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Acetaminophen production in the edible, filamentous cyanobacterium Arthrospira platensis. 可食用丝状蓝藻 Arthrospira platensis 中的对乙酰氨基酚生产。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-11 DOI: 10.1002/bit.28858
Jacob M Hilzinger, Skyler Friedline, Divya Sivanandan, Ya-Fang Cheng, Shunsuke Yamazaki, Douglas S Clark, Jeffrey M Skerker, Adam P Arkin

Spirulina is the common name for the edible, nonheterocystous, filamentous cyanobacterium Arthrospira platensis that is grown industrially as a food supplement, animal feedstock, and pigment source. Although there are many applications for engineering this organism, until recently no genetic tools or reproducible transformation methods have been published. While recent work showed the production of a diversity of proteins in A. platensis, including single-domain antibodies for oral delivery, there remains a need for a modular, characterized genetic toolkit. Here, we independently establish a reproducible method for the transformation of A. platensis and engineer this bacterium to produce acetaminophen as proof-of-concept for small molecule production in an edible host. This work opens A. platensis to the wider scientific community for future engineering as a functional food for nutritional enhancement, modification of organoleptic traits, and production of pharmaceuticals for oral delivery.

螺旋藻是一种可食用的非单胞丝状蓝藻的俗称,在工业上被用作食品补充剂、动物饲料和色素来源。虽然这种生物的工程应用很多,但直到最近,还没有遗传工具或可重复的转化方法问世。虽然最近的研究表明,A. platensis 可以生产多种蛋白质,包括用于口服给药的单域抗体,但仍然需要一个模块化、特征化的基因工具包。在这里,我们独立地建立了一种可重复的方法来转化 A. platensis,并设计这种细菌来生产对乙酰氨基酚,作为在可食用宿主中生产小分子的概念验证。这项工作为更广泛的科学界打开了大门,未来可将 Platensis 改造成一种功能性食品,用于增强营养、改变感官性状和生产口服给药的药物。
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引用次数: 0
Online monitoring of methane transfer rates unveils nitrogen fixation dynamics in Methylococcus capsulatus. 甲烷转移率的在线监测揭示了荚膜甲球菌的固氮动态。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-11 DOI: 10.1002/bit.28855
Dominik Engel, Maximilian Hoffmann, Udo Kosfeld, Marcel Mann

This study explores methane utilization by the methanotrophic microorganism Methylococcus capsulatus (Bath) for biomass production, presenting a promising approach to mitigate methane emissions and foster the development sustainable biomaterials. Traditional screening methods for gas cultivations involve either serum flasks without online monitoring or costly, low-throughput fermenters. To address these limitations, the Respiration Activity MOnitoring System was augmented with methane sensors for real-time methane transfer rate (MTR) monitoring in shake flasks. Utilizing online monitoring of the MTR in shake flasks results in enhanced throughput and cost-effectiveness for cultivating M. capsulatus. Simultaneous monitoring of transfer rates for oxygen, methane, and carbon dioxide was conducted in up to eight shake flasks, ensuring the success of the cultivation process. Alterations in methane-to-oxygen transfer rate ratios and carbon fixation rates reveal the impact of transfer limitations on microbial growth. Detection of gas transfer limitations, exploration of process parameter influences, and investigations of medium components were enabled by the introduced method. Optimal nitrogen concentrations could be determined to ensure optimal growth. This streamlined approach accelerates the screening process, offering efficient investigations into metabolic effects, limitations, and parameter influences in gas fermentations without the need for elaborate offline sampling, significantly reducing costs and enhanced reproducibility.

