Biosensing estrogenic endocrine disruptors in human blood and urine: A RAPID cell-free protein synthesis approach

Salehi, Amin S. M.; Yang, Seung Ook; Earl, Conner C.; Tang, Miriam J. Shakalli; Hunt, J. Porter; Smith, Mark T.; Wood, David; Bundy, Bradley C.

doi:10.1016/j.taap.2018.02.016

Cited by 56 publications

(39 citation statements)

References 47 publications

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“…Several of the aptamers showed the ability to 191 bind a wider range of fluorophores than what has previously been reported; for example, 192 Broccoli2X binds 4 out of the 5 fluorophores analysed (Figure 2a and S1), surprisingly 193 including ROX (5-Carboxy-X-rhodamine N-succinimidyl ester) commonly used for basal 194 line generation during routine fluorescent assays, such as qPCR. Using TXO systems 195 fluorescent responses can be generated in a matter of minutes, as compared to an hour or 196 more for reported TX-TL systems based on generation of fluorescent proteins or enzymes 197 with chromogenic substrates [6,[23][24][25][26][27]. Thus we conclude that aptamer-fluorophore 198 complexes are a suitable output for TXO synthetic biology systems.…”

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confidence: 83%

“…25 Considering the need for in-situ field analysis, an improvement of cell-free systems 26 response time is necessary [9,10]. 27 Since CFS are open and easily modifiable systems [11], time-consuming translation 28 and post-translational modification can be removed, allowing novel approaches with 29 faster signal generation. Alternative output signals independent of translation and 30 post-translational modification can provide the system with a simpler composition, 31 lower resource requirements and, most importantly, a shorter response time.…”

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confidence: 99%

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TXO: Transcription-Only genetic circuits as a novel cell-free approach for Synthetic Biology

Millacura

Valenzuela-Ortega

et al. 2019

Preprint

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While synthetic biology represents a promising approach to solve real-world problems, the use of genetically modified organisms is a cause of legal and environmental concerns. Cellfree systems have emerged as a possible solution but much work is needed to optimize their functionality and simplify their usage for Synthetic Biology. Here we present TXO, transcription-only genetic circuits, independent of translation or post-translation maturation. RNA aptamers are used as reaction output allowing the generation of fast, reliable and simple-to-design transcriptional units. TXO cell-free reactions and their possible applications are a promising new tool for fast and simple bench-to-market genetic circuit and biosensor applications. Introduction 1 Cells, including microbial, plant and mammalian cells, have been long engineered for 2 responding to a plethora of environmental factors, benefiting from their intrinsic ability 3 to process the entire cycle from the recognition of a specific target, to analysis of 4 the information, and generation of an output signal [1]. However, cell-based systems 5 present also unavoidable disadvantages during their usage, including the risk of releasing 6 genetically modified organisms (GMOs) into the environment, accumulation of mutations 7 and genetic instability, uncontrollable side-reactions within the cell, and issues with 8 viability maintenance during long-term storage [2-5]. 9In order to solve these issues, cell-free systems (CFS), also known as transcription-10 translation (TX-TL) systems, have emerged as a promising alternative. CFS not only 11 inherit most of the benefits from cell-based systems, but also avoid major challenges 12 that they face. Typically CFS are based on non-living cell-extracts or reconstituted 13 systems that by not presenting a living prospect solve problems related to biosafety, 14 cross-reactivity and long-term functionality maintenance. CFS are rather robust to 15 factors that pose cellular stress, including sensitivity to toxic pollutants and to the 16 typical "overload problem" observed when foreign "genetic circuits" are introduced into 17 the cell [6]. 18TX-TL systems share with cells a problem hindering their usage in real-time detection. 19 Response time in TX-TL systems may need a long time for transcription, translation and 20 post-translational maturation to occur, in order to finally produce a detectable signal. 21

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confidence: 83%

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confidence: 99%

TXO: Transcription-Only genetic circuits as a novel cell-free approach for Synthetic Biology

Millacura

Valenzuela-Ortega

et al. 2019

Preprint

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“…(d) CFPS for biosensing estrogenic endocrine disruptors in human blood and urine. Reprinted from Toxicology and Applied Pharmacology (Salehi et al, 2018), Biosensing estrogenic endocrine disruptors in human blood and urine: A RAPID cell‐free protein synthesis approach, 345:19–25, Doi:10.1016/j.taap.2018.02.016, Copyright (2018) with permission from Elsevier. CFPS, cell‐free protein synthesis; IY, 3‐iodo‐ l ‐tyrosine; nnAA, nonnatural amino acid; TX‐TL, transcription‐translation [Color figure can be viewed at wileyonlinelibrary.com]…”

