Department of Energy, Environmental, and Chemical Engineering, Washington University, St. Louis, MO 63130, USA.
J Microbiol Methods. 2011 Feb;84(2):228-33. doi: 10.1016/j.mimet.2010.11.022. Epub 2010 Dec 7.
In this study, we developed a technique for delivering genes to microorganisms via electrospray of gold nanoparticles. During the electrospray process, charged monodisperse nano-droplets (a mixture of pET30a-GFP plasmid and nano-sized gold particles) were accelerated and deposited on a thin layer of non-competent Escherichia coli cells. Via antibiotic selection, transformed cells containing green fluorescent protein appeared on the agar plates. PCR amplification and restriction enzyme analysis further confirmed that pET30a-GFP plasmid had successfully been delivered into the non-competent E. coli cells. The transformation efficiencies were optimized under different electrospray conditions. Among several electrospray buffer solutions, CaCl(2) (0.01M) was found to be the best for gene delivery. Furthermore, gold nanoparticles (NPs, 50 nm diameter) significantly improved plasmid transformation efficiency by 5-7 fold (up to 2×10(6) CFU/μg plasmid) compared with that obtained using naked plasmid. Electronic microscopy images and gel electrophoresis showed that the morphology of plasmids remained unchanged during the electrospray process, but cellular membrane integrity was reduced after being electrosprayed with gold NPs and CaCl(2) buffer solutions. This gene delivery method has the potential to work for many other microorganisms.
在这项研究中，我们开发了一种通过金纳米粒子电喷雾将基因递送至微生物的技术。在电喷雾过程中，带电荷的单分散纳米液滴（pET30a-GFP 质粒和纳米级金颗粒的混合物）被加速并沉积在一层薄的非感受态大肠杆菌细胞上。通过抗生素选择，在琼脂平板上出现了含有绿色荧光蛋白的转化细胞。PCR 扩增和限制性内切酶分析进一步证实了 pET30a-GFP 质粒已成功递送至非感受态大肠杆菌细胞中。在不同的电喷雾条件下优化了转化效率。在几种电喷雾缓冲溶液中，发现 CaCl2（0.01M）最适合基因传递。此外，与使用裸露质粒相比，金纳米颗粒（NPs，50nm 直径）使质粒转化效率显著提高了 5-7 倍（高达 2×106CFU/μg 质粒）。电子显微镜图像和凝胶电泳显示，在电喷雾过程中质粒的形态保持不变，但在用金纳米颗粒和 CaCl2 缓冲溶液电喷雾后细胞膜完整性降低。这种基因传递方法有可能适用于许多其他微生物。
