DNA Shuffling

DNA shuffling is a multifunctional tool that can be applied to a wide range of industries and applications. Creative BioMart can provide customers with yeast DNA shuffling services. Our expert team can assist in every step of the process, from gene separation to optimization of DNA shuffling strategies, and ultimately to the selection of optimized mutants.

Technology Roadmap of Yeast DNA Shuffling

Figure 1. One-pot strategy for yeast in vivo DNA shuffling. (Martin-Diaz et al., 2018)

Design and optimization of DNA restructuring strategies.
Isolation of genes of interest.
Generate a random pool of GOI DNA fragments.
Using yeast as the host to assemble DNA fragments through homologous recombination.
Screening for mutants generated for the desired phenotype.
Iterative evolution to further improve the selected phenotype.

Generation of Yeast DNA Random Pools

Requirements for parental strains

We use at least 3 to 5 parental yeast strains with genomic DNA fragments ranging from 200bp to 5kb in length to create a random library of DNA fragments.

Methods for generating random fragment pools

We can use different PCR methods according to the needs of customers and the size of target fragments, such as overlapping extension PCR, error-prone PCR, site-directed mutation PCR, and large primer PCR.

Library size

The size of the random library we constructed can reach the order of magnitude of 10⁸ or even 10⁹ variants, including 3-5 parental strains.

However, customers please note that the size of the library is often limited by the transformation efficiency of yeast cells or the ability to select the desired phenotype during the screening process.

Service Process of Yeast DNA Shuffling

Steps	Description
Plan development	Our team will consult with you to understand your project requirements and objectives and provide you with the best possible DNA restructuring strategy.
Acquisition of GOI	We will use various techniques to isolate genes of interest, such as PCR, RT-PCR, or cDNA cloning.
Random DNA fragment generation	We will use the selected PCR strategy to generate a DNA fragment library and introduce mutations into the genes of interest.
DNA reorganization	We will use yeast as the host for homologous recombination and assemble the DNA fragment library into different mutant libraries.
Screening and selection of mutants	We will use various techniques, such as functional analysis, colorimetry or flow cytometry, to screen mutants with the desired phenotype or function.
Iterative evolution	We will use the mutants selected in the previous step for recombination and further evolution, creating additional diversity in the mutant library and improving the selected phenotype.
Data analysis and reporting	We will provide a comprehensive results report, including detailed analysis of the mutant, optimization process, and suggestions for further research.

Achievable Goals of Yeast DNA Shuffling Service

Through our yeast DNA restructuring service, we are expected to achieve the following goals for our customers.

Protein engineering

We can create new enzymes or modify existing enzymes for customers to improve their properties, such as activity, stability, and substrate specificity. This can be applied to a wide range of industries, including biotechnology, pharmaceuticals, and food production.

Metabolic engineering

We can optimize the metabolic pathways of yeast for customers to produce new compounds such as biofuels, drugs, and other high-value chemicals.

Drug discovery

We can generate new candidate drugs for customers by creating and screening large libraries of mutated proteins.

Directed evolution

We can evolve enzymes and metabolic pathways for specific applications for customers, or improve existing processes.

High throughput screening

We can combine yeast DNA shuffling with high-throughput screening methods for customers to quickly identify improved enzyme variants or metabolic pathways.

Creative BioMart's yeast DNA reorganization aims to provide customers with the most accurate, reliable and efficient methods of gene evolution and directed evolution. With our professional knowledge and state-of-the-art facilities, we can help you confidently achieve your research goals. Please feel free to contact us for more information.

Reference

Martin-Diaz, J., Paret, C., García-Ruiz, E., Molina-Espeja, P., & Alcalde, M. (2018). Shuffling the neutral drift of unspecific peroxygenase in saccharomyces cerevisiae. Applied and Environmental Microbiology, 84(15).