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bioRxiv · 10.1101/2025.08.28.672974

One-pot cloning and protein expression platform for genetic engineering

Sato, W.·Sharon, J.·Cash, B.·Deich, C.·Gaut, N. J.·Heili, J. M.·Engelhart, A. E.·Adamala, K. P.
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Abstract

In this work, we present a streamlined one-pot cloning and protein expression platform that integrates mutagenesis, plasmid assembly, and functional protein testing in a single reaction. By combining Golden Gate cloning with cell-free transcription-translation, we demonstrate efficient generation and screening of genetic variants without the need for intermediate purification or bacterial amplification. Using fluorescent proteins, luciferase enzymes, antibiotic-converting enzymes, and the violacein biosynthetic pathway, we validate the versatility of this approach for single-and multi-site mutagenesis, combinatorial variant libraries, metabolic pathway programming, and whole-plasmid assembly. By demonstrating compatibility with multiplexed reactions and multi-cistronic constructs, we establish this approach as a generalizable and automatable method for high-throughput cloning and protein engineering in synthetic biology.

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Sato, W., Sharon, J., Cash, B., Deich, C., Gaut, N. J., Heili, J. M., Engelhart, A. E., Adamala, K. P.. 2025-08-29. One-pot cloning and protein expression platform for genetic engineering. https://doi.org/10.1101/2025.08.28.672974

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