Chemical and Biological Engineering, Iowa State University.
Chemical and Biological Engineering, Iowa State University;
J Vis Exp. 2021 Jun 14(172). doi: 10.3791/62728.
This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 10x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 µg of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.
本方案描述了最小 DNA 模板的设计和酶促扩增步骤，使用无细胞表达可在不到 24 小时内快速原型化可检测蛋白质。从供应商收到 DNA 后，通过 PCR 扩增、切割、环化和低温保存基因片段。然后，从皮克级别的起始材料（如果使用所有起始合成片段，则为毫克级）中少量稀释并显著扩增（高达 10 倍）使用等温滚环扩增（RCA）。RCA 可以从微克级别的起始材料中获得微克量的最小表达模板（如果使用所有起始合成片段，则为毫克级）。在这项工作中，起始量为 20pg，最终产物为 4μg。所得 RCA 产物（最小模板的连接体）可直接添加到无细胞反应中，无需纯化步骤。由于该方法完全基于 PCR，因此当与自动化液体处理系统结合使用时，可能会为未来的高通量筛选工作提供便利。
