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   https://doi.org/10.1038/nchem.2927  

   Yang, Hao; Swartz, Alan M.; Park, Hyun June; Srivastava, Poonam; Ellis-Guardiola, Ken; Upp, David M.; Lee, Gihoon; Belsare, Ketaki; Gu, Yifan; Zhang, Chen; et al (January 2018, Nature Chemistry)
2. Laboratory Culture and Mutagenesis of Amphioxus (Branchiostoma floridae).

   https://doi.org/10.1007/978-1-0716-0974-3_1  

   Holland LZ; Li G. (January 2021, Methods in molecular biology)

   Cephalochordates (amphioxus) are invertebrate chordates closely related to vertebrates. As they are evolving very slowly, they are proving to be very appropriate for developmental genetics studies aimed at understanding how vertebrates evolved from their invertebrate ancestors. To date, techniques for gene knockdown and overexpression have been developed, but methods for continuous breeding cultures and generating germline mutants have been developed only recently. Here we describe methods for continuous laboratory breeding cultures of the cephalochordate Branchiostoma floridae and the TALEN and Tol2 methods for mutagenesis. Included are strategies for analyzing the mutants and raising successive generations to obtain homozygotes. These methods should be applicable to any warm water species of cephalochordates with a relatively short generation time of 3-4 months and a life span of 3 years or more. 

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3. Standardizing cassette‐based deep mutagenesis by Golden Gate assembly

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   Daffern, Nicolas; Francino‐Urdaniz, Irene_M; Baumer, Zachary_T; Whitehead, Timothy_A (September 2023, Biotechnology and Bioengineering)

   Abstract Protocols for the construction of large, deeply mutagenized protein encoding libraries via Golden Gate assembly of synthetic DNA cassettes employ disparate, system‐specific methodology. Here we present a standardized Golden Gate method for building user‐defined libraries. We demonstrate that a 25 μL reaction, using 40 fmol of input DNA, can generate a library on the order of 1 × 10⁶members and that reaction volume or input DNA concentration can be scaled up with no losses in transformation efficiency. Such libraries can be constructed from dsDNA cassettes generated either by degenerate oligonucleotides or oligo pools. We demonstrate its real‐world effectiveness by building custom, user‐defined libraries on the order of 10⁴–10⁷unique protein encoding variants for two orthogonal protein engineering systems. We include a detailed protocol and provide several general‐use destination vectors. 

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4. Large-scale mapping and mutagenesis of human transcriptional effector domains

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5. Protocol for the generation of Symbiodiniaceae mutants using UV mutagenesis

   https://doi.org/10.1016/j.xpro.2023.102627  

   Russo, Joseph A; Xiang, Tingting; Jinkerson, Robert E (December 2023, STAR Protocols)

   Genetic approaches are limited in the dinoflagellate family, Symbiodiniaceae, causing a bottleneck in the discovery of useful mutants toward the goal of preventing future coral bleaching events. In this protocol, we demonstrate the application of UV exposure, coupled with downstream phenotypic screening and mutant isolation, to form a UV mutagenesis pipeline. This pipeline provides an avenue to generate Symbiodiniaceae mutants to help link genotype to phenotype, as well as address previously unanswered questions surrounding relationships with host organisms, like coral. 

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