Accurate phenotype-to-genotype mapping of high-diversity yeast libraries by heat-shock-electroporation (HEEL) DOI Creative Commons
Marcus Wäneskog,

Emma Elise Hoch-Schneider,

Shilpa Garg

и другие.

mBio, Год журнала: 2024, Номер unknown

Опубликована: Дек. 20, 2024

High-throughput DNA transformation techniques are invaluable when generating high-diversity mutant libraries, a cornerstone of successful protein engineering. However, efficiencies have direct correlation with the probability introducing multiple molecules into each cell, although reliable library screenings require cells that contain single unique genotype. Thus, methods yield high multiplicity transformations unsuitable for screenings. Here, we describe an innovative yeast method is both simple and highly efficient. Our dual heat-shock electroporation approach (HEEL) creates high-quality libraries by increasing fraction mono-transformed from 20% to over 70% all transformed cells, thus allowing near-perfect phenotype-to-genotype associations. HEEL also allows more than 10

Язык: Английский

Labels as a feature: Network homophily for systematically annotating human GPCR drug-target interactions DOI Creative Commons
Frederik G. Hansson, Niklas Gesmar Madsen, Lea G. Hansen

и другие.

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Май 3, 2025

Abstract Machine learning has revolutionized drug discovery by enabling the exploration of vast, uncharted chemical spaces essential for discovering novel patentable drugs. Despite critical role human G protein-coupled receptors in FDA-approved drugs, exhaustive in-distribution drug-target interaction testing across all pairs and known drugs is rare due to significant economic technical challenges. This often leaves off-target effects unexplored, which poses a considerable risk safety. In contrast traditional focus on out-of-distribution (drug discovery), we introduce neighborhood-to-prediction model termed Chemical Space Neural Networks that leverages network homophily training-free graph neural networks with labels as features. We show Networks’ ability make accurate predictions strongly correlates homophily. Thus, features increase machine model’s capacity enhance prediction accuracy, integrating labeled data during inference. validate these advancements high-throughput yeast biosensing system (3773 interactions, 539 compounds, 7 receptors) discover interactions expand general understanding how build reliable predictors guide experimental verification.

Язык: Английский

Процитировано

0
Heat pre-treatment reduces multiplicity of plasmid transformations in yeast during electroporation, without diminishing the transformation efficiency DOI
Marcus Wäneskog,

Emma Elise Hoch-Schneider,

Shilpa Garg

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Июль 3, 2024

Abstract High-throughput DNA transformation techniques are invaluable when creating high-diversity mutant libraries, and the success rate of any protein engineering endeavors is directly dependent on both size diversity library that to be screened. It also widely accepted in bacteria yeast there an inverse correlation between efficiency likelihood transforming multiple molecules into each cell. However, most successful high-throughput screening efforts require high quality i.e., libraries comprised cells with a clear phenotype-to-genotype relationship (one genotype/cell). Thus, methods multiplicity highly undesirable detrimental assays. Here we describe simple, robust, efficient plasmid methodology, using dual heat-shock electroporation approach (HEEL) generates more than 2 x 10 7 plasmid-transformed per reaction, while simultaneously increasing fraction mono-transformed from 20% 70% transformed population. By automated genotyping workflow coupled dual-barcoding approach, consisting SNP barcode (10N), can consistently identify enumerate unique genotypes within heterogeneous population merely through Sanger sequencing. We demonstrate here no longer need inversely correlated large methods. Significance With recent expansion artificial intelligence field synthetic biology has never been greater for high-quality data reliable measurements relationships. one major obstacle accurate computer-based models current abundance low-quality phenotypic originating numerous high-throughput, but low-resolution Rather quantity measurements, new studies should aim generate as possible. The HEEL methodology presented aims address this issue by minimizing problem multi-plasmid uptake during transformations, which leads creation cellular genotypes. enable going forward, could used construct better models.

Язык: Английский

Процитировано

1
Engineered yeast cells simulating CD19+ cancers to control CAR T cell activation DOI Creative Commons
Marcus Deichmann, Giovanni Schiesaro, Keerthana Ramanathan

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Окт. 26, 2023

Abstract Chimeric antigen receptor (CAR) T cells have become an established immunotherapy and show promising results for the treatment of hematological cancers. However, modulation surface levels targeted in cancer affects quality safety CAR cell therapy. Here we present S ynthetic C ellular A dvanced ignal dapter (SCASA) system, based on successful engineering yeast to simulate with tunable surface-antigen densities, as a tool controlled activation responses assessment density effects. Specifically, demonstrate I) controllable antigen-densities CD19 using G protein-coupled receptors (GPCRs), II) customizable system allowing choice signal input modular pathway precise fine-tuning output, III) synthetic cell-cell communication application CD19-displaying characterization designs, IV) more efficient robust activational control clinically-derived comparison NALM6 line. Based this yeast-based antigen-presenting envision how varying densities affect ultimately support development safer better personalized therapies.

