Variability in cell-free expression reactions can impact qualitative genetic circuit characterization

Rhea, Katherine; McDonald, Nathan D.; Cole, Stephanie D.; Noireaux, Vincent; Lux, Matthew W.; Buckley, Patricia E.

doi:10.1093/synbio/ysac011

Cited by 7 publications

(7 citation statements)

References 52 publications

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“…Batch-to-batch variability is a known issue for cell-free studies, and differences are expected between each batch of lysate grown and processed on different days. 43 The results here show a range of viable conditions for productive lysates and similar trends across the process conditions for all three species. This wide range of useable variations with this protocol demonstrates a degree of robustness to variation in lysate processing and reaction preparation.…”

Section: ■ Results and Discussionsupporting

confidence: 61%

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

Meyerowitz,

Larsson,

Murray

2024

ACS Synth. Biol.

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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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Section: ■ Results and Discussionsupporting

confidence: 61%

“…Batch-to-batch variability is a known issue for cell-free studies, and differences are expected between each batch of lysate grown and processed on different days . The results here show a range of viable conditions for productive lysates and similar trends across the process conditions for all three species.…”

Section: Resultssupporting

confidence: 52%

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

Meyerowitz,

Larsson,

Murray

2024

ACS Synth. Biol.

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“…Rhea et al proved that the quality and variability of cell-free assays are dependent on the complexity of genetic circuits, reaction conditions, and molecular concentration. And they proposed that a controller circuit could help to subtract variability [7].…”

Section: Cell-free Characterizationmentioning

confidence: 99%

Synthetic cell-based biosensors and methods for discovering novel sensing elements

Zhan

2024

Third International Conference on Biological Engineering and Medical Science (ICBioMed2023)

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With the advancement of synthetic biology, cells can be modified and engineered through gene recombination, gene editing and protein engineering. Given that many elements in gene expression are environmental-sensitive, synthetic biosensor based on cells has been proposed and many studies have validated this concept. Synthetic biosensors can facilitate research on the revelation of biomolecular activity in living systems. And it has the advantages of easy operation, energy economy, and adaptability. This review introduces three types of synthetic biosensors including transcriptional biosensors, translational biosensors, and post-translational biosensors. Then the topic will focus on approaches that help discovery of biosensing elements in cells, which is vital but still challenging for the development of synthetic biosensors. Cell-free assays, directed revolution, high through-put screening and computational methods are discussed in this review. Subsequently, the application of synthetic biosensors is summarized, presenting utility and advantages of them. At last, the future direction is discussed, hoping to provide prospects or advice for research in this field.

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“…Although cell lysate-based TX-TL systems have many uses, their performance and applications are limited by issues such as batch-to-batch variability [9], lack of predictability for complex circuits [23], and limited lifetime [24]. These issues arise from the fact that cell lysate contains many proteins that are not necessary for cell-free protein synthesis.…”

Section: Introductionmentioning

confidence: 99%

Metabolic perturbations to anE. coli-based cell-free system reveal a trade-off between transcription and translation

Kapasiawala

Murray

2023

Preprint

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Cell-free transcription-translation (TX-TL) systems have been used for diverse applications, from prototyping gene circuits to providing a platform for the development of synthetic life, but their performance is limited by issues such as batch-to-batch variability, poor predictability, and limited lifetime. These issues stem largely from the fact that cell lysate contains an active and complex metabolism whose effect on TX-TL has remained largely uncharacterized. Motivated by a minimal model of cell-free metabolism, this work explored the effects of energy molecules, which power TX-TL, and fuel molecules, which regenerate energy by harnessing core metabolism, on anE. coli-based TX-TL system. This work reports a compensatory interaction between TX-TL components Mg+2and 3-phosphoglyceric acid (3-PGA, used to regenerate ATP), where if one component's concentration is increased, the other's must likewise be increased to maintain optimal translation. Furthermore, maximum total mRNA and protein production occur in different and opposite concentration regimes of Mg+2and 3-PGA. To explore the observed phenomenon, transcription and translation were decoupled. Under translation inhibition, transcriptional output was uniform across Mg+2and 3-PGA concentrations, but in a translation-only system, maximum protein production occurred in the previously found optimal regime of Mg+2and 3-PGA, suggesting a TX-TL trade-off. Using alternative fuels to regenerate energy, this work found that the trade-off is universal across the different fuel sources, and that a system's position along the trade-off is determined strongly by Mg+2and DNA concentrations. In systems where additional energy is supplied and where a fuel source is absent, the trade-off is absent, suggesting the trade-off arises from limitations in the regulation of translation and efficient energy regeneration. This work represents a significant advancement in understanding the effects of fuel and energy metabolism on TX-TL in cell-free systems and lays the foundation for improving TX-TL performance, lifetime, standardization, and prediction.

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Variability in cell-free expression reactions can impact qualitative genetic circuit characterization

Cited by 7 publications

References 52 publications

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

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

Synthetic cell-based biosensors and methods for discovering novel sensing elements

Metabolic perturbations to anE. coli-based cell-free system reveal a trade-off between transcription and translation

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