Department of Environmental Health Science, Konkuk University, Seoul 05029, Republic of Korea.
Biosensors (Basel). 2024 Nov 12;14(11):547. doi: 10.3390/bios14110547.
Multiple antibiotic resistance regulators (MarRs) control the transcription of genes in the operon of in the presence of salicylic acid (SA). The interaction with SA induces conformational changes in the MarR released from the promoter of the operon, turning on transcription. We constructed an SA-specific cell-based biosensor by fusing the promoter of the operon (P) and the gene that encodes an enhanced green fluorescent protein (). Because SA and aspirin are structurally similar, a biosensor for monitoring aspirin can be obtained by genetically engineering MarR to be aspirin (ASP)-responsive. To shift the selectivity of MarR toward ASP, we changed the residues around the ligand-binding sites by site-directed mutagenesis. We examined the effects of genetic engineering on MarR by introducing MarRs with P- into . Among the tested mutants, MarR T72A improved the ASP responses by approximately 3 times compared to the wild-type MarR, while still showing an SA response. Although the MarR T72A biosensor exhibited mutual interference between SA and ASP, it accurately determined the ASP concentration in spiked water and medicine samples with over 90% accuracy. While the ASP biosensors still require improvement, our results provide valuable insights for developing cell-based biosensors for ASP and transcription factor-based biosensors in general.
多重抗生素耐药调节剂 (MarR) 在水杨酸 (SA) 的存在下控制 操纵子中基因的转录。与 SA 的相互作用诱导从 操纵子启动子释放的 MarR 发生构象变化，从而开启转录。我们通过融合 操纵子的启动子 (P) 和编码增强型绿色荧光蛋白 () 的基因，构建了一种基于 SA 的特异性细胞生物传感器。由于 SA 和阿司匹林在结构上相似，通过基因工程使 MarR 对阿司匹林 (ASP) 产生反应，可以获得用于监测阿司匹林的生物传感器。为了将 MarR 的选择性转向 ASP，我们通过定点突变改变了配体结合位点周围的残基。我们通过将含有 P-的 MarRs 引入 来检查遗传工程对 MarR 的影响。在测试的突变体中，MarR T72A 与野生型 MarR 相比，ASP 反应提高了约 3 倍，同时仍显示出 SA 反应。虽然 MarR T72A 生物传感器显示出 SA 和 ASP 之间的相互干扰，但它仍能准确测定加标水样和药物样品中的 ASP 浓度，准确率超过 90%。虽然 ASP 生物传感器仍需要改进，但我们的结果为开发基于 ASP 的基于细胞的生物传感器和基于转录因子的生物传感器提供了有价值的见解。
