Changes in Coding and Efficiency through Modular Modifications to a One Pot PURE System for <i>In Vitro</i> Transcription and Translation

Ngo, Phuoc H. T.; Ishida, Satoshi; Busogi, Bianca B.; Do, Hannah; Ledesma, Maximiliano A.; Kar, Shaunak; Ellington, Andrew

doi:10.1021/acssynbio.3c00461

Cited by 2 publications

(4 citation statements)

References 33 publications

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“…Nonetheless, the composition of the ultimate in vitro transcription and translation factor mixure can be controlled by controlling the composition of the strains introduced into the original culture. This system can be readily used to change the genetic code, via the introduction of strains expressing orthogonal aaRS and their accompanying purified, cognate tRNAs …”

Section: Engineering the Core Components Of Information Decoding For ...mentioning

confidence: 99%

Engineering Ribosomal Machinery for Noncanonical Amino Acid Incorporation

Ishida,

Ngo,

Gundlach

et al. 2024

Chem. Rev.

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The introduction of noncanonical amino acids into proteins has enabled researchers to modify fundamental physicochemical and functional properties of proteins. While the alteration of the genetic code, via the introduction of orthogonal aminoacyl-tRNA synthetase:tRNA pairs, has driven many of these efforts, the various components involved in the process of translation are important for the development of new genetic codes. In this review, we will focus on recent advances in engineering ribosomal machinery for noncanonical amino acid incorporation and genetic code modification. The engineering of the ribosome itself will be considered, as well as the many factors that interact closely with the ribosome, including both tRNAs and accessory factors, such as the all-important EF-Tu. Given the success of genome re-engineering efforts, future paths for radical alterations of the genetic code will require more expansive alterations in the translation machinery.

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Section: Engineering the Core Components Of Information Decoding For ...mentioning

confidence: 99%

Engineering Ribosomal Machinery for Noncanonical Amino Acid Incorporation

Ishida,

Ngo,

Gundlach

et al. 2024

Chem. Rev.

Self Cite

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“…This has been observed in another work, where background protein expression burden led to promoter mutations, resulting in the loss of protein expression. 5 To reduce background protein expression, we investigated whether catabolite repression, where the addition of a preferred carbon source (glucose), could upregulate the intracellular concentration of repressors (LacI) that block secondary carbon source utilization. 26 With upregulated LacI repressor production and reduced background protein expression from PT7-lacO and PT5-lacO-lacO promoters, we expect more stable plasmid maintenance and protein expression.…”

Section: Enhancing Protein Expression Stability For the One-pot Pure ...mentioning

confidence: 99%

“…The open nature of these expression platforms allows direct manipulation of the reaction environment. Applications of these platforms span high-throughput screening, [1][2][3] novel protein modifications, 4,5 interfacing biomolecules with synthetic materials, [6][7][8][9] on-demand biosensing [10][11][12] and biomanufacturing, 13,14 and understanding the rules of life by building it from scratch. [15][16][17][18] Nearly all in vitro protein expression platforms can be categorized into two classes -crude lysatebased and reconstituted systems.…”

Section: Introductionmentioning

confidence: 99%

“…20 On the other hand, the PURE (Protein synthesis Using Recombinant Elements) system isolates the necessary transcription, translation, and energy regeneration proteins to support gene expression by protein purification and reconstitution. This creates a more defined reaction environment, enabling the construction of predictive models for protein expression, 23,24 allowing for facile removal and replacement of reaction enzymes for customization, 5 and maintaining a minimal reaction proteome toward a self-regenerating synthetic cell. 16 However, traditional PURE systems are expensive to purchase ($0.64 -1.00 per microliter of reaction) and laborious to prepare in-house, posing a significant bottleneck in the wide adoption of PURE systems.…”

Section: Introductionmentioning

confidence: 99%

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Optimizing protein expression in the One-Pot PURE System: Insights into reaction composition and translation efficiency

Zhang,

Qiu,

Deveikis

et al. 2024

Preprint

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The One-Pot PURE system forin vitroprotein expression, which results from a co-culture and one-step purification of 36 essential proteins to support gene transcription and translation, can significantly improve the accessibility and affordability of PURE systems. However, replicating this protocol to match the productivity of the traditional PURE system can take considerable time and effort due to variability in the expression level of individual proteins. In this work, we observed unstable PURE protein expression in twoE. coliprotein expression strains, M15/pREP4 and BL21(DE3), and addressed this instability using catabolite repression. We identified key proteins whose concentration in the One-Pot PURE mixture significantly impacted the reaction’s protein expression capacity. Compared to the original method using twoE. coliprotein expression strains, we found that consolidating all expression vectors onto one BL21 (DE3) strain led to more uniform cell growth at the time of protein induction, thereby improving the composition of critical translation initiation factors in the purified mixture for efficient translation. We also found that variations in commercial energy solution formulations could compensate for deficiencies in the One-Pot PURE protein composition. Additionally, our study revealed significant differences in the translation capacity of commercially availableE. colitRNAs, suggesting the potential of optimizing tRNA composition to improve protein translation. Taken together, this work highlights the intricate biochemical interplay influencing protein expression capacity in the One-Pot PURE system and presents strategies to improve its robustness and productivity.

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Changes in Coding and Efficiency through Modular Modifications to a One Pot PURE System for In Vitro Transcription and Translation

Cited by 2 publications

References 33 publications

Engineering Ribosomal Machinery for Noncanonical Amino Acid Incorporation

Engineering Ribosomal Machinery for Noncanonical Amino Acid Incorporation

Optimizing protein expression in the One-Pot PURE System: Insights into reaction composition and translation efficiency

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