合成生物学视角下的基因功能探索与酵母工程菌株文库构建

章益蜻, 刘高雯

Exploration of gene functions and library construction for engineering strains from a synthetic biology perspective

ZHANG Yiqing, LIU Gaowen

图1 经典文库构建方法（本图由biorender绘制） ［（a）基因缺失文库中，目的基因ORF（橘色片段）被替换成卡那霉素抗性筛选标签KanMX（黄色片段），两侧伴有“分子条形码”barcode（蓝色片段）；Chr.DNA：染色体DNA。（b）Bar-seq原理示意图。在特定条件处理下，目标菌株（红色）的生长量相对低（淡粉色），表现为测序时相应barcode的读数量相对低（红色线条比其他颜色少）。（c）基因过表达文库中，目的基因ORF（橘色片段）除了在基因组上正常表达之外，还额外在质粒上由通用型启动子（淡绿色片段）驱动表达。（d）ORF-Tag文库中，在删除终止密码子的目的基因ORF（橘色片段）C端插入荧光蛋白GFP（深绿色片段）和筛选标签KanMX（黄色片段）］

Fig. 1 Schematics for constructing classical libraries (created by biorender) [(a) In the gene deletion library, the ORF (orange fragment) of target gene is replaced by the KanMX (kanamycin resistance selection marker, yellow fragment), flanked by unique barcodes (blue fragments). Chr.DNA: chromosomal DNA.(b) Schematic diagram of the Bar-seq principle. Under specific treatment conditions, the target strain (red) shows reduced growth (light pink), which is reflected by a lower read count of its corresponding barcode during sequencing (fewer red lines compared to others).(c) In the gene overexpression library, the ORF (orange fragment) of target gene is not only expressed from its endogenous locus but is also additionally expressed from a plasmid, driven by a constitutive promoter (light green fragment).(d) In the ORF-Tag library, the C-terminus of the target gene ORF (orange fragment), with its stop codon removed, is fused to the fluorescent protein GFP (dark green fragment) and the selection marker KanMX (yellow fragment).]