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bioRxiv · 10.1101/2023.06.09.544292

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

Meyerowitz, J. T.·Larsson, E. M.·Murray, R. M.
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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 M 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.

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Meyerowitz, J. T., Larsson, E. M., Murray, R. M.. 2023-06-09. Development of cell-free transcription-translation systems in three soil Pseudomonads. https://doi.org/10.1101/2023.06.09.544292

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