本研究探讨了甲烷营养微生物荚膜甲烷球菌(Bath)利用甲烷生产生物质的情况,为减少甲烷排放和促进可持续生物材料的发展提供了一种前景广阔的方法。传统的气体培养筛选方法涉及没有在线监测的血清烧瓶或昂贵的低通量发酵罐。为了解决这些局限性,呼吸活动监测系统增加了甲烷传感器,用于实时监测摇瓶中的甲烷转移率(MTR)。利用在线监测摇瓶中的甲烷转移率可提高培养蝙蝠蛾的产量和成本效益。在多达八个摇瓶中同时监测氧气、甲烷和二氧化碳的转移率,确保了培养过程的成功。甲烷与氧气的转移率比率和碳固定率的变化揭示了转移限制对微生物生长的影响。采用这种方法可以检测气体转移限制、探索过程参数的影响以及研究培养基成分。可以确定最佳氮浓度,以确保最佳生长。这种简化方法加快了筛选过程,可有效研究气体发酵中的代谢作用、限制和参数影响,而无需进行复杂的离线采样,从而大大降低了成本并提高了可重复性。
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引用次数: 0
The proliferation and differentiation of skeletal muscle stem cells are enhanced in a bioreactor. 骨骼肌干细胞的增殖和分化在生物反应器中得到增强。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-06 DOI: 10.1002/bit.28857
Wei-Hsuan Lin, Chung-Yuh Tzeng, Fan-Che Kao, Chia-Wen Tsao, Ning Li, Chuan-Che Wu, Sheng-Huei Lee, Kai-Fan Huang, Wei-Wen Hu, Shen-Liang Chen

Skeletal muscle (SKM) is the largest organ in mammalian body and it can repair damages by using the residential myogenic stem cells (MuSC), but this repairing capacity reduces with age and in some genetic muscular dystrophy. Under these circumstances, artificial amplification of autologous MuSC in vitro might be necessary to repair the damaged SKM. The amplification of MuSC is highly dependent on myogenic signals, such as sonic hedgehog (Shh), Wnt3a, and fibroblast growth factors, so formulating an optimum myogenic kit composed of specific myogenic signals might increase the proliferation and differentiation of MuSC efficiently. In this study, various myogenic signals have been tested on C2C12 myoblasts and primary MuSC, and a myogenic kit consists of insulin, lithium chloride, T3, and retinoic acid has been formulated, and we found it significantly increased the fusion index and MHC expression level of both C2C12 and MuSC myotubes. A novel bioreactor providing cyclic stretching (CS) and electrical stimulation (ES) has been fabricated to enhance the myogenic differentiation of both C2C12 and MuSC. We further found that coating the bioreactor substratum with collagen gave the best effect on proliferation and differentiation of MuSC. Furthermore, combining the collagen coating and physical stimuli (CS + ES) in the bioreactor can generate more proliferative primary MuSC cells. Our results have demonstrated that the combination of myogenic kit and bioreactor can provide environment for efficient MuSC proliferation and differentiation. These MuSC and mature myotubes amplified in the bioreactor might be useful for clinical grafting into damaged SKM in the future.

骨骼肌(SKM)是哺乳动物体内最大的器官,它可以利用体内的肌原干细胞(MuSC)修复损伤,但这种修复能力会随着年龄的增长和某些遗传性肌肉萎缩症的发生而减弱。在这种情况下,可能需要在体外人工扩增自体MuSC来修复受损的SKM。MuSC的扩增高度依赖于致肌信号,如声波刺猬(Shh)、Wnt3a和成纤维细胞生长因子,因此配制由特定致肌信号组成的最佳致肌试剂盒可能会有效地增加MuSC的增殖和分化。本研究在C2C12肌母细胞和原代MuSC上测试了各种致肌信号,并配制了由胰岛素、氯化锂、T3和维甲酸组成的致肌试剂盒,结果发现它能显著提高C2C12和MuSC肌管的融合指数和MHC表达水平。我们制作了一种新型生物反应器,提供循环拉伸(CS)和电刺激(ES),以增强 C2C12 和 MuSC 的成肌分化。我们进一步发现,在生物反应器的基底上涂覆胶原蛋白对 MuSC 的增殖和分化效果最佳。此外,在生物反应器中结合使用胶原涂层和物理刺激(CS + ES)可以产生更多增殖的原代MuSC细胞。我们的研究结果表明,成肌试剂盒与生物反应器的结合可为成肌细胞的增殖和分化提供有效的环境。这些在生物反应器中扩增的MuSC和成熟的肌小管将来可能会用于临床移植到受损的SKM中。
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引用次数: 0
Biointerface engineering through amalgamation of gene technology and site-specific growth factor conjugation for efficient osteodifferentiation. 通过基因技术与特定位点生长因子结合的生物界面工程,实现高效骨分化。
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-19 DOI: 10.1002/bit.28852
Zhenxu Wu,Li Mo,Zongliang Wang,Liangsong Song,Eiry Kobatake,Yoshihiro Ito,Yi Wang,Peibiao Zhang
The development of bone implants through bioinspired immobilization of growth factors remains a key issue in the generation of biological interfaces, especially in enhancing osteodifferentiation ability. In this study, we developed a strategy for surface functionalization of poly(lactide-glycolide) (PLGA) and hydroxyapatite (HA) composite substrates through site-specific conjugation of bone morphogenetic protein 2 containing 3,4-hydroxyphenalyalanine (DOPA-BMP2) mediated by tyrosinase and sortase A (SrtA). Firstly, the growth factor BMP2-LPETG containing LPETG motif was successfully expressed in Escherichia coli through recombinant DNA technology. The excellent binding affinity of binding growth factor (DOPA-BMP2) was achieved by converting the tyrosine residue (Y) of YKYKY-GGG peptide into DOPA (X) by tyrosinase, which bound to the substrates. Then its GGG motif was specifically bound to the end of BMP2-LPETG mediated by SrtA. Therefore, the generated bioactive DOPA-BMP2/PLGA/HA substrates significantly promoted the osteogenic differentiation of MC3T3-E1 cells. Thanks to this microbial-assisted engineering approach, our work presents a facile and highly site-specific strategy to engineer biomimetic materials for orthopedics and dentistry by effectively delivering growth factors, peptides, and other biomacromolecules.
