Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli

Horvath, Nicholas; Vilkhovoy, Michael; Wayman, Joseph A.; Calhoun, Kara; Swartz, James R.; Varner, Jeffrey D.

doi:10.1016/j.mec.2019.e00113

Cited by 26 publications

(33 citation statements)

References 59 publications

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“…Equally important, we identified transcription from the σ 70 promoter to be a key limiting step in CFE operating at non-saturating plasmid DNA concentrations. This result opposes a common prevailing notion, mainly borne from experiments with bacteriophage polymerases, that energy regeneration for translation strictly determines final protein titer in in vitro transcription-translation reactions [79][80][81]. With our optimized extracts, we were able to activate a diverse array of genetic circuitry in CFE, including RNA transcriptional activators, RNA translational activators, protein transcription factors, CRISPR-Cas9 nuclease activity, RNA translational repressors, and a translational riboswitch.…”

Section: Discussionmentioning

confidence: 57%

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Silverman¹,

Kelley‐Loughnane

Lucks³

et al. 2018

Preprint

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Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits designed as biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the method by which an extract is prepared affects its functionality. Specifically, a key goal toward developing cell-free biosensors based on native genetic regulators is activating the transcriptional machinery present in bacterial extracts for protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are quite low in conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial σ 70 promoters is constrained by the rate of transcription in crude extracts and that processing the extract with a ribosomal run-off reaction and subsequent dialysis can alleviate this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture, but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing and will encourage the further proliferation of cell-free gene expression technology for new applications.

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Section: Discussionmentioning

confidence: 57%

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Silverman¹,

Kelley‐Loughnane

Lucks³

et al. 2018

Preprint

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show abstract

“…This has been demonstrated by a number of modeling studies of increasing sophistication (Karzbrun et al, 2011 ; Stögbauer et al, 2012 ; Tuza et al, 2015 ; Gyorgy and Murray, 2016 ; Nieß et al, 2017 ; Marshall and Noireaux, 2019 ), as well as notable examples of model-guided forward engineering of genetic circuits (Hu et al, 2015 , 2018 ; Agrawal et al, 2019 ; Lehr et al, 2019 ; Westbrook et al, 2019 ). Recent development of integrated gene expression and metabolic models have elucidated the factors limiting CFPS (Wayman et al, 2015 ; Vilkhovoy et al, 2018 , 2019 ; Horvath et al, 2020 ), suggesting that combined computational and experimental metabolomic studies are poised to contribute significantly to our understanding of CFPS in lysates.…”

Section: Rational Biodesign Strategies For Cell-free Synthetic Biomentioning

confidence: 99%

Cell-Free Systems: A Proving Ground for Rational Biodesign

Laohakunakorn

2020

Front. Bioeng. Biotechnol.

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Cell-free gene expression systems present an alternative approach to synthetic biology, where biological gene expression is harnessed inside non-living, in vitro biochemical reactions. Taking advantage of a plethora of recent experimental innovations, they easily overcome certain challenges for computer-aided biological design. For instance, their open nature renders all their components directly accessible, greatly facilitating model construction and validation. At the same time, these systems present their own unique difficulties, such as limited reaction lifetimes and lack of homeostasis. In this Perspective, I propose that cell-free systems are an ideal proving ground to test rational biodesign strategies, as demonstrated by a small but growing number of examples of model-guided, forward engineered cell-free biosystems. It is likely that advances gained from this approach will contribute to our efforts to more reliably and systematically engineer both cell-free as well as living cellular systems for useful applications.

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“…Although transcription-translation systems can be described at varying levels of granularity, e.g., ref. [42][43][44][45] , we chose to model the processes at the most coarse-grained level using coupled ordinary differential equations (ODEs) ( Fig. 3f, Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

A partially self-regenerating synthetic cell

2020

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Self-regeneration is a fundamental function of all living systems. Here we demonstrate partial molecular self-regeneration in a synthetic cell. By implementing a minimal transcription-translation system within microfluidic reactors, the system is able to regenerate essential protein components from DNA templates and sustain synthesis activity for over a day. By quantitating genotype-phenotype relationships combined with computational modeling we find that minimizing resource competition and optimizing resource allocation are both critically important for achieving robust system function. With this understanding, we achieve simultaneous regeneration of multiple proteins by determining the required DNA ratios necessary for sustained self-regeneration. This work introduces a conceptual and experimental framework for the development of a self-replicating synthetic cell.

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Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli

Cited by 26 publications

References 59 publications

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Cell-Free Systems: A Proving Ground for Rational Biodesign

A partially self-regenerating synthetic cell

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