关键词: 合成生物学, 单细胞拉曼光谱技术, 细胞工厂, 单细胞表型识别, 高通量分选

Abstract:

The advancement of synthetic biology depends on the breakthroughs in the "design", "build", "test", and "learn" (DBTL) stages. With the rapid methodological innovations in genome sequencing, editing, synthesis, and artificial intelligence, the industry has made remarkable progress in designing and building mutants and even artificial cell factories. However, the "unmanageable complexity of large systems" remains one of the ongoing challenges for synthetic biology. As the cell mutant libraries get larger, the process of testing becomes more tedious and even impossible. Hence, there is an urgent need to develop high throughput sorting platforms. SCRS (single-cell Raman spectrum)technology can identify panoramic information at the single-cell level in a non-labeled state, distinguishing complex functional phenotypes. It has advantages such as being fast, low-cost, and capable of being coupled with downstream omics research, thus making it a novel technology for single-cell phenotype identification. At present, based on the powerful ability of SCRS in phenotyping, a series of synthetic phenotypic testing and cell sorting equipment have been developed and a wide range of application demonstrations have been carried out, which demonstrates its enormous potential in accelerating the phenotypic testing and cell sorting in synthetic biology. In this review, we selected the self-developed Raman-activated cell sorting coupled sequencing system (RACS-Seq), single-cell microfluidic droplet sorting system (EasySort), and high-throughput flow cytometry Raman-activated cell sorting (FlowRACS) as the typical equipment, by introducing their technical principles, technical iterations and characteristic application cases. Despite the advances in SCRS-based synthetic phenotypic testing and cell sorting equipment, there are still challenges to overcome. For example, there is a need for improving automation and standardizing protocols to ensure reproducibility and scalability. The development of more powerful artificial intelligence algorithms for dissecting SCRS is also required to exploit more complicated phenometypes. Finally, the throughput and sensitivity still need to be improved significantly. In conclusion, in spite of some disadvantages, the SCRS-based equipment has shown great promise in accelerating the phenotypic testing and cell sorting in synthetic biology.

Key words: synthetic biology, Raman spectroscopy, cell factory, single-cell phenotyping, high-throughput sorting