Transforming Synthetic Biology with Cell-Free Systems

Ranji, Arnaz; Wu, Jeffrey C.; Bundy, Bradley C.; Jewett, Michael C.

doi:10.1016/b978-0-12-394430-6.00015-7

Cited by 9 publications

(3 citation statements)

References 142 publications

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“…Equally important, the reported tRNA emancipated cell‐free system is capable of producing protein at significant yields (∼300 μg/mL) and reasonable costs (∼$6–9/mg) . For example, these results show that the presented method can approximate the near tRNA‐free environment of a customized PURE system, but with higher yields and at a 1,000 fold reduction in cost . In conclusion, quantification of tRNA removal is essential to verifying near 100% tRNA removal and such an optimized cell‐free protein synthesis platform holds great promise for widespread use of codon reassigned systems.…”

Section: Resultsmentioning

confidence: 79%

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

Salehi

Smith

Schinn

et al. 2017

Biotechnology Progress

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Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate-based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase-coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real-time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1401-1407, 2017.

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Section: Resultsmentioning

confidence: 79%

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

Salehi

Smith

Schinn

et al. 2017

Biotechnology Progress

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“…Third, in vitro approaches offer rapid prototyping environments (i.e., hours to days) that are faster than in vivo standard testing settings that require time consuming cloning work (i.e., days to weeks). [3,6,49] Given these advantages and recent technical improvements, CFPS is increasingly adapted for use in new application areas. In particular, the approach has been leveraged to advance efforts to expand the chemistry of life via ncAA incorporation into proteins.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An efficient cell‐free protein synthesis platform for producing proteins with pyrrolysine‐based noncanonical amino acids

Charna

Soye

Ntai

et al. 2022

Biotechnology Journal

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Incorporation of noncanonical amino acids (ncAAs) into proteins opens new opportunities in biotechnology and synthetic biology. Pyrrolysine (Pyl)-based ncAAs are some of the most predominantly used, but expression systems suffer from low yields. Here, we report a highly efficient cell-free protein synthesis (CFPS) platform for site-specific incorporation of Pyl-based ncAAs into proteins using amber suppression. This platform is based on cellular extracts derived from genomically recoded Escherichia coli lacking release factor 1 and enhanced through deletion of endonuclease A. To enable ncAA incorporation, orthogonal translation system (OTS) components (i.e., the orthogonal transfer RNA [tRNA] and orthogonal aminoacyl tRNA synthetase) were coexpressed in the source strain prior to lysis and the orthogonal tRNA CUA Pyl that decodes the amber codon was further enriched in the CFPS reaction via co-synthesis with the product.Using this platform, we demonstrate production of up to 442 ± 23 μg/mL modified superfolder green fluorescent protein (sfGFP) containing a single Pyl-based ncAA at high (>95%) suppression efficiency, as well as sfGFP variants harboring multiple, identical ncAAs. Our CFPS platform can be used for the synthesis of modified proteins containing multiple precisely positioned, genetically encoded Pyl-based ncAAs. We anticipate that it will facilitate more general use of CFPS in synthetic biology.

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Cell‐Free Protein Synthesis: An Emerging Technology for Understanding, Harnessing, and Expanding the Capabilities of Biological Systems

Schoborg

Jewett

2018

Synthetic Biology

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Transforming Synthetic Biology with Cell-Free Systems

Cited by 9 publications

References 142 publications

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

An efficient cell‐free protein synthesis platform for producing proteins with pyrrolysine‐based noncanonical amino acids

Cell‐Free Protein Synthesis: An Emerging Technology for Understanding, Harnessing, and Expanding the Capabilities of Biological Systems

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