Development of cell-free transcription-translation systems in three soil Pseudomonads

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Title Development of cell-free transcription-translation systems in three soil Pseudomonads
Authors Joseph T. Meyerowitz, Elin M. Larsson and Richard M. Murray
Source To appear, ACS Synthetic Biology, 2024
Abstract In vitro transcription-translation (TX-TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis. This work shows the successful development of TX-TL systems using three soil-derived wild-type Pseudomonads known to promote plant growth: Pseudomonas synxantha, Pseudomonas chlororaphis, and Pseudomonas aureofaciens. One, P. synxantha, was further characterized. A lysate test of P. synxantha showed a maximum protein yield of 2.5 μM at 125 proteins per DNA template and a maximum protein synthesis rate of 20 nM/min. A set of different constitutive promoters driving mNeonGreen expression were tested in TX-TL and integrated into the genome, showing similar normalized strengths for in vivo and in vitro fluorescence. This correspondence between the TX-TL derived promoter strength and the in vivo promoter strength indicates these lysate-based cell-free systems can be used to characterize and engineer biological parts without genome integration, enabling a faster designbuild-test cycle.
Type Journal paper
URL https://www.biorxiv.org/content/10.1101/2023.06.09.544292v1.abstract
DOI 10.1101/2023.06.09.544292
Tag MLM23-ACSSynBio
ID 2023f
Funding ARL Cell Free
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