AUTHOR=Zibulski Darius Leon , Schlichting Niels , Kabisch Johannes 

TITLE=HyperXpress: Rapid Single Vessel DNA Assembly and Protein Production in Microliterscale

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=Volume 10 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.832176

DOI=10.3389/fbioe.2022.832176

ISSN=2296-4185

ABSTRACT=Rapid prototyping of biological functions has the common aim to generate, screen and select variant libraries as quickly as possible. This approach is now to be extended by the HyperXpress workflow, which connects ligase cycling reaction for DNA assembly, multiply-primed rolling circle amplification for signal amplification and cell-free protein synthesis to a single vessel reaction in the lower μl scale. After substantial optimization of the method a proof-of-principle demonstrating the high flexibility of HyperXpress for semi-rational protein engineering by expanding, reducing and replacing β-strands of three different green fluorescent proteins is described. These single-day experiments resulted in six functional, new-to-nature GFP prototypes.