{"doi":"10.1101/2025.08.28.672974","title":"One-pot cloning and protein expression platform for genetic engineering","abstract":"In this work, we present a streamlined one-pot cloning and protein expression platform that integrates mutagenesis, plasmid assembly, and functional protein testing in a single reaction. By combining Golden Gate cloning with cell-free transcription-translation, we demonstrate efficient generation and screening of genetic variants without the need for intermediate purification or bacterial amplification. Using fluorescent proteins, luciferase enzymes, antibiotic-converting enzymes, and the violacein biosynthetic pathway, we validate the versatility of this approach for single- and multi-site mutagenesis, combinatorial variant libraries, metabolic pathway programming, and whole-plasmid assembly. By demonstrating compatibility with multiplexed reactions and multi-cistronic constructs, we establish this approach as a generalizable and automatable method for high-throughput cloning and protein engineering in synthetic biology.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":558927,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9495,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":809092,"name":"Judee Sharon","orcid":"0000-0001-5691-0407","position":1,"is_corresponding":false},{"id":809654,"name":"Brock Cash","orcid":null,"position":2,"is_corresponding":false},{"id":809653,"name":"Christopher Deich","orcid":null,"position":3,"is_corresponding":false},{"id":809094,"name":"Nathaniel J. Gaut","orcid":"0000-0002-7979-0759","position":4,"is_corresponding":false},{"id":1460155,"name":"J. Heili","orcid":null,"position":5,"is_corresponding":false},{"id":809095,"name":"Aaron E. Engelhart","orcid":"0000-0002-1849-7700","position":6,"is_corresponding":false},{"id":303913,"name":"Katarzyna P. Adamala","orcid":"0000-0003-1066-7207","position":7,"is_corresponding":false},{"id":648048,"name":"Wakana Sato","orcid":"0000-0002-5997-4438","position":0,"is_corresponding":true}],"reference_count":47,"raw_metadata":null,"created_at":"2026-07-19T02:55:30.312295Z","pmid":"40909620","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}