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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.

2025-08-29 bioengineering
10.1101/2025.08.28.672974 bioRxiv
Show 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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