Delgadillo-Guevara Mario, Halte Manuel, Erhardt Marc, Popp Philipp F
Institute of Biology/Molecular Microbiology, Humboldt-Universität zu Berlin, Berlin, 10115, Germany.
Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC, Australia.
J Biol Eng. 2024 Apr 8;18(1):25. doi: 10.1186/s13036-024-00420-9.
Standardized and thoroughly characterized genetic tools are a prerequisite for studying cellular processes to ensure the reusability and consistency of experimental results. The discovery of fluorescent proteins (FPs) represents a milestone in the development of genetic reporters for monitoring transcription or protein localization in vivo. FPs have revolutionized our understanding of cellular dynamics by enabling the real-time visualization and tracking of biological processes. Despite these advancements, challenges remain in the appropriate use of FPs, specifically regarding their proper application, protein turnover dynamics, and the undesired disruption of cellular functions. Here, we systematically compared a comprehensive set of 15 FPs and assessed their performance in vivo by focusing on key parameters, such as signal over background ratios and protein stability rates, using the Gram-negative model organism Salmonella enterica as a representative host. We evaluated four protein degradation tags in both plasmid- and genome-based systems and our findings highlight the necessity of introducing degradation tags to analyze time-sensitive cellular processes. We demonstrate that the gain of dynamics mediated by the addition of degradation tags impacts the cell-to-cell heterogeneity of plasmid-based but not genome-based reporters. Finally, we probe the applicability of FPs for protein localization studies in living cells using standard and super-resolution fluorescence microscopy. In summary, our study underscores the importance of careful FP selection and paves the way for the development of improved genetic reporters to enhance the reproducibility and reliability of fluorescence-based research in Gram-negative bacteria and beyond.
标准化且经过充分表征的遗传工具是研究细胞过程的先决条件，以确保实验结果的可重复性和一致性。荧光蛋白（FPs）的发现是用于监测体内转录或蛋白质定位的遗传报告基因发展中的一个里程碑。荧光蛋白通过实现生物过程的实时可视化和追踪，彻底改变了我们对细胞动态的理解。尽管取得了这些进展，但在荧光蛋白的合理使用方面仍然存在挑战，特别是在其正确应用、蛋白质周转动力学以及对细胞功能的意外干扰方面。在这里，我们系统地比较了一组全面的15种荧光蛋白，并以革兰氏阴性模式生物肠炎沙门氏菌作为代表性宿主，通过关注关键参数，如信号与背景比率和蛋白质稳定率，评估了它们在体内的性能。我们在基于质粒和基于基因组的系统中评估了四种蛋白质降解标签，我们的研究结果突出了引入降解标签以分析对时间敏感的细胞过程的必要性。我们证明，添加降解标签介导的动态变化会影响基于质粒的报告基因的细胞间异质性，但不会影响基于基因组的报告基因。最后，我们使用标准和超分辨率荧光显微镜探究了荧光蛋白在活细胞中进行蛋白质定位研究的适用性。总之，我们的研究强调了谨慎选择荧光蛋白的重要性，并为开发改进的遗传报告基因铺平了道路，以提高革兰氏阴性细菌及其他领域基于荧光的研究的可重复性和可靠性。