通过生物启发固定生长因子来开发骨植入物仍然是生物界面生成过程中的一个关键问题,尤其是在增强骨分化能力方面。在这项研究中,我们开发了一种在酪氨酸酶和分选酶 A(SrtA)的介导下,通过特定位点共轭含有 3,4-羟基苯丙氨酸的骨形态发生蛋白 2(DOPA-BMP2),实现聚(乳糖-乙二醇)(PLGA)和羟基磷灰石(HA)复合基底表面功能化的策略。首先,通过 DNA 重组技术在大肠杆菌中成功表达了含有 LPETG 矩阵的生长因子 BMP2-LPETG。通过酪氨酸酶将 YKYKY-GGG 肽中的酪氨酸残基(Y)转化为 DOPA(X),与底物结合,实现了生长因子(DOPA-BMP2)的优异结合亲和力。然后,在 SrtA 的介导下,其 GGG 基序与 BMP2-LPETG 的末端特异性结合。因此,生成的生物活性 DOPA-BMP2/PLGA/HA 基质能显著促进 MC3T3-E1 细胞的成骨分化。得益于这种微生物辅助工程方法,我们的工作提出了一种简便且高度定点的策略,通过有效传递生长因子、肽和其他生物大分子,为整形外科和牙科设计生物仿生材料。
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引用次数: 0
Buffer system improves the removal of host cell protein impurities in monoclonal antibody purification 缓冲液系统可改善单克隆抗体纯化过程中宿主细胞蛋白质杂质的去除效果
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-18 DOI: 10.1002/bit.28844
Dániel Lakatos, Martina Idler, Selina Stibitzky, Jennifer Amann, Jakob Schuschkewitz, Dominik Krayl, Judith Liebau, Jan-Hendrik Grosch, Erik Arango Gutierrez, Simon Kluters
Polysorbates (PS) are commonly used as stabilizers of biopharmaceuticals such as monoclonal antibodies (mAbs). However, they are prone to chemical and enzymatic degradation. The latter can be caused by residual host cell proteins (HCPs) in the drug substance. Degradation affects the functionality of the PS surfactant which can lead to formation of particles. An increasing number of publications describe enzymatic PS degradation. Significant efforts have been made to characterize HCP removal during Downstream Processing (DSP) of mAbs and to develop mitigation strategies. Here we describe the use of glycine buffer for acidic elution in Protein A affinity chromatography compared to acetate buffer, which is more commonly used in the biopharmaceutical industry. Increased turbidity was observed during pH re-adjustment after low pH virus inactivation when using glycine buffer. Analytical data suggests that this turbidity is caused by the formation of precipitates which include HCP and DNA impurities. Additionally, as a zwitterion, glycine does not contribute to conductivity; this further enhances HCP removal during anion-exchange flow-through chromatography. Although glycine is well known as a possible elution buffer for Protein A affinity chromatography, its positive impact on HCP removal and PS stability have not yet been described in literature.
