Assessing translational efficiency by a reporter protein co-expressed in a cell-free synthesis system

Park, Yu Jin; Lee, Kyung-Ho; Kim, Dong‐Myung

doi:10.1016/j.ab.2016.11.019

Cited by 6 publications

(4 citation statements)

References 14 publications

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“…To accommodate a variety of curricular limitations, the Genetic Code Kit can be completed within a single 3-h laboratory period, and does not require instructors to dedicate time in a subsequent day to collect data. The kit utilizes crude, E. coli-based extract and a DNA template encoding sfGFP, which together have been broadly demonstrated to support robust and reliable protein expression (Park et al, 2017;Levine et al, 2019a,b). Importantly, the sfGFP product resulting from a successful CFPS reaction is easy to visualize in real-time with minimal equipment or processing, and introduces students to a workhorse reporter broadly used in research and industry.…”

Section: Introductionmentioning

confidence: 99%

The Genetic Code Kit: An Open-Source Cell-Free Platform for Biochemical and Biotechnology Education

Williams

Gregorio

et al. 2020

Front. Bioeng. Biotechnol.

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Teaching the processes of transcription and translation is challenging due to the intangibility of these concepts and a lack of instructional, laboratory-based, active learning modules. Harnessing the genetic code in vitro with cell-free protein synthesis (CFPS) provides an open platform that allows for the direct manipulation of reaction conditions and biological machinery to enable inquiry-based learning. Here, we report our efforts to transform the research-based CFPS biotechnology into a hands-on module called the "Genetic Code Kit" for implementation into teaching laboratories. The Genetic Code Kit includes all reagents necessary for CFPS, as well as a laboratory manual, student worksheet, and augmented reality activity. This module allows students to actively explore transcription and translation while gaining exposure to an emerging research technology. In our testing of this module, undergraduate students who used the Genetic Code Kit in a teaching laboratory showed significant score increases on transcription and translation questions in a post-lab questionnaire compared with students who did not participate in the activity. Students also demonstrated an increase in self-reported confidence in laboratory methods and comfort with CFPS, indicating that this module helps prepare students for careers in laboratory research. Importantly, the Genetic Code Kit can accommodate a variety of learning objectives beyond transcription and translation and enables hypothesis-driven science. This opens the possibility of developing Course-Based Undergraduate Research Experiences (CUREs) based on the Genetic Code Kit, as well as supporting next-generation science standards in 8-12th grade science courses. Keywords: biochemical education, learn by doing, cell-free protein synthesis (CFPS), in vitro transcription and translation, synthetic biology (synbio), central dogma of molecular biology (CDMB), chemical education and teaching, augmented reality (AR) Abbreviations: CFPS, cell-free protein synthesis; CUREs, course-based undergraduate research experiences; sfGFP, superfolder green fluorescent protein.

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Section: Introductionmentioning

confidence: 99%

The Genetic Code Kit: An Open-Source Cell-Free Platform for Biochemical and Biotechnology Education

Williams

Gregorio

et al. 2020

Front. Bioeng. Biotechnol.

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“…Karim and Jewett observed a similar decreasing effect on the protein expression levels when they tried the cell-free coexpression of several enzymes. According to Park et al, mRNA species must compete for a finite pool of ribosomes and aminoacyl-tRNAs when CFPS reaction mixtures are primed with several DNA templates, leading to unequal expression levels of different proteins. Indeed, the sGFP fluorescence was reported to be inversely proportional to the translation rates of the coexpressed genes.…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, sGFP fluorescence was reported to be inversely proportional to translation rates of the co-expressed genes. 48 Beside the fluorescence of the immobilized Cys-sGFP that reveals its proper folding and structurally innocuous immobilization, His-ADHBs in vitro synthesized and immobilized was catalytically active after CFPS-i ( Figure 7C). The multimeric nature of this enzyme was not a hurdle for its successful expression and in situ immobilization by using the CFPS-i system, validating the feasibility of our technology for fabrication of solid platforms harboring more complex biological systems.…”

Section: Cell-free Synthesis and Immobilization Of His-sgfp In One-potmentioning

confidence: 99%

Expanding One-Pot Cell-Free Protein Synthesis and Immobilization for On-Demand Manufacturing of Biomaterials

et al. 2018

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Fabrication of protein-based biomaterials is an arduous and time-consuming procedure with multiple steps. In this work, we describe a portable toolkit that integrates both cell-free protein synthesis (CFPS) and protein immobilization in one pot just by mixing DNA, solid materials, and a CFPS system. We have constructed a modular set of plasmids that fuse the N-terminus of superfolded green fluorescent protein (sGFP) with different peptide tags (poly(6X)Cys, poly(6X)His, and poly(6X)Lys), which drive the immobilization of the protein on the tailored material (agarose beads with different functionalities, gold nanorods, and silica nanoparticles). This system also enables the incorporation of azide-based amino acids into the nascent protein for its selective immobilization through copper-free click reactions. Finally, this technology has been expanded to the synthesis and immobilization of enzymes and antibody-binding proteins for the fabrication of functional biomaterials. This synthetic biological platform has emerged as a versatile tool for on-demand fabrication of therapeutic, diagnostic, and sensing biomaterials.

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“…PCR primers listed in Supplementary Table S1 were synthesized by Macrogen (Seoul, Korea). E. coli S12 extracts were prepared from the BL21 Star (DE3) strain as described previously ( Park et al, 2017 ). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, United States) and used without further purification.…”

Section: Methodsmentioning

confidence: 99%

Production of Recombinant Horseradish Peroxidase in an Engineered Cell-free Protein Synthesis System

Park

Kim

2021

Front. Bioeng. Biotechnol.

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One of the main advantages of a cell-free synthesis system is that the synthetic machinery of cells can be modularized and re-assembled for desired purposes. In this study, we attempted to combine the translational activity of Escherichia coli extract with a heme synthesis pathway for the functional production of horseradish peroxidase (HRP). We first optimized the reaction conditions and the sequence of template DNA to enhance protein expression and folding. The reaction mixture was then supplemented with 5-aminolevulinic acid synthase to facilitate co-synthesis of the heme prosthetic group from glucose. Combining the different synthetic modules required for protein synthesis and cofactor generation led to successful production of functional HRP in a cell-free synthesis system.

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Assessing translational efficiency by a reporter protein co-expressed in a cell-free synthesis system

Cited by 6 publications

References 14 publications

The Genetic Code Kit: An Open-Source Cell-Free Platform for Biochemical and Biotechnology Education

The Genetic Code Kit: An Open-Source Cell-Free Platform for Biochemical and Biotechnology Education

Expanding One-Pot Cell-Free Protein Synthesis and Immobilization for On-Demand Manufacturing of Biomaterials

Production of Recombinant Horseradish Peroxidase in an Engineered Cell-free Protein Synthesis System

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