生物技术通报2023，Vol.39Issue(12)：90-98,9.DOI:10.13560/j.cnki.biotech.bull.1985.2023-0129
荧光素酶辅助定量大肠杆菌破碎效果的方法
Establishment of a Luciferase-assisted Quantitative Method for Measuring Ultrasonic Disruption of Escherichia coli Cells
摘要
Abstract
In this study,we present a novel and effective method for measuring ultrasonic disruption of Escherichia coli cells based on firefly luciferase.This quantitative approach provides high sensitivity and rapid detection of cell disruption,by which the disruption was characterized.To evaluate this method,we employed an E.coli strain expressing firefly luciferase as an internal reference,which was mixed with the target bacterial suspension at a specific ratio before sonication.The release of both luciferase and target protein activities into the extracellular solution under different ultrasonic conditions was then investigated,and the assisted quantitative effect was evaluated.The results demonstrated that:A ratio of 1∶500(volume ratio)E.coli strain expressing firefly luciferase added to the target bacterial suspension was sufficient for sonication quantification.The effect of sonication was calculated by the change of luciferase activity within the supernatant.Importantly,luciferase activity also provided clues for maintaining target protein activity during sonication.The luciferase protein incorporated into the final supernatant showed no effect on the purification of the target protein by nickel beads.Bacteria containing firefly luciferase can be stored at-80℃for at least 90 d without significant loss of luciferase activity.Moreover,the disruption resistance was not changed during the long-term cryo-storage process.Therefore,freezing bacterial suspension can be thawed and directly mixed with target cells for assisting quantification without losing accuracy.In conclusion,the luciferase-assisted quantification of E.coli ultrasonic disruption is convenient,robust,and efficient.关键词
荧光素酶/超声破碎/大肠杆菌/定量分析Key words
luciferase/ultrasonic disruption/Escherichia coli/quantitative analysis引用本文复制引用
李奕雅,吴一凡,丁能水,范小萍,陈凡..荧光素酶辅助定量大肠杆菌破碎效果的方法[J].生物技术通报,2023,39(12):90-98,9.基金项目
福建省自然科学基金项目(2021J01998),福建省中青年教师教育科研项目(JAT200314) （2021J01998）