Section: Cfps: From Test Tube Reactions To Cell‐free Expression In MImentioning

confidence: 99%

“…Notably, genomic material withdrawal from chassis organism bestows circumstances in which desired synthesis reactions are achievable at high rates . Thus, such features enable prevailing utilization of CFPS systems, which are also complementary to in vivo expression for various applications such as biocatalyst development (Rolf, Rosenthal, & Lütz, 2019), complex product fabrication (Chi, Wang, Li, Ren, & Huang, 2015), disease detection (Soltani, Davis, Ford, Nelson, & Bundy, 2018), glycoprotein synthesis (Jaroentomeechai et al, 2018), prototyping minimal cells (Yue, Zhu, & Kai, 2019), synthetic gene networks (Dubuc et al, 2019), as well as protein engineering (Hong, Kwon, & Jewett, 2014;Venkat, Chen, Gan, & Fan, Sheng, Lei, Yuan and Feng (2017) Functional CRISPR gene editing toolkit Marshall et al (2018) Pathway/network prototyping UDP-N-acetylglucosamine and UDP-GlcNAc pathway Zhou et al (2010) CRISPR-mediated gene activation and repression of the reporter and endogenous genes in mammalian cells Nakamura et al (2019) Protein engineering E. coli strain mutant for release factor 1 allows turning UAG termination codon into a sense codon for site-specific incorporation of nonnatural amino acids into proteins for selective conjugation with different biomolecules Adachi et al (2019) Incorporation of uAA AzF allows for site-specific mono and dipegylation of T4 Lysozyme Wilding et al (2018) Incorporating optimized nonnatural amino acids site-specifically, the feasibility of conjugating a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug by para-azidomethyl-L-phenylalanine (pAMF) to the tumor-specific, Her2-binding IgG Trastuzumab Zimmerman et al (2014) Biosensing Cheomogenic detection of estrogenic endocrine disruptors (hERb) in human blood and urine Salehi et al (2018) Glycoengineering Site-specific controlled glycosylation of proteins (glycoproteins) using E. coli extracts enriched for oligosaccharyltransferases and lipidlinked oligosaccharides Jaroentomeechai et al (2018) Site-directed incorporation of AzF, as well as ppropargyloxyphenylalanine from Sf21 insect cell, extracts for engineered O-glycosylation site of EPO Zemella et al (2018) Protein evolution: ribosome display Protein-tRNA-ribosome-mRNA complex for affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin Mattheakis, Bhatt and Dower (1994) Protein evolution: mRNA display Protein-puromycin-mRNA complex Roberts...…”

Section: Simple Standard Batch Reactions In Test Tubesmentioning

confidence: 99%

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Ayoubi‐Joshaghani

Dianat‐Moghadam

Seidi

et al. 2020

Biotech & Bioengineering

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Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab‐on‐chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell‐free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell‐cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell‐free protein synthesis (CFPS) formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed.

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“…Cell free protein synthesis (CFPS) has seen many recent applications in prototyping gene circuits, 1-3 sensors, [4][5][6][7][8] and therapies. [9][10][11] Cell extract derived from E. coli is the most widely used with efficient extract protocols, multiple supplement recipes, and clear experimental protocols available in literature.…”

Section: Introductionmentioning

confidence: 99%

Simple, Functional, Inexpensive Cell Extract forin vitroPrototyping of Proteins with Disulfide Bonds

Dopp

Reuel

2019

Preprint

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In vitro expression of proteins from E. coli extract is a useful method for prototyping and production of cytotoxic or unnatural products. However, proteins that have multiple disulfide bonds require custom extract that to date requires careful addition of exogenous, purified isomerase enzymes. Many important proteins such as enzymes, cytokines, and monoclonal antibodies require disulfide bonds for stabilization and/or proper activity. Currently the only solution to expressing these products is to purchase commercial kits (such as PUREfrex® (GeneFrontier) or PURExpress® (New England BioLabs)) or to grow up the KGK10 strain and supplement with purified enzymes (T7 RNA polymerase and disulfide bond isomerase C). This multistep process can limit the accessibility of such extract to some groups that wish to rapidly prototype proteins with disulfide bonds. In this work, we present a "one-pot" solution that does not require addition of supplemental enzymes. This is done using a commercially available SHuffle® T7 Express lysY strain of E. coli that can express both the needed T7 polymerase and DsbC isomerase enzymes. We find optimal growth conditions for our 1 L cultures in 2.5 L shake flasks (5.6 and 3.9 hr for harvest and induction respectively) using a luciferase (from Gaussia princeps) that contains 5 disulfide bonds as our reporter protein. The method presented here uses a continuous pass homogenizer and pilot scale lyophilizer to produce large volumes of extract; also, optimal reconstitution ratios of the dried extract are found. To show the broad applicability of the extract, three other enzymes containing ≥ 3 disulfide bonds (hevamine, endochitinase A, and periplasmic AppA) were also expressed from minimal genetic templates that had undergone rolling circle amplification.

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Biosensing estrogenic endocrine disruptors in human blood and urine: A RAPID cell-free protein synthesis approach

Cited by 56 publications

References 47 publications

TXO: Transcription-Only genetic circuits as a novel cell-free approach for Synthetic Biology

TXO: Transcription-Only genetic circuits as a novel cell-free approach for Synthetic Biology

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Simple, Functional, Inexpensive Cell Extract forin vitroPrototyping of Proteins with Disulfide Bonds

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