia composition for bioproduction, synthetic ecology, and engineered living therapies.

synthetic biology↗

bioRxiv2025

CF2H: a Cell-Free Two-Hybrid platform for rapid protein binder screening

Protein binders that detect, activate, inhibit or otherwise modulate their targets are pivotal for biomedical applications. With the increasing accuracy and accessibility of de novo protein design, faster and cheaper experimental screening methods would democratize and accelerate the identification of high-affinity binders. Here we present Cell-Free Two-Hybrid (CF2H), a rapid and sensitive method for detecting high-affinity protein-protein interactions (PPI) that does not require cloning, protein purification nor high-end laboratory equipment. CF2H uses a dimerization-activated DNA binding domain (DBD) fused to prey and bait proteins to trigger transcription upon protein-protein interaction. We demonstrate that CF2H enables the detection of interactions between various types of target and binder proteins such as single-domain antibodies, DARPins and de novo designed binders. We benchmark CF2H as a screening platform by validating previously reported binders for Mdm2 and discover high-affinity binders targeting the checkpoint inhibitor PD-L1 in less than 24 hours. Finally, we show that CF2H can be used to characterize small-molecules modulators of PPI and detect protein biomarkers, opening the door for a new class of cell-free biosensors.

synthetic biology↗

bioRxiv2024

Split minireporters facilitate monitoring of gene expression and peptide production in linear cell-free transcription-translation systems

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present minireporters based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that minireporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP, and matching with in vivo expression measurements. We also use minireporters as small fusion tags to measure TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent minireporter based on split-nanoluciferase. The [~]80% gene sequence length reduction afforded by minireporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of minireporters compared to full-length GFP is less likely to bias peptide solubility assays. We anticipate that minireporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

synthetic biology↗

bioRxiv2023

Engineering the ADDobody protein scaffold for generation of high-avidity ADDomer super-binders

Adenovirus-derived dodecamer (ADDomer) nanoparticles comprise 60 copies of Adenovirus penton base protein (PBP). ADDomer is thermostable, rendering the storage, transport and deployment of ADDomer-based therapeutics independent of a cold-chain. To expand the scope of ADDomer nanoparticles for new applications, we engineered ADDobodies. ADDobodies represent the crown domain of the PBP, genetically separated from its multimerization domain. We inserted heterologous sequences into hyper-variable loops in the crown domain. The resulting ADDobodies were expressed at high yields in Escherichia coli, are monomeric and maintain thermostability. We solved the X-ray structure of an ADDobody prototype validating our design. We demonstrated that ADDobodies can be used to select a specific binder against a target in in vitro selection experiments using ribosome display, with an enrichment factor of [~]104-fold in one selection round. We show that ADDobodies can be converted back into ADDomers by genetically reconnecting the selected ADDobody with the PBP multimerization domain from a different species, giving rise to a multivalent nanoparticle, called Chimera, confirmed by a 2.2 [A] structure determined by cryogenic electron microscopy (cryo-EM). Chimera comprises 60 binding sites, resulting in ultra-high, picomolar avidity to the target.

synthetic biology↗

bioRxiv2023

Antibodies generated in vitro and in vivo elucidate design of a thermostable ADDomer COVID-19 nasal nanoparticle vaccine

COVID-19 continues to damage populations, communities and economies worldwide. Vaccines have reduced COVID-19-related hospitalisations and deaths, primarily in developed countries. Persisting infection rates, and highly transmissible SARS-CoV-2 Variants of Concern (VOCs) causing repeat and breakthrough infections, underscore the ongoing need for new treatments to achieve a global solution. Based on ADDomer, a self-assembling protein nanoparticle scaffold, we created ADDoCoV, a thermostable COVID-19 candidate vaccine displaying multiple copies of a SARS-CoV-2 receptor binding motif (RBM)-derived epitope. In vitro generated neutralising nanobodies combined with molecular dynamics (MD) simulations and electron cryo-microscopy (cryo-EM) established authenticity and accessibility of the epitopes displayed. A Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Antibodies generated by immunising mice cross-reacted with VOCs including Delta and Omicron. Our study elucidates nasal administration of ADDomer-based nanoparticles for active and passive immunisation against SARS-CoV-2 and provides a blueprint for designing nanoparticle reagents to combat respiratory viral infections.

biochemistry↗

bioRxiv2020

Baculovirus-vectored precision delivery of large DNA cargoes in human genomes

Precise gene editing and genome engineering by CRISPR technology requires simultaneous delivery of multiple DNA-encoded components into living cells rapidly exceeding the cargo capacity of currently utilized viral vector systems. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck. We implement hybrid DNA techniques (MultiMate) for rapid and error-free assembly of currently up to 25 functional DNA modules in a single baculoviral vector enabling CRISPR-based genome engineering. Utilizing homology-independent targeted integration (HITI), we achieve up to 30% correct genome interventions in human cells, including precision docking of large DNA payloads in the ACTB locus. We demonstrate baculovirus-vectored delivery of prime-editing toolkits for seamless DNA search-and-replace interventions achieving, with a single vector, highly efficient cleavage-free trinucleotide insertion in the HEK3 locus without any detectable indels. Our approach thus unlocks a wide range of editing and engineering applications in human cell genomes.

synthetic biology↗

bioRxiv2020

Unexpected free fatty acid binding pocket in the cryo-EM structure of SARS-CoV-2 spike protein

COVID-19, caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms driving high infectivity, broad tissue tropism and severe pathology. Our cryo-EM structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains (RBDs) tightly and specifically bind the essential free fatty acid (FFA) linoleic acid (LA) in three composite binding pockets. The pocket also appears to be present in the highly pathogenic coronaviruses SARS-CoV and MERS-CoV. Lipid metabolome remodeling is a key feature of coronavirus infection, with LA at its core. LA metabolic pathways are central to inflammation, immune modulation and membrane fluidity. Our structure directly links LA and S, setting the stage for interventions targeting LA binding and metabolic remodeling by SARS-CoV-2. One Sentence SummaryA direct structural link between SARS-CoV-2 spike and linoleic acid, a key molecule in inflammation, immune modulation and membrane fluidity.

biochemistry↗