Язык: Английский

Процитировано

3
Engineered yeast multicellularity via synthetic cell-cell adhesion and direct-contact signalling DOI Creative Commons
Fankang Meng, W. M. Shaw,

Yui Kei Keith Kam

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Июнь 24, 2024

Abstract Coordination of behaviour in multicellular systems is one the main ways that nature increases complexity biological function organisms and communities. While Saccharomyces cerevisiae yeast used extensively research biotechnology, it a unicellular organism capable only limited states. Here we expand possibilities for engineering behaviours by developing modular toolkits two key mechanisms seen multicellularity, contact-dependent signalling specific cell-to-cell adhesion. MARS ( M ating-peptide A nchored R esponse S ystem) toolkit based on surface-displayed fungal mating peptides G protein-coupled receptor (GPCR) which can mimic juxtacrine between yeasts. SATURN accharomyces dhesion T oolkit multicell U lar patte RN ing) surface displays adhesion-proteins pairs yeasts facilitates creation cell aggregation patterns. Together they be to create logic circuits, equivalent developmental programs lead differentiation local population. Using SATURN, further developed JUPITER JU xtacrine sensor P rotein-protein In TER action), genetic assaying protein-protein interactions culture, demonstrating this as tool select high affinity binders among population mutated nanobodies. Collectively, MARS, present valuable tools facilitate complex multicellularity with scope its biotechnological applications.

Язык: Английский

Процитировано

0
High‐throughput G protein‐coupled receptor‐based autocrine screening for secondary metabolite production in yeast DOI Creative Commons
Tatyana E. Saleski, Huadong Peng, Bettina Lengger

и другие.

Biotechnology and Bioengineering, Год журнала: 2024, Номер 121(10), С. 3283 - 3296

Опубликована: Июль 7, 2024

Abstract Biosensors are valuable tools in accelerating the test phase of design‐build‐test‐learn cycle cell factory development, as well bioprocess monitoring and control. G protein‐coupled receptor (GPCR)‐based biosensors enable cells to sense a wide array molecules environmental conditions specific manner. Due extracellular nature their sensing, GPCR‐based require compartmentalization distinct genotypes when screening production levels strain library ensure that detected originate exclusively from under assessment. Here, we explore integration sensing modalities into single Saccharomyces cerevisiae using three different methods: (1) cultivation microtiter plates, (2) spatial separation on agar (3) encapsulation water‐in‐oil‐in‐water double emulsion droplets, combined with analysis sorting via fluorescence‐activated machine. Employing tryptamine serotonin proof‐of‐concept target molecules, optimize biosensing demonstrate ability autocrine method enrich for high producers, showing enrichment serotonin‐producing over nonproducing strain. These findings illustrate workflow can be adapted range complex chemistry at throughput commercially available microfluidic systems.

Язык: Английский

Процитировано

0
Optimized single-cell gates for yeast display screening DOI
Xiaoli Pan, Matheus Oliveira de Souza,

Francisco M Figueiras

и другие.

Protein Engineering Design and Selection, Год журнала: 2024, Номер 38

Опубликована: Дек. 11, 2024

Abstract Yeast display is a widely used technology in antibody discovery and protein engineering. The cell size of yeast enables fluorescence-activated sorting (FACS) to precisely screen gene libraries, including for multi-parameter selection phenotypes. However, cells show broader distribution than mammalian that complicates single-cell gate determination FACS. In this report, we analyze several gating options detail present an optimized strategy select single via flow cytometry. These data reveal strategies support robust high-efficiency studies.

Язык: Английский

Процитировано

0
Accurate phenotype-to-genotype mapping of high-diversity yeast libraries by heat-shock-electroporation (HEEL) DOI Creative Commons
Marcus Wäneskog,

Emma Elise Hoch-Schneider,

Shilpa Garg

и другие.

mBio, Год журнала: 2024, Номер unknown

Опубликована: Дек. 20, 2024

High-throughput DNA transformation techniques are invaluable when generating high-diversity mutant libraries, a cornerstone of successful protein engineering. However, efficiencies have direct correlation with the probability introducing multiple molecules into each cell, although reliable library screenings require cells that contain single unique genotype. Thus, methods yield high multiplicity transformations unsuitable for screenings. Here, we describe an innovative yeast method is both simple and highly efficient. Our dual heat-shock electroporation approach (HEEL) creates high-quality libraries by increasing fraction mono-transformed from 20% to over 70% all transformed cells, thus allowing near-perfect phenotype-to-genotype associations. HEEL also allows more than 10

Язык: Английский

Процитировано

0