Back to Basics: Creating Genetic Diversity

Tee, Kang Lan; Wong, Tuck Seng

doi:10.1007/978-3-319-50413-1_8

Cited by 8 publications

(7 citation statements)

References 66 publications

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“…We used QuikChange-HT mutagenesis, an oligo array-based strategy that provides rationally chosen mutants and offers the following advantages: 1) a simple experimental procedure, thus increasing throughput; 2) the ability to selectively amplify distinct mutagenic oligo subsets from the same array, permitting the use of the same source array for different experiments and targets; and 3) the ability to implement a design strategy that disfavors the production of double and higher-order mutants during pooled mutagenesis reactions, reducing otherwise-costly downstream sampling of clones to identify the desired single mutants. 20 Other previously reported methods can generate large libraries of 1C) divides the ORF from the protein of interest into mutagenic sublibrary regions, with sublibrary oligo length constrained by DNA synthesis limits. Each window contains unique forward and reverse priming sites (dark shading, here ~25 nt each) at the 5'-and 3'-termini surrounding a mutational region (light shading, here ~150 nt).…”

Section: Design Of Tiling Orf Windows Allows Selective Mutagenesis From Oligo Arraysmentioning

confidence: 99%

uPIC–M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry

Appel

Longwell

Morri

et al. 2021

Preprint

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New high-throughput biochemistry techniques complement selection-based approaches and provide quantitative kinetic and thermodynamic data for thousands of protein variants in parallel. With these advances, library generation rather than data collection has become rate limiting. Unlike pooled selection approaches, high-throughput biochemistry requires mutant libraries in which individual sequences are rationally designed, efficiently recovered, sequence-validated, and separated from one another, but current strategies are unable to produce these libraries at the needed scale and specificity at reasonable cost. Here, we present a scalable, rapid, and inexpensive approach for creating User-designed Physically Isolated Clonal–Mutant (uPIC–M) libraries that utilizes recent advances in oligo synthesis, high-throughput sample preparation, and next-generation sequencing. To demonstrate uPIC–M, we created a scanning mutant library of SpAP, a 541 amino acid alkaline phosphatase, and recovered 94% of desired mutants in a single iteration. uPIC–M uses commonly available equipment and freely downloadable custom software and can produce a 5000 mutant library at 1/3 the cost and 1/5 the time of traditional techniques.

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Section: Design Of Tiling Orf Windows Allows Selective Mutagenesis From Oligo Arraysmentioning

confidence: 99%

uPIC–M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry

Appel

Longwell

Morri

et al. 2021

Preprint

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“…Plant biosynthetic pathways can require up to 20-30 enzymes for the synthesis of phytochemicals in heterologous hosts. S. cerevisiae features efficient homologous recombination, and a number of cloning techniques have been developed based on this feature, 119 which makes the reconstruction of pathways of such complexity feasible. Recently, yeast recombination has been adopted to assemble complex pathways in yeast on a plasmid or on the chromosome, an approach referred to as "DNA assembler".…”

Section: Genetic Modication Strategies For Multi-gene Pathway Assemblymentioning

confidence: 99%

Synthetic biology strategies toward heterologous phytochemical production

Kotopka

Smolke

2018

Nat. Prod. Rep.

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Covering: 2006 to 2018 Phytochemicals are important sources for the discovery and development of agricultural and pharmaceutical compounds, such as pesticides and medicines. However, these compounds are typically present in low abundance in nature, and the biosynthetic pathways for most phytochemicals are not fully elucidated. Heterologous production of phytochemicals in plant, bacterial, and yeast hosts has been pursued as a potential approach to address sourcing issues associated with many valuable phytochemicals, and more recently has been utilized as a tool to aid in the elucidation of plant biosynthetic pathways. Due to the structural complexity of certain phytochemicals and the associated biosynthetic pathways, reconstitution of plant pathways in heterologous hosts can encounter numerous challenges. Synthetic biology approaches have been developed to address these challenges in areas such as precise control over heterologous gene expression, improving functional expression of heterologous enzymes, and modifying central metabolism to increase the supply of precursor compounds into the pathway. These strategies have been applied to advance plant pathway reconstitution and phytochemical production in a wide variety of heterologous hosts. Here, we review synthetic biology strategies that have been recently applied to advance complex phytochemical production in heterologous hosts.

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“…Over the past 30 years, numerous techniques and commercial kits have been developed to assemble novel genes and systematically mutate genes at random or specific sites (Mate et al, 2016; Neylon, 2004; Qu et al, 2020; Tee and Wong, 2017). Site Saturation Mutagenesis (SSM) is a widely used technique for construction of a gene library in which a single residue is randomly substituted with all possible amino acids at a single or at multiple positions (Arnold et al, 2003; Miyazaki and Arnold, 1999; Siloto and Weselake, 2012).…”

Section: Introductionmentioning

confidence: 99%

Mutation Maker, An Open Source Oligo Design Platform for Protein Engineering

Hiraga

Mejzlík²,

Marcisin³

et al. 2020

Preprint

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Founded on principles of natural selection, directed evolution techniques harness high-throughput gene diversification methodologies to generate proteins with optimized properties for applications in research, therapeutic and industrial processes. However, design of gene libraries is typically hampered by scale and complexity, necessitating development of advanced automation and optimization tools that can improve efficiency and accuracy. At present, automated library design tools are obsolete, functionally limited or not freely available for commercial use. To address these issues, we developed Mutation Maker, an open source mutagenic oligo design software for large-scale protein engineering experiments. Mutation Maker is not only specifically tailored to multi-site random and directed mutagenesis protocols, but also pioneers bespoke mutagenic oligo design for de novo gene synthesis workflows. Enabled by a novel bundle of orchestrated heuristics, optimization, constraint-satisfaction and backtracking algorithms, Mutation Maker offers a versatile toolbox for gene diversification design at industrial scale. Supported by in-silico simulations and compelling experimental validation data, Mutation Maker oligos produce diverse gene libraries at high success rates irrespective of genes or vectors used. Finally, Mutation Maker was created as an extensible platform on the notion that directed evolution techniques will continue to evolve and revolutionize current and future-oriented applications.

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Back to Basics: Creating Genetic Diversity

Cited by 8 publications

References 66 publications

uPIC–M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry

uPIC–M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry

Synthetic biology strategies toward heterologous phytochemical production

Mutation Maker, An Open Source Oligo Design Platform for Protein Engineering

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