Biology subjects

Plesa, C.

Publications and source records attributed to Plesa, C..

9 recordsLinked to original sources

bioRxiv2026

Characterizing Sequence-Function Relationships in Chimeric DcuS/EnvZ Histidine Kinases at Scale

While bacterial sensor histidine kinases (SHKs) are widespread as natural molecular biosensors, tools for high-throughput characterization of SHK signaling phenotypes are limited, hindering wide scale implementation of bacterial-based sensing. Here, we developed a synthetic two-component signaling system that reports chimeric SHK signaling via a standardized fluorescence readout. With this synthetic system, we screened a library of chimeric DcuS/EnvZ SHKs to characterize sequence-function relationships within in the DcuS sensory and transmembrane domains. We quantified the effects of 1,173 mutations on signaling outputs in the presence of fumarate, a native DcuS ligand, as well as aspartate for which DcuS has minimal affinity for. We identified eleven positions across the DcuS domains which significantly alter aspartate responsiveness and selectivity and further observed a role for cytoplasmic N-terminal residues in determining signaling outputs. In future studies, this framework will expedite design of biosensors for novel ligands by enabling high-throughput screening of mutagenized libraries of natural SHKs.

synthetic biology↗

bioRxiv2026

DropSynth-Gold: Golden Gate Assembly in Emulsions Extends Multiplexed Gene Libraries to Greater Lengths

The ability to synthesize longer genes at scale remains a central challenge in multiplexed gene synthesis. DropSynth is a pooled gene synthesis platform that enables highly multiplexed, compartmentalized assembly from microarray-derived oligonucleotides, but current implementations rely on polymerase cycling assembly (PCA), which constrains fragment number, construct length, and assembly fidelity. Here we present DropSynth-Gold, an evolution of the DropSynth platform that replaces PCA with Golden Gate assembly (GGA) performed within emulsion droplets. This modification preserves the core workflow, including bead-linked oligonucleotide capture and pooled processing, while altering only the assembly chemistry and computational oligo design strategy. Emulsion-based Golden Gate assembly enables directional, multi-fragment ligation within isolated droplets, followed by recovery and amplification of full-length constructs. As a proof of concept, we constructed six 384-member libraries spanning increasing construct lengths and fragment counts, including designs from 5x300-mer fragments to 12x350-mer architectures ([~]3 kb). DropSynth-Gold reliably assembled full-length constructs across all libraries. A direct comparison of a shared 5x300-mer library demonstrated comparable recovery and fidelity to PCA-based DropSynth, indicating that Golden Gate assembly can replace PCA without compromising assembly performance. These gains were achieved without increasing cost, workflow complexity, or turnaround time, expanding the accessible design space for multiplexed gene synthesis.

synthetic biology↗

bioRxiv2026

High Diversity Gene Libraries Facilitate Machine Learning Guided Exploration of Fluorescent Protein Sequence Space

While protein language models (PLMs) have shown great promise for protein design, their performance is fundamentally constrained by the diversity and completeness of available training data. In particular, PLMs often struggle to extrapolate to sequences that fall outside the distribution spanned by their training sets, limiting their ability to discover proteins in sparsely sampled regions of sequence space. Here we test the hypothesis that experimentally expanding training diversity can convert extrapolation into interpolation and thereby enable discovery of functional sequences beyond natural protein manifolds. Using large-scale gene synthesis and DNA shuffling, we generate libraries that span a broad region of fluorescent protein sequence space and create chimeric variants that bridge between distant homologs. Functional screening for blue fluorescence yields thousands of active variants distributed across diverse sequence lineages. Fine-tuning ProtGPT2 on this expanded dataset enables generation of diverse fluorescent proteins, including designs that extend beyond the regions occupied by known natural sequences while retaining function. This work illustrates how synthetic approaches can help address key limitations in machine learning-guided protein design, especially for small or sparsely populated protein families, by actively creating novel sequences across unexplored but functional regions of sequence space.

synthetic biology↗

bioRxiv2025

BAR-CAT: Targeted Recovery of Synthetic Genes via Barcode-Directed CRISPR-dCas9 Enrichment

Modern gene-synthesis platforms let us probe protein function and genome biology at unprecedented scale. Yet in large, diverse gene libraries the proportion of error-free constructs decreases with length due to the propagation of oligo synthesis errors. To rescue these rare, error-free molecules we developed BAR-CAT (Barcode-Assisted Retrieval CRISPR-Activated Targeting), an in-vitro enrichment method that couples unique PAM-adjacent 20-nt barcodes to each library member and uses multiplexed dCas9-sgRNA complexes to fish out the barcodes corresponding to perfect assemblies. After a single 15-min reaction and optimized wash regime (BAR-CAT v1.0), three low-abundance targets in a 300, 000-member test library were enriched 600-fold, greatly reducing downstream requirements. When applied to 384x and 1, 536x member DropSynth gene libraries, BAR-CAT retrieved up to 122-fold enrichment for 12 targets and revealed practical limits imposed by sgRNA competition and library complexity, which now guide ongoing protocol scaling. By eliminating laborious clone-by-clone validation and working directly on plasmid libraries, BAR-CAT provides a versatile platform for recovering perfect synthetic genes, subsetting large libraries, and ultimately lowering the cost of functional genomics at scale.

synthetic biology↗

bioRxiv2025

Optimizing in-vitro Transcribed CRISPR-Cas9 single-guide RNA Libraries for Improved Uniformity and Affordability