聚山梨醇酯(PS)通常用作单克隆抗体(mAbs)等生物制药的稳定剂。然而,它们容易发生化学和酶降解。后者可由药物中残留的宿主细胞蛋白(HCPs)引起。降解会影响 PS 表面活性剂的功能,从而导致颗粒的形成。越来越多的出版物对 PS 的酶降解进行了描述。为了描述 mAbs 下游加工 (DSP) 过程中 HCP 的去除情况并制定缓解策略,人们付出了巨大的努力。与生物制药行业更常用的醋酸盐缓冲液相比,我们在此介绍在蛋白 A 亲和层析中使用甘氨酸缓冲液进行酸性洗脱的情况。在使用甘氨酸缓冲液进行低 pH 值病毒灭活后的 pH 值再调整过程中,观察到浑浊度增加。分析数据表明,这种浑浊是由包括 HCP 和 DNA 杂质在内的沉淀物形成造成的。此外,作为一种齐聚物,甘氨酸不会对电导率产生影响;这进一步提高了阴离子交换流动色谱法对 HCP 的去除率。虽然甘氨酸作为蛋白质 A 亲和层析的洗脱缓冲液已广为人知,但其对 HCP 去除和 PS 稳定性的积极影响尚未在文献中有所描述。
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引用次数: 0
3D printed gelatin methacryloyl hydrogels for perfusion culture of human trabecular meshwork cells and glaucoma studies 用于人类小梁网细胞灌注培养和青光眼研究的三维打印明胶甲基丙烯酰水凝胶
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-18 DOI: 10.1002/bit.28849
Bikram Adhikari, Prasanga Barakoti, Mina B. Pantcheva, Melissa D. Krebs
Glaucoma, a progressive eye disease leading to irreversible blindness, currently affects over 70 million people globally. Elevated intraocular pressure (IOP) is implicated in its development. IOP is carefully regulated by the trabecular meshwork (TM). However, studying TM behavior has been limited to traditional tissue culture studies or costly ex vivo cultures of animal and donor eyes. Developing novel functional TM models could enhance cell/tissue behavior understanding and aid therapeutic development for glaucoma. In this study, we 3D printed a simplified and reproducible model of the human TM (hTM) and studied hTM cell behavior under static and dynamic cultures. Gelatin Methacryloyl bioinks proved suitable for printing with viable and proliferative hTM cells expressing crucial marker genes in response to glucocorticoid induction. This, to our knowledge, is the first functional 3D printed hTM model and aims to facilitate TM research. Moreover, this easily reproducible model could also be applicable in the study of numerous other cell types throughout the body.
青光眼是一种渐进性眼病,会导致不可逆转的失明,目前全球有 7000 多万人患有青光眼。眼压升高与青光眼的发病有关。眼压受小梁网(TM)的严格调节。然而,对小梁啮合行为的研究一直局限于传统的组织培养研究或昂贵的动物和供体眼球体外培养。开发新型功能性小梁网模型可以加深对细胞/组织行为的理解,有助于青光眼的治疗开发。在这项研究中,我们用三维打印技术打印了一个简化的、可重复的人类 TM(hTM)模型,并研究了 hTM 细胞在静态和动态培养下的行为。事实证明,明胶甲基丙烯酰生物链接适合打印有活力和增殖的 hTM 细胞,这些细胞在糖皮质激素诱导下表达重要的标记基因。据我们所知,这是首个功能性三维打印 hTM 模型,旨在促进 TM 研究。此外,这种易于复制的模型还可用于研究全身其他多种细胞类型。
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引用次数: 0
Culture system for longitudinal monitoring of bone dynamics ex vivo 用于纵向监测体内外骨骼动态的培养系统
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-18 DOI: 10.1002/bit.28848
Esther E.A. Cramer, Kim C.J. Hermsen, Linda M. Kock, Keita Ito, Sandra Hofmann
To quantify and visualize both bone formation and resorption within osteochondral explants cultured ex vivo is challenging with the current analysis techniques. An approach that enables monitoring of bone remodeling dynamics is longitudinal microcomputed tomography (µCT), a non-destructive technique that relies on repeated µCT scanning and subsequent registration of consecutive scans. In this study, a two-compartment culture system suitable for osteochondral explants that allowed for µCT scanning during ex vivo culture was established. Explants were scanned repeatedly in a fixed orientation, which allowed assessment of bone remodeling due to adequate image registration. Using this method, bone formation was found to be restricted to the outer surfaces when cultured statically. To demonstrate that the culture system could capture differences in bone remodeling, explants were cultured statically and under dynamic compression as loading promotes osteogenesis. No quantitative differences between static and dynamic culture were revealed. Still, only in dynamic conditions, bone formation was visualized on trabecular surfaces located within the inner cores, suggesting enhanced bone formation towards the center of the explants upon mechanical loading. Taken together, the ex vivo culture system in combination with longitudinal µCT scanning and subsequent registration of images demonstrated potential for evaluating bone remodeling within explants.
