Cell-free supplement mixtures: Elucidating the history and biochemical utility of additives used to support in vitro protein synthesis in E. coli extract

Dopp, Jared L.; Tamiev, Denis; Reuel, Nigel F.

doi:10.1016/j.biotechadv.2018.12.006

Cited by 78 publications

(103 citation statements)

References 138 publications

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“…The pioneering work by BioBits and myTXTL have provided the proof-of-concept in adapting CFPS to classroom settings and engaging students at various grade levels (Huang et al, 2018;Stark et al, 2018Stark et al, , 2019Collias et al, 2019). Additionally, CFPS remains robust in a variety of chemical environments (Yin and Swartz, 2004;Seki et al, 2008;Dopp et al, 2019;Gregorio et al, 2019;Karim et al, 2020) providing extensive flexibility in accommodating a broad range of learning objectives. These advantages make CFPS a next-generation educational technology to help meet the next-generation science standards.…”

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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“…In addition to determining the optimal Mg 2+ concentration for a given lysate and construct, it is customary to optimize the K + concentration [ 61 ]. Figure 2 d displays the total production of H16 when simultaneously varying the Mg 2+ (5–20 mM, magnesium acetate, Mg(OAc) 2 ) and K + (5–200 mM potassium glutamate, KGlu) concentrations.…”

Section: Resultsmentioning

confidence: 99%

Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins

et al. 2020

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The high-resolution structural study of huntingtin exon-1 (HttEx1) has long been hampered by its intrinsic properties. In addition to being prone to aggregate, HttEx1 contains low-complexity regions (LCRs) and is intrinsically disordered, ruling out several standard structural biology approaches. Here, we use a cell-free (CF) protein expression system to robustly and rapidly synthesize (sub-) pathological HttEx1. The open nature of the CF reaction allows the application of different isotopic labeling schemes, making HttEx1 amenable for nuclear magnetic resonance studies. While uniform and selective labeling facilitate the sequential assignment of HttEx1, combining CF expression with nonsense suppression allows the site-specific incorporation of a single labeled residue, making possible the detailed investigation of the LCRs. To optimize CF suppression yields, we analyze the expression and suppression kinetics, revealing that high concentrations of loaded suppressor tRNA have a negative impact on the final reaction yield. The optimized CF protein expression and suppression system is very versatile and well suited to produce challenging proteins with LCRs in order to enable the characterization of their structure and dynamics.

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“…Cell-free systems contain many proteins and metabolites required for complex functions such 22 as transcription and translation or multi-step metabolic conversions. Research into expanding 23 the delivery of these systems by drying or by embedding into other materials is enabling new 24 applications in sensing, point-of-need manufacturing, and responsive materials. Meanwhile, silk 25 fibroin from the silk worm, Bombyx mori, has received attention as a protective additive for 26 dried enzyme formulations and as a material to build biocompatible hydrogels for controlled 27 localization or delivery of biomolecular cargoes.…”

Section: Abstract 21mentioning

confidence: 99%

“…444 Choosing a more targeted enzymatic crosslinking method could be critical to successful 445 implementation in the complex CFPS reaction environment. Other factors that could influence 446 HRP and tyrosinase activity may be tweaked in a cell-free system, including ionic strength or 447 redox-related additives or enzymes such as reductases and dismutases [23]. The difficulty of 448 reformulating the CFPS environment for each target enzyme is a significant hurdle to 449 potentiating some sensitive enzymatic outputs via CFPS.…”

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confidence: 99%

Silk Fibroin as an Additive for Cell-Free Protein Synthesis

Lee

Hung

Phillips

et al. 2020

Preprint

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17 18 19 20 34 enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are 35inhibited in the presence of the CFPS reaction mixture. Crosslinking attempts did not impact 36 CFPS activity, but did yield localized protein aggregates rather than a hydrogel. We discuss the 37 mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for 38

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Cell-free supplement mixtures: Elucidating the history and biochemical utility of additives used to support in vitro protein synthesis in E. coli extract

Cited by 78 publications

References 138 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

Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins

Silk Fibroin as an Additive for Cell-Free Protein Synthesis

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