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Constructing Cell-Free Expression Systems for Low-Cost Access
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2022-03-08 , DOI: 10.1021/acssynbio.1c00342 Fernando Guzman-Chavez 1 , Anibal Arce 2 , Abhinav Adhikari 3 , Sandra Vadhin 3 , Jose Antonio Pedroza-Garcia 4 , Chiara Gandini 5 , Jim W Ajioka 6 , Jenny Molloy 5 , Sobeida Sanchez-Nieto 4 , Jeffrey D Varner 3 , Fernan Federici 2 , Jim Haseloff 1
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2022-03-08 , DOI: 10.1021/acssynbio.1c00342 Fernando Guzman-Chavez 1 , Anibal Arce 2 , Abhinav Adhikari 3 , Sandra Vadhin 3 , Jose Antonio Pedroza-Garcia 4 , Chiara Gandini 5 , Jim W Ajioka 6 , Jenny Molloy 5 , Sobeida Sanchez-Nieto 4 , Jeffrey D Varner 3 , Fernan Federici 2 , Jim Haseloff 1
Affiliation
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Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203–424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.
中文翻译：
构建低成本的无细胞表达系统
用于基因表达的无细胞系统作为简单研究遗传回路的平台和高效的教学工具而受到关注。尽管最近取得了进展，但由于制造所需的昂贵试剂以及分配和储存所需的专用设备，许多低收入和中等收入国家仍无法获得该技术。为了应对这些挑战，我们解构了无细胞混合物制备所需的工艺，并开发了一套替代的低成本策略，以便于提取物的生产和共享。首先，我们探索了通过基于二氧化硅珠的低成本设备干燥的无细胞反应的稳定性，作为商业自动冷冻干燥机的替代品。其次，我们报告了乳糖作为添加剂在使用环状或线性 DNA 模板的基于麦芽糖糊精的无细胞反应中增加蛋白质合成的积极作用。这些修饰用于产生活性量的两种高价值试剂：等温聚合酶Bst和限制酶Bsa I。第三，我们证明了基于无细胞系统 (CFS) 中核苷三磷酸的内源再生和丙酮酸合成磷酸烯醇式丙酮酸（PEP）和麦芽糖糊精（MDX）。我们利用这一新发现来证明使用完全不含任何外源三磷酸核苷酸 (NTP) 的无细胞混合物来产生高产量的 sfGFP 表达。总之，这些修改可以生产比商业版本便宜 203-424 倍的干燥提取物。这些改进将促进 CFS 更广泛地用于研究和教育目的。
更新日期：2022-03-08
中文翻译：
构建低成本的无细胞表达系统
用于基因表达的无细胞系统作为简单研究遗传回路的平台和高效的教学工具而受到关注。尽管最近取得了进展，但由于制造所需的昂贵试剂以及分配和储存所需的专用设备，许多低收入和中等收入国家仍无法获得该技术。为了应对这些挑战，我们解构了无细胞混合物制备所需的工艺，并开发了一套替代的低成本策略，以便于提取物的生产和共享。首先，我们探索了通过基于二氧化硅珠的低成本设备干燥的无细胞反应的稳定性，作为商业自动冷冻干燥机的替代品。其次，我们报告了乳糖作为添加剂在使用环状或线性 DNA 模板的基于麦芽糖糊精的无细胞反应中增加蛋白质合成的积极作用。这些修饰用于产生活性量的两种高价值试剂：等温聚合酶Bst和限制酶Bsa I。第三，我们证明了基于无细胞系统 (CFS) 中核苷三磷酸的内源再生和丙酮酸合成磷酸烯醇式丙酮酸（PEP）和麦芽糖糊精（MDX）。我们利用这一新发现来证明使用完全不含任何外源三磷酸核苷酸 (NTP) 的无细胞混合物来产生高产量的 sfGFP 表达。总之，这些修改可以生产比商业版本便宜 203-424 倍的干燥提取物。这些改进将促进 CFS 更广泛地用于研究和教育目的。
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