Pub Date : 2024-10-04DOI: 10.1016/j.jbiosc.2024.09.001
Céleste Caffin, Lhéa Milhamont, Eva Duriez, Agathe Hembert, Pauline Huzet, Camille Lerouge, Marie Deblieck, Denis Watier
The growing use of bacteriophages in the fields of agriculture, agri-food, veterinary treatments, and medicine involves the quantitative production of these bacteriophages. In this study, we propose a bacteriophage production protocol that can easily be transposed to industry. We used a cellstat production system because the latest studies have shown that it is the most suitable process for the production of phages due to volumetric productivity, safety (limitation of co-evolution), and flexibility (choice of growth rate criteria). Sizing of the assembly used makes it possible to extrapolate the results to industrial production. The production conditions are indicated precisely, which would allow manufacturers to adapt the protocol to their own equipment. We propose experimental conditions in order to obtain a stable Escherichia coli population, qualitatively and over time, and production of high-titer T7 bacteriophages. The optimized production conditions (yield, cost and simplicity of the process) are: a buffered peptone water medium concentration of 11 g L-1 and a dilution rate of 1.6 h-1. Under these conditions, we obtained a production of 7.35×1016 plaque-forming units (PFU) L-1 day-1 with a concentration of 9.8×1012 PFU mL-1. The strength of this work lies in its focus on industrial applicability.
噬菌体在农业、农业食品、兽医治疗和医药领域的应用日益广泛,这涉及到这些噬菌体的定量生产。在本研究中,我们提出了一种噬菌体生产方案,该方案可以很容易地移植到工业领域。我们使用了细胞培养生产系统,因为最新研究表明,由于体积生产率、安全性(限制共同进化)和灵活性(选择生长速度标准),细胞培养生产系统是最适合噬菌体生产的工艺。对所使用的组件进行选型,可以将结果推断为工业化生产。对生产条件进行了精确说明,使制造商能够根据自己的设备调整方案。我们提出了一些实验条件,以获得稳定的大肠杆菌种群,并生产高滴度的 T7 噬菌体。优化的生产条件(产量、成本和工艺的简易性)是:缓冲蛋白胨水培养基浓度为 11 g L-1,稀释率为 1.6 h-1。在这些条件下,我们获得了 7.35×1016 plaque-forming units (PFU) L-1 day-1 的产量和 9.8×1012 PFU mL-1 的浓度。这项工作的优势在于其对工业应用的关注。
{"title":"Optimization of bacteriophage propagation in high-yield continuous culture (cellstat) meeting the constraints of industrial manufacturing processes.","authors":"Céleste Caffin, Lhéa Milhamont, Eva Duriez, Agathe Hembert, Pauline Huzet, Camille Lerouge, Marie Deblieck, Denis Watier","doi":"10.1016/j.jbiosc.2024.09.001","DOIUrl":"10.1016/j.jbiosc.2024.09.001","url":null,"abstract":"<p><p>The growing use of bacteriophages in the fields of agriculture, agri-food, veterinary treatments, and medicine involves the quantitative production of these bacteriophages. In this study, we propose a bacteriophage production protocol that can easily be transposed to industry. We used a cellstat production system because the latest studies have shown that it is the most suitable process for the production of phages due to volumetric productivity, safety (limitation of co-evolution), and flexibility (choice of growth rate criteria). Sizing of the assembly used makes it possible to extrapolate the results to industrial production. The production conditions are indicated precisely, which would allow manufacturers to adapt the protocol to their own equipment. We propose experimental conditions in order to obtain a stable Escherichia coli population, qualitatively and over time, and production of high-titer T7 bacteriophages. The optimized production conditions (yield, cost and simplicity of the process) are: a buffered peptone water medium concentration of 11 g L<sup>-1</sup> and a dilution rate of 1.6 h<sup>-1</sup>. Under these conditions, we obtained a production of 7.35×10<sup>16</sup> plaque-forming units (PFU) L<sup>-1</sup> day<sup>-1</sup> with a concentration of 9.8×10<sup>12</sup> PFU mL<sup>-1</sup>. The strength of this work lies in its focus on industrial applicability.</p>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142377847","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-28DOI: 10.1016/j.jbiosc.2024.08.002
Della Rahmawati, Mary Faith Yamballa Adan, Muhammad Maulana Malikul Ikram, Marvin Nathanael Iman, Eiichiro Fukusaki, Sastia Prama Putri
Young coconuts (Cocos nucifera L.) used for export are trimmed to reduce their size and weight to lower transport costs. However, trimmed coconuts have a shorter shelf life due to microbial spoilage and surface discoloration caused by enzymatic browning. To minimize these effects, trimmed coconuts were dipped in an anti-browning agent, sodium metabisulfite (SMB), and stored under ambient conditions. However, there have been no reports on the effects of SMB treatment on metabolome changes in the flesh and water of young coconuts. Hence, this study investigated the metabolite changes in trimmed young coconuts after SMB treatment under different storage conditions using a gas chromatography (GC)/mass spectrometry (MS) metabolomic profiling approach. Tall young coconut samples were trimmed and treated with a 2% SMB solution for 5 min before storage at 25 °C or 4 °C for 2-4 weeks. Coconut flesh and water samples were collected after storage for 0, 2, and 4 weeks, and were subjected to GC-MS analysis. The results showed that the major metabolites affected by coconut deterioration were amino acids, sugars, and sugar alcohols. SMB treatment and/or refrigeration can help prevent metabolite changes in the flesh and water of young coconuts. In the future, improvements in storage conditions based on metabolite profiles should be explored.
