Data_Sheet_7_Blasticidin S Deaminase: A New Efficient Selectable Marker for Chlamydomonas reinhardtii.docx

Chlamydomonas reinhardtii is a model unicellular organism for basic or biotechnological research, such as the production of high-value molecules or biofuels thanks to its photosynthetic ability. To enable rapid construction and optimization of multiple designs and strains, our team and collaborators have developed a versatile Chlamydomonas Modular Cloning toolkit comprising 119 biobricks. Having the ability to use a wide range of selectable markers is an important benefit for forward and reverse genetics in Chlamydomonas. We report here the development of a new selectable marker based on the resistance to the antibiotic blasticidin S, using the Bacillus cereus blasticidin S deaminase (BSR) gene. The optimal concentration of blasticidin S for effective selection was determined in both liquid and solid media and tested for multiple laboratory strains. In addition, we have shown that our new selectable marker does not interfere with other common antibiotic resistances: zeocin, hygromycin, kanamycin, paromomycin, and spectinomycin. The blasticidin resistance biobrick has been added to the Chlamydomonas Modular Cloning toolkit and is now available to the entire scientific community.

莱茵衣藻（Chlamydomonas reinhardtii）是用于基础研究或生物技术研究的模式单细胞生物，可依托其光合能力生产高价值分子或生物燃料。为实现多种设计与菌株的快速构建与优化，本团队与合作者开发了一套多功能衣藻模块化克隆（Chlamydomonas Modular Cloning）工具箱，内含119个生物砖（biobrick）。可使用多样化筛选标记是衣藻正向与反向遗传学研究的重要优势。本文报道了一种基于杀稻瘟菌素S（blasticidin S）抗性的新型筛选标记，其采用了蜡样芽孢杆菌（Bacillus cereus）的杀稻瘟菌素S脱氨酶（BSR）基因。研究人员在液体与固体培养基中均测定了实现有效筛选的最优杀稻瘟菌素S浓度，并在多个实验室菌株中完成了测试验证。此外，本研究证实该新型筛选标记不会与其他常见抗生素抗性：博来霉素（zeocin）、潮霉素（hygromycin）、卡那霉素（kanamycin）、巴龙霉素（paromomycin）、壮观霉素（spectinomycin）产生交叉干扰。该杀稻瘟菌素抗性生物砖已被加入衣藻模块化克隆工具箱，目前已面向全球科研社群开放使用。