ArticleACS synthetic biology2017
Rapid and Programmable Protein Mutagenesis Using Plasmid Recombineering.
Article in ACS synthetic biology, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed.
- MOSAIC: A Highly Efficient, One-Step Recombineering Approach to Plasmid Editing and Diversification.ACS synthetic biology · 2025Article
- Exploring the deletion landscape ofbioRxiv : the preprint server for biology · 2025Article
- CRISPR/Cas system-guided plasmid mutagenesis without sequence restriction.Fundamental research · 2025Article
- ORBIT for E. coli: kilobase-scale oligonucleotide recombineering at high throughput and high efficiency.Nucleic acids research · 2024Article
- Reengineering of a flavin-binding fluorescent protein using ProteinMPNN.Protein science : a publication of the Protein Society · 2024Article
- Deep mutational scanning reveals the molecular determinants of RNA polymerase-mediated adaptation and tradeoffs.Nature communications · 2023Article
- Oligo Pools as an Affordable Source of Synthetic DNA for Cost-Effective Library Construction in Protein- and Metabolic Pathway Engineering.Chembiochem : a European journal of chemical biology · 2022Review
- Engineering ligand-specific biosensors for aromatic amino acids and neurochemicals.Cell systems · 2022Article
- SpeedyGenesXL: an Automated, High-Throughput Platform for the Preparation of Bespoke Ultralarge Variant Libraries for Directed Evolution.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Extreme dependence of Chloroflexus aggregans LOV domain thermo- and photostability on the bound flavin species.Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology · 2021Article
- Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules.Nature communications · 2021Article
- Recombineering and MAGE.Nature reviews. Methods primers · 2021Article
- CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli.Molecular systems biology · 2020Article
- Beyond the Green Fluorescent Protein: Biomolecular Reporters for Anaerobic and Deep-Tissue Imaging.Bioconjugate chemistry · 2020Review
- Modern methods for laboratory diversification of biomolecules.Current opinion in chemical biology · 2017Review
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Authors and funding
3 authors.
Funding
Abstract
Comprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. There is thus a need for robust and unbiased molecular biological approaches for the construction of the requisite comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space. Using the fluorescent protein iLOV as a model target, we build a complete mutagenesis library and find it to be specific and comprehensive, detecting 99.8% of our intended mutations. We then develop a thermostability screen and utilize our comprehensive mutation data to rapidly construct a targeted and multiplexed library that identifies significantly improved variants, thus demonstrating rapid protein engineering in a simple protocol.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.