Development of Cell-Free Transcription–Translation Systems in Three Soil Pseudomonads

Creators: Meyerowitz, Joseph T.; Larsson, Elin M.; Murray, Richard M.¹

1. California Institute of Technology

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. All three species demonstrated multiple sonication, runoff, and salt conditions producing detectable protein synthesis. One of these new TX–TL systems, P. synxantha, demonstrated a maximum protein yield of 2.5 μM at 125 proteins per DNA template, a maximum protein synthesis rate of 20 nM/min, and a range of DNA concentrations with a linear correspondence with the resulting protein synthesis. 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 that these lysate-based cell-free systems can be used to characterize and engineer biological parts without genomic integration, enabling a faster design–build–test cycle.

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Acknowledgement

With thanks to M. Prator for performing the Bradford assays of the lysates, to Z. Jurado for purifying the mNeonGreen fluorescence standard, to Prof. Dianne Newman for the Pseudomonas strains and R. Alcalde for the Pa10403-mNeonGreen plasmid and the P. synxantha strain, to Dr. R. Sidney Cox III for assistance with data visualization, to Dr. A. Pandey for assistance with data processing, and to Dr. Dmitri Mavrodi for assistance with the genomic integration protocol for P. synxantha. This research is supported by the Institute for Collaborative Biotechnologies through contract W911NF-19-D-0001, cooperative agreement W911NF-19-2-0026, and grant W911NF-09-0001 from the U.S. Army Research Office, the National Science Foundation through grant CBET-1903477, and the International Human Frontiers Science Program. The content of this paper does not necessarily reflect the position or the policy of the U.S. Government, and no official endorsement should be inferred.

Contributions

J.T.M. and E.M.L. equally contributed. J.T.M., E.M.L., and R.M.M. conceptualized the project. J.T.M. and E.M.L. designed the experiments and analyzed the data. J.T.M. performed TX–TL experiments. E.M.L. performed in vivo experiments, with the exception of the syringaldehyde experiment which was performed by J.T.M. E.M.L. did the plasmid construction and integration. J.T.M. and E.M.L. wrote the manuscript with input from R.M.M.

Conflict of Interest

The authors declare no competing financial interest.

Data Availability

(ZIP)
Phylogenetic relationships and genome-to-genome distances for species in this study; growth rates of the three Pseudomonads and E. coli; details of the growth inhibition and dose–response to tested growth inhibitors; protein concentrations of clarified lysates; negative controls for selected reaction conditions; time course data for in vitro TX–TL promoter panel reactions; time course data for in vitro promoter panel measurements; schematic representation of plasmids used in this study; and illustration of the lysate production protocol (PDF)
Sequences of primers, promoters, and Addgene deposition information for strains used in this study (XLSX)

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