We describe a scalable and cost-effective sgRNA synthesis workflow that reduces costs by over 70% through the use of large pools of microarray-derived oligos encoding unique sgRNA spacers. These sub-pool oligos are assembled into full-length dsDNA templates via Golden Gate Assembly before in vitro transcription with T7 RNA polymerase. RNA-seq analysis reveals severe biases in spacer representation, with some spacers being highly overrepresented while others are completely absent. Consistent with previous studies, we identify guanine-rich sequences within the first four nucleotides of the spacer, immediately downstream of the T7 promoter, as the primary driver of this bias. To address this issue, we introduced a guanine tetramer upstream of all spacers, which reduced bias by an average of 19% in sgRNA libraries containing 389 spacers. However, this modification also increased the presence of high-molecular-weight RNA species after transcription. We also tested two alternative bias-reduction strategies: compartmentalizing spacers within emulsions and optimizing DNA input and reaction volumes. Both methods independently reduced bias in 2,626-plex sgRNA libraries, though to a lesser extent than the guanine tetramer approach. These advancements enhance both the affordability and uniformity of sgRNA libraries, with broad implications for improving CRISPR-Cas9 screens and optimizing guide RNA design for other CRISPR and nuclease systems.

molecular biology↗

bioRxiv2025

Exploring Antibiotic Resistance in Diverse Homologs of the Dihydrofolate Reductase Protein Family through Broad Mutational Scanning

Current antibiotic resistance studies often focus on individual protein variants, neglecting broader protein family dynamics. Dihydrofolate reductase (DHFR), a key antibiotic target, has been extensively studied using deep mutational scanning, yet resistance mechanisms across this diverse protein family remain poorly understood. Using DropSynth, a scalable gene synthesis platform, we designed a library of 1,536 synthetic DHFR homologs representing 778 species of bacteria, archaea, and viruses, including clinically relevant pathogens. A multiplexed in vivo assay tested their ability to restore metabolic function and confer trimethoprim resistance in an E. coli {Delta}folA strain. Over half of the synthetic homologs rescued the phenotype without supplementation, and mutants with up to five amino acid substitutions increased the rescue rate to 90%, highlighting DHFRs evolutionary resilience. Broad Mutational Scanning (BMS) of homologs and 100,000 mutants provided critical insights into DHFRs fitness landscape and resistance pathways, representing the most extensive analysis of homolog complementation and inhibitor tolerance to date and advancing our understanding of antibiotic resistance mechanisms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/634126v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@165b786org.highwire.dtl.DTLVardef@f3cc77org.highwire.dtl.DTLVardef@1f4b2c3org.highwire.dtl.DTLVardef@432a6_HPS_FORMAT_FIGEXP M_FIG C_FIG TeaserDropSynth technology enables scalable and cost-effective exploration of antibiotic resistance across the DHFR protein family.

synthetic biology↗

bioRxiv2023

Degenerate DropSynth for Simultaneous Assembly of Diverse Gene Libraries and Local Designed Mutants

Protein engineering efforts often involve the creation of hybrid or chimeric proteins, where functionality critically hinges on the precise design of linkers and fusion points. Traditional methods have been constrained by a focus on single genes or the random selection of fusion points. Here we introduce an approach which enables the creation of large gene libraries where each library comprises a multitude of diverse, specifically designed genes, each with a corresponding set of programmatically designed fusion points or linkers. When combined with multiplex functional assays, these libraries facilitate the derivation of generalized engineering principles applicable across whole protein families or domain types. Degenerate DropSynth is a multiplex gene synthesis technique which allows for the assembly of up to eight distinct variants for each of the 1,536 designed parent genes in a single reaction. We assemble chimeric sensor histidine kinases and demonstrate the assembly of genes up to 1 kbp in length with an 8% rate of perfect assemblies per gene. Our findings indicate that incorporating an increased number of variants in droplets containing barcoded beads does not significantly affect the rate of perfect assemblies. However, maintaining a consistent level of degeneracy across the library is important to ensure good coverage and reduce inequality. The results suggest the potential for scaling this process to assemble at least 8,000 distinct variants in a single reaction. Degenerate DropSynth enables the systematic exploration of protein families through large-scale, programmable assembly of chimeric proteins, moving beyond the limitations of individual protein studies.

synthetic biology↗

bioRxiv2019

DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions

Multiplexed assays allow functional testing of large synthetic libraries of genetic elements, but are limited by the designability, length, fidelity and scale of the input DNA. Here we improve DropSynth, a low-cost, multiplexed method which builds gene libraries by compartmentalizing and assembling microarray-derived oligos in vortexed emulsions. By optimizing enzyme choice, adding enzymatic error correction, and increasing scale, we show that DropSynth can build thousands of gene-length fragments at >20% fidelity.

synthetic biology↗

bioRxiv2017

Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

Next-generation sequencing has engendered an expanding suite of functional assays that can test sequence-function relationships at unprecedented scales in pooled formats (multiplex). Such assays are currently constrained by the short length of oligonucleotide (oligo) pools, which limit potential applications. Here we report a simple, low-cost, and scalable method called DropSynth that assembles gene libraries from oligo pools for use in multiplexed functional assays. DropSynth utilizes a library of barcoded beads to isolate and concentrate oligos needed for a genes synthesis in a pooled format. These bead-bound oligos are then emulsified, processed, and assembled into genes within the emulsion droplets. We synthesized ~1000 phylogenetically diverse orthologs of phosphopantetheine adenylyltransferase (PPAT) and tested their fitness in a multiplexed functional assay. While the majority of orthologs complement, those that do not are broadly distributed across the phylogenetic tree. Synthetic errors in our assemblies allow us to explore local landscapes around the designed orthologs revealing constrained mutations for complementing orthologs as well as gain-of-function mutations for low-fitness orthologs. This broad mutational scanning approach is complementary to deep mutational scanning and helps us understand proteins by probing evolutionarily divergent sequences that share function.

biochemistry↗