Key Lab of Dairy Biotechnology and Safety Control, College of Food Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225127, China.
Bloomage Biotechnology Corporation Limited, Ji'nan, Shandong 250000, China.
J Microbiol Methods. 2020 Jan;168:105798. doi: 10.1016/j.mimet.2019.105798. Epub 2019 Nov 30.
The promoter of the srf operon (P) had been used to construct a cell-density-dependent expression system in B. subtilis in our previous work. The P and its derivative P12 showed good performance of heterologous protein expression in B. subtilis. In this work, using green fluorescent protein (GFP) and β-galactosidase (LacZ) as the reporter proteins, the host feasibility and expression characteristics of the P and P12 in E. coli were identified. The prominent green fluorescence shooted by laser scanning confocal microscope, fluorescence intensity measured by spectrophotometer and the distinct protein bands detected by SDS-PAGE demonstrated that the GFP could be largely expressed under the control of the P and P12 in the E. coli host strain of BL21 (DE3) and JM109 and the expression of GFP in strain BL21 (DE3) was much higher than that of in strain JM109. Meanwhile, the promoter P 12 was much stronger than P to the extent that the GFP controlled by P12 in strain BL21 (DE3) was leaked into the supernatant. And the fluorescence intensity detected in the supernatant of the recombinant strain BL21 (DE3) containing P12 was 10.25-fold higher than that of strain JM109 containing P. Moreover, the LacZ could also be produced by P and P12 in strain BL21 (DE3) and JM109 and the strain JM109 showed better performance than BL21 (DE3) in expressing LacZ. The LacZ activity controlled by P and P12 in JM109 were separately 2.47-fold and 2.36-fold higher than that of in strain BL21 (DE3). This work will broaden the applied range of the P and enrich the efficient toolbar for cross-species gene expression or module construction in E. coli and B. subtilis.
在我们之前的工作中，使用 srf 操纵子（P）的启动子构建了枯草芽孢杆菌中的细胞密度依赖性表达系统。P 和其衍生物 P12 在枯草芽孢杆菌中表现出良好的异源蛋白表达性能。在这项工作中，使用绿色荧光蛋白（GFP）和β-半乳糖苷酶（LacZ）作为报告蛋白，确定了 P 和 P12 在大肠杆菌中的宿主可行性和表达特性。激光扫描共聚焦显微镜拍摄的显著绿色荧光、分光光度计测量的荧光强度和 SDS-PAGE 检测的明显蛋白条带表明，GFP 可以在大肠杆菌宿主菌株 BL21（DE3）和 JM109 中由 P 和 P12 控制下大量表达，并且 GFP 在菌株 BL21（DE3）中的表达量要高于菌株 JM109。同时，启动子 P12 比 P 要强得多，以至于由 P12 控制的 GFP 在菌株 BL21（DE3）中泄漏到上清液中。含有 P12 的重组菌株 BL21（DE3）上清液中的荧光强度比含有 P 的菌株 JM109 高 10.25 倍。此外，P 和 P12 也可以在菌株 BL21（DE3）和 JM109 中产生 LacZ，并且菌株 JM109 在表达 LacZ 方面表现优于 BL21（DE3）。在 JM109 中，由 P 和 P12 控制的 LacZ 活性分别比菌株 BL21（DE3）高 2.47 倍和 2.36 倍。这项工作将拓宽 P 的应用范围，丰富大肠杆菌和枯草芽孢杆菌中跨物种基因表达或模块构建的有效工具。
