Open AccessRapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems
Author(s) -
Jared L. Dopp,
Nigel F. Reuel
Publication year - 2021
Publication title -
journal of visualized experiments
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.596
H-Index - 91
ISSN - 1940-087X
DOI - 10.3791/62728
Subject(s) - concatemer , template , rolling circle replication , cell free protein synthesis , dna , synthetic biology , chemistry , computational biology , loop mediated isothermal amplification , microbiology and biotechnology , chromatography , gene , biology , biochemistry , nanotechnology , polymerase , materials science , protein biosynthesis , genome
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 10 6 x) 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.