Department of Integrated Applied Life Science, Integrated Graduate School of Medicine, Engineering, and Agricultural Sciences, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan.
Department of Integrated Applied Life Science, Integrated Graduate School of Medicine, Engineering, and Agricultural Sciences, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan.
J Microbiol Methods. 2024 Jan;216:106859. doi: 10.1016/j.mimet.2023.106859. Epub 2023 Nov 22.
Botryococcus braunii, a colonial alga, is known for notably slow growth; however, the growth rate and hydrocarbon productivity are expected to be improved using genetic modification techniques. Nevertheless, B. braunii has a hydrocarbon-rich extracellular matrix (ECM), and the ECM is a major barrier to DNA transformation. To analyse and utilize genetically modified B. braunii, it is essential to regenerate genetically homogeneous colonies derived from single cells. In this study, we developed a novel, simple method for harvesting viable single cells of B. braunii by centrifugation of the culture and subsequent filtration alone. The harvest of single cells was made possible by culturing B. braunii colonies in AF6 medium until the depletion of nitrogen and phosphorus sources and then releasing the single cells in colonies into the medium. Twenty-day culture of single cells in a 96-well plate resulted in 96% regeneration of colonies, and the regeneration of colonies was also confirmed on agar medium. This is the first report of colony regeneration from single cells of B. braunii. We believe that our method developed in this study will contribute greatly to the advancement of genetic modification techniques for B. braunii.
胶球藻是一种具有明显缓慢生长特性的群居藻类，但是通过遗传修饰技术有望提高其生长速度和烃类产量。然而，胶球藻具有富含烃类的细胞外基质（ECM），而 ECM 是 DNA 转化的主要障碍。为了分析和利用遗传修饰的胶球藻，必须从单细胞再生遗传同质的菌落。在这项研究中，我们开发了一种新颖、简单的方法，通过单独离心培养物和过滤来收获胶球藻的活单细胞。通过在 AF6 培养基中培养胶球藻菌落，直到耗尽氮和磷源，然后将菌落中的单细胞释放到培养基中，从而实现了单细胞的收获。在 96 孔板中培养单细胞 20 天可使菌落再生率达到 96%，在琼脂培养基上也可确认菌落的再生。这是首次从胶球藻单细胞中再生出菌落的报道。我们相信，我们在这项研究中开发的方法将极大地促进胶球藻遗传修饰技术的发展。
