Functional genomics of drug resistance genes in Escherichia coli

In order to expedite gene discovery coding for either drug targets or antibiotic resistance, we have developed a functional genomic strategy termed Plas-Seq. This technique is coupling muticopy suppressor library to next generation sequencing. We generated an Escherichia coli plasmid genomic library that was transformed into E. coli. These transformants were selected step by step using 0.25× to 2× minimum inhibitory concentrations for ceftriaxone, gentamicin, levofloxacin, tetracycline and trimethoprim. Plasmids were isolated at each selection steps and subjected to Illumina sequencing. This has allowed to observe an increase in reads abundance and fold enrichment for 48 different genomic loci.

为加速靶向药物靶点或抗生素耐药性相关编码基因的发现，我们开发了一种名为Plas-Seq的功能基因组学策略。该技术将多拷贝抑制子文库与下一代测序相结合。我们构建了大肠杆菌（Escherichia coli）质粒基因组文库，并将其转化至大肠杆菌（E. coli）中。我们以0.25×至2×的头孢曲松、庆大霉素、左氧氟沙星、四环素及甲氧苄啶的最低抑菌浓度（minimum inhibitory concentrations, MIC），逐步筛选获得这些转化子。在每一轮筛选步骤中分离质粒，并进行Illumina测序。该流程得以观测到48个不同基因组位点的测序读段丰度与富集倍数均出现上升。