Wäneskog Marcus, Hoch-Schneider Emma Elise, Garg Shilpa, Kronborg Cantalapiedra Christian, Schäfer Elena, Krogh Jensen Michael, Damgaard Jensen Emil
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.
mBio. 2025 Feb 5;16(2):e0319724. doi: 10.1128/mbio.03197-24. Epub 2024 Dec 20.
High-throughput DNA transformation techniques are invaluable when generating high-diversity mutant libraries, a cornerstone of successful protein engineering. However, transformation efficiencies have a direct correlation with the probability of introducing multiple DNA molecules into each cell, although reliable library screenings require cells that contain a single unique genotype. Thus, transformation methods that yield a high multiplicity of transformations are unsuitable for high-diversity library screenings. Here, we describe an innovative yeast library transformation method that is both simple and highly efficient. Our dual heat-shock and electroporation approach (HEEL) creates high-quality DNA libraries by increasing the fraction of mono-transformed yeast cells from 20% to over 70% of all transformed cells, thus allowing for near-perfect phenotype-to-genotype associations. HEEL also allows more than 10 yeast cells per reaction to be transformed with a circular plasmid molecule, which corresponds to an almost 100-fold improvement compared with current yeast transformation methods. To further refine our library screening approach, we integrated an automated yeast genotyping workflow with a dual-barcode design that employs both a single nucleotide polymorphism and a high-diversity region. This design allows for robust identification and quantification of unique genotypes within a heterogeneous population using standard Sanger sequencing. Our findings demonstrate that the longstanding trade-off between the size and quality of transformed yeast libraries can be overcome. By employing the HEEL method, large DNA libraries can be transformed into yeast with high-efficiency, while maintaining high library quality, essential for successful mutant screenings. This advancement holds significant promise for the fields of molecular biology and protein engineering.IMPORTANCEWith the recent expansion of artificial intelligence in the field of synthetic biology, there has never been a greater need for high-quality data and reliable measurements of phenotype-to-genotype relationships. However, one major obstacle to creating accurate computer-based models is the current abundance of low-quality phenotypic measurements originating from numerous high-throughput but low-resolution assays. Rather than increasing the quantity of measurements, new studies should aim to generate as accurate measurements as possible. The HEEL methodology presented here aims to address this issue by minimizing the problem of multi-plasmid uptake during high-throughput yeast DNA transformations, which leads to the creation of heterogeneous cellular genotypes. HEEL should enable highly accurate phenotype-to-genotype measurements going forward, which could be used to construct better computer-based models.
在生成高度多样化的突变文库(成功进行蛋白质工程的基石)时,高通量DNA转化技术非常宝贵。然而,转化效率与将多个DNA分子导入每个细胞的概率直接相关,尽管可靠的文库筛选需要包含单一独特基因型的细胞。因此,产生高转化复数的转化方法不适合用于高度多样化的文库筛选。在此,我们描述了一种既简单又高效的创新型酵母文库转化方法。我们的双热休克和电穿孔方法(HEEL)通过将单转化酵母细胞在所有转化细胞中的比例从20%提高到70%以上,创建了高质量的DNA文库,从而实现了近乎完美的表型与基因型关联。HEEL还允许每个反应中用环状质粒分子转化超过10个酵母细胞,与当前的酵母转化方法相比,这几乎提高了100倍。为了进一步优化我们的文库筛选方法,我们将自动化酵母基因分型工作流程与双条形码设计相结合,该设计同时采用了单核苷酸多态性和高多样性区域。这种设计允许使用标准的桑格测序在异质群体中对独特基因型进行可靠的识别和定量。我们的研究结果表明,长期存在的转化酵母文库大小与质量之间的权衡可以被克服。通过采用HEEL方法,可以将大型DNA文库高效地转化到酵母中,同时保持高文库质量,这对于成功的突变体筛选至关重要。这一进展为分子生物学和蛋白质工程领域带来了巨大的希望。
重要性
随着人工智能最近在合成生物学领域的扩展,对高质量数据以及表型与基因型关系的可靠测量的需求从未如此迫切。然而,创建基于计算机的准确模型的一个主要障碍是目前大量来自众多高通量但低分辨率测定的低质量表型测量。新的研究不应增加测量数量,而应旨在尽可能生成准确的测量结果。本文介绍的HEEL方法旨在通过最小化高通量酵母DNA转化过程中多质粒摄取的问题来解决这一问题,多质粒摄取会导致异质细胞基因型的产生。未来,HEEL应该能够实现高度准确的表型与基因型测量,可用于构建更好的基于计算机的模型。