要量化和可视化体外培养骨软骨外植体的骨形成和骨吸收,目前的分析技术还很难做到。纵向微计算机断层扫描(µCT)是一种能够监测骨重塑动态的方法,它是一种非破坏性技术,依赖于µCT的重复扫描和连续扫描的后续注册。本研究建立了一种适合骨软骨外植体的两室培养系统,可在体外培养过程中进行 µCT 扫描。外植体以固定的方向反复扫描,通过适当的图像配准可以评估骨重塑情况。使用这种方法发现,在静态培养时,骨形成仅限于外表面。为了证明该培养系统能捕捉到骨重塑的差异,对外植体进行了静态培养和动态压缩培养,因为加载会促进骨生成。结果显示,静态培养和动态培养之间没有数量上的差异。不过,只有在动态条件下,位于内核的骨小梁表面才能看到骨形成,这表明在机械加载时,外植体中心的骨形成会增强。综上所述,体内外培养系统与纵向 µCT 扫描及随后的图像配准相结合,证明了评估外植体内骨重塑的潜力。
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引用次数: 0
Physics-informed neural networks for biopharmaceutical cultivation processes: Consideration of varying process parameter settings 用于生物制药培养过程的物理信息神经网络:考虑不同的工艺参数设置
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-18 DOI: 10.1002/bit.28851
Niklas Adebar, Sabine Arnold, Liliana M. Herrera, Victor N. Emenike, Thomas Wucherpfennig, Jens Smiatek
We present a new modeling approach for the study and prediction of important process outcomes of biotechnological cultivation processes under the influence of process parameter variations. Our model is based on physics-informed neural networks (PINNs) in combination with kinetic growth equations. Using Taylor series, multivariate external process parameter variations for important variables such as temperature, seeding cell density and feeding rates can be integrated into the corresponding kinetic rates and the governing growth equations. In addition to previous approaches, PINNs also allow continuous and differentiable functions as predictions for the process outcomes. Accordingly, our results show that PINNs in combination with Taylor-series expansions for kinetic growth equations provide a very high prediction accuracy for important process variables such as cell densities and concentrations as well as a detailed study of individual and combined parameter influences. Furthermore, the proposed approach can also be used to evaluate the outcomes of new parameter variations and combinations, which enables a saving of experiments in combination with a model-driven optimization study of the design space.
我们提出了一种新的建模方法,用于研究和预测生物技术培养过程在工艺参数变化影响下的重要工艺结果。我们的模型基于物理信息神经网络(PINN)与动力学生长方程的结合。利用泰勒级数,可以将温度、播种细胞密度和喂食率等重要变量的多变量外部过程参数变化整合到相应的动力学速率和支配生长方程中。与之前的方法相比,PINN 还允许用连续和可微分函数来预测过程结果。因此,我们的研究结果表明,将 PINNs 与泰勒级数展开相结合用于动力学生长方程,可为细胞密度和浓度等重要过程变量提供极高的预测精度,并可对单个参数和组合参数的影响进行详细研究。此外,建议的方法还可用于评估新参数变化和组合的结果,从而节省实验时间,并结合模型驱动对设计空间进行优化研究。
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引用次数: 0
Clear insight into complex multimodal resins and impurities to overcome recombinant protein purification challenges: A review 清晰洞察复杂的多模式树脂和杂质,克服重组蛋白纯化难题:综述
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-18 DOI: 10.1002/bit.28846
Maryam Moazami Goodarzi, Reza Jalalirad
Increasing attention has been paid to the purity of therapeutic proteins imposing extensive costs and challenges to the downstream processing of biopharmaceuticals. One of the efforts, that has been exerted to overcome such limitations, was developing multimodal or mixed‐mode chromatography (MMC) resins for launching selective, orthogonal, non‐affinity purification platforms. Despite relatively extensive usage of MMC resins, their real potential and fulfillment have not been extensively reviewed yet. In this work, the explanation of practical and key aspects of downstream processing of recombinant proteins with or without MMC resins was debated, as being useful for further purification process development. This review has been written as a step‐by‐step guide to deconvolute both inherent protein purification and MMC complexities. Here, after complete elucidation of the potential of MMC resins, the effects of frequently used additives (mobile phase modifiers) and their possible interactions during the purification process, the critical characteristics of common product‐related impurities (e.g., aggregates, charge variants, fragments), host‐related impurities (e.g., host cell protein and DNA) and process related impurities (e.g., endotoxin, and viruses) with solved or unsolved challenges of traditional and MMC resins have been discussed. Such collective experiences which are reported in this study could be considered as an applied guide for developing successful downstream processing in challenging conditions by providing a clear insight into complex MMC resins and impurities.
