Pardatscher Günther, Schwarz-Schilling Matthaeus, Sagredo Sandra, Simmel Friedrich C
Physics Department, Technical University of Munich.
Physics Department, Technical University of Munich;
J Vis Exp. 2018 Oct 25(140):58634. doi: 10.3791/58634.
Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.
将基因固定在光刻结构化表面上，能够在开放式微流控生物反应器系统中研究分隔的基因表达过程。与其他构建人工细胞系统的方法不同，这种设置允许持续供应基因表达试剂并同时排出废物。这有助于长时间实施无细胞基因表达过程，这对于实现动态基因调控反馈系统很重要。在此，我们提供了一份详细方案，用于使用基于DNA链置换反应的简单光刻技术制造基因生物芯片，该技术仅使用市售组件。我们还提供了一份关于将分隔的基因与基于聚二甲基硅氧烷（PDMS）的微流控系统整合的方案。此外，我们表明该系统与全内反射荧光（TIRF）显微镜兼容，可用于直接观察DNA与表达混合物中所含分子之间的分子相互作用。