人们越来越重视治疗蛋白质的纯度,这给生物制药的下游加工带来了巨大的成本和挑战。为克服这些限制,人们一直在努力开发多模式或混合模式色谱(MMC)树脂,以推出选择性、正交、非亲和纯化平台。尽管 MMC 树脂的应用相对广泛,但其真正的潜力和作用尚未得到广泛的研究。在这项工作中,对使用或不使用 MMC 树脂进行重组蛋白下游处理的实用性和关键方面进行了讨论,这对进一步的纯化工艺开发非常有用。这篇综述的写作是为了逐步解开蛋白质纯化和 MMC 的内在复杂性。在全面阐明了 MMC 树脂的潜力之后,还讨论了常用添加剂(流动相改性剂)的影响及其在纯化过程中可能产生的相互作用,以及常见的产品相关杂质(如聚集体、电荷变异体、片段)、宿主相关杂质(如宿主细胞蛋白和 DNA)和工艺相关杂质(如内毒素和病毒)的关键特征,以及传统树脂和 MMC 树脂已解决或未解决的难题。本研究报告中介绍的这些集体经验可作为应用指南,通过提供对复杂的 MMC 树脂和杂质的清晰认识,在具有挑战性的条件下开发成功的下游加工工艺。
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引用次数: 0
Active diagnostic ingredients (ADIs) for PCR: A mini-bioreactor producing dNTPs with silica immobilized R5-kinases 用于 PCR 的活性诊断成分 (ADI):用硅胶固定 R5 激酶生产 dNTPs 的微型生物反应器
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-18 DOI: 10.1002/bit.28837
Anna R. Bird, Elizabeth A. H. Hall
Low availability of routine nucleic acid amplification testing (NAAT) during infection outbreaks, especially in less resourced environments, was highlighted by the Covid pandemic. One of the barriers lies with the supply chain and cost of the active diagnostic ingredients (ADIs) that are the reagents for NAATs. This work explores a novel synthesis method to produce a key NAAT reagent, namely the 2′-deoxynucleoside 5′-triphosphate (dNTPs), via a reusable enzyme bioreactor, that can be integrated into a NAAT workflow. A self-immobilizing R5-silaffin kinase fusion enzyme was designed for immobilization on silica, converting dNMPs to their respective dNTP ADIs for PCR in a R5-kinase mini-bioreactor, designed to be implemented in a reusable device, stable over 2 months, when stored at 4°C. The performance is demonstrated for PCR reactions of the lambda genome and showed successful amplification up to 7.5 kb. In comparison with commercial dNTPs, in Plasmodium malariae NAATs, a high linear correlation was shown between the Ct value and the log(Copy Number), with lower incidence of false positives than with the commercial dNTPs. Overall a pathway to generate deoxynucleotides from monophosphate precursors was demonstrated, and an immobilized enzyme mini-bioreactor investigated as a proof-of-principle for work-flow integration with NAAT in low-resource research and diagnostics labs.
Covid 大流行凸显了在感染爆发期间,特别是在资源较少的环境中,常规核酸扩增检测(NAAT)的可用性较低。障碍之一在于作为 NAAT 试剂的活性诊断成分(ADI)的供应链和成本。这项研究探索了一种新的合成方法,通过可重复使用的酶生物反应器生产一种关键的 NAAT 试剂,即 2′-脱氧核苷酸 5′-三磷酸酯(dNTPs),该方法可集成到 NAAT 工作流程中。设计了一种固定在二氧化硅上的自固定 R5-丝氨酸激酶融合酶,在 R5 激酶微型生物反应器中将 dNMPs 转化为 PCR 所需的相应 dNTP ADIs。在对λ基因组进行 PCR 反应时证明了其性能,并成功扩增了 7.5 kb。与商用 dNTP 相比,在疟疾疟原虫 NAATs 中,Ct 值与 log(拷贝数)之间呈高度线性相关,假阳性发生率低于商用 dNTP。总之,从单磷酸前体生成脱氧核苷酸的途径已得到证实,并对固定化酶微型生物反应器进行了研究,作为低资源研究和诊断实验室与 NAAT 工作流程整合的原理验证。
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