{"title":"Effect of sodium metabisulfite treatment and storage condition on metabolic profile of young coconut (Cocos nucifera L.).","authors":"Della Rahmawati, Mary Faith Yamballa Adan, Muhammad Maulana Malikul Ikram, Marvin Nathanael Iman, Eiichiro Fukusaki, Sastia Prama Putri","doi":"10.1016/j.jbiosc.2024.08.002","DOIUrl":"https://doi.org/10.1016/j.jbiosc.2024.08.002","url":null,"abstract":"<p><p>Young coconuts (Cocos nucifera L.) used for export are trimmed to reduce their size and weight to lower transport costs. However, trimmed coconuts have a shorter shelf life due to microbial spoilage and surface discoloration caused by enzymatic browning. To minimize these effects, trimmed coconuts were dipped in an anti-browning agent, sodium metabisulfite (SMB), and stored under ambient conditions. However, there have been no reports on the effects of SMB treatment on metabolome changes in the flesh and water of young coconuts. Hence, this study investigated the metabolite changes in trimmed young coconuts after SMB treatment under different storage conditions using a gas chromatography (GC)/mass spectrometry (MS) metabolomic profiling approach. Tall young coconut samples were trimmed and treated with a 2% SMB solution for 5 min before storage at 25 °C or 4 °C for 2-4 weeks. Coconut flesh and water samples were collected after storage for 0, 2, and 4 weeks, and were subjected to GC-MS analysis. The results showed that the major metabolites affected by coconut deterioration were amino acids, sugars, and sugar alcohols. SMB treatment and/or refrigeration can help prevent metabolite changes in the flesh and water of young coconuts. In the future, improvements in storage conditions based on metabolite profiles should be explored.</p>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347274","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The fibroblast growth factor-2 (FGF-2) is a critical protein for biological processes such as angiogenesis and tissue regeneration. Recently, hydrogels based on semi-synthetic sulfated polysaccharides have been developed for the controlled delivery of FGF-2. These affinity-based FGF-2 carriers utilizing hydrogels based on sulfated polysaccharides enable sustained delivery of FGF-2, yet choice of materials is limited. Here, we demonstrate a novel synthetic sulfated polysaccharide-based hydrogel based on bacterial polyglucuronic acid (PGU). We synthesized phenol-grafted sulfated PGU (PGUS-Ph), an enzymatically cross-linkable PGU derivative that exhibited an enhanced affinity for FGF-2. The aqueous solution of PGUS-Ph, when combined with FGF-2, could be injected into affected sites and form a hydrogel in a minimally invasive manner. The FGF-2 released from the PGUS-Ph hydrogel induced blood vessel formation, as proven by a chick embryo-based angiogenesis assay. Our results indicate that the PGUS-Ph has the potential as an enzymatically cross-linkable and minimally invasively injectable affinity-based FGF-2 delivery system.
{"title":"Enzymatically cross-linkable sulfated bacterial polyglucuronic acid as an affinity-based carrier of FGF-2 for therapeutic angiogenesis.","authors":"Ryota Goto, Shinji Sakai, Cédric Delattre, Emmanuel Petit, Redouan El Boutachfaiti, Masaki Nakahata","doi":"10.1016/j.jbiosc.2024.08.011","DOIUrl":"https://doi.org/10.1016/j.jbiosc.2024.08.011","url":null,"abstract":"<p><p>The fibroblast growth factor-2 (FGF-2) is a critical protein for biological processes such as angiogenesis and tissue regeneration. Recently, hydrogels based on semi-synthetic sulfated polysaccharides have been developed for the controlled delivery of FGF-2. These affinity-based FGF-2 carriers utilizing hydrogels based on sulfated polysaccharides enable sustained delivery of FGF-2, yet choice of materials is limited. Here, we demonstrate a novel synthetic sulfated polysaccharide-based hydrogel based on bacterial polyglucuronic acid (PGU). We synthesized phenol-grafted sulfated PGU (PGUS-Ph), an enzymatically cross-linkable PGU derivative that exhibited an enhanced affinity for FGF-2. The aqueous solution of PGUS-Ph, when combined with FGF-2, could be injected into affected sites and form a hydrogel in a minimally invasive manner. The FGF-2 released from the PGUS-Ph hydrogel induced blood vessel formation, as proven by a chick embryo-based angiogenesis assay. Our results indicate that the PGUS-Ph has the potential as an enzymatically cross-linkable and minimally invasively injectable affinity-based FGF-2 delivery system.</p>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347276","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-23DOI: 10.1016/j.jbiosc.2024.08.010
Masahiro Watanabe, Yusuke Nakamichi, Shohei Mine
d-Allulose is a low-calorie sweetener with multiple nutritional functions that can be produced through d-fructose isomerization by ketose 3-epimerase (KEase). l-Ribulose 3-epimerase from Arthrobacterglobiformis (AgLRE) is one of the most important enzymes that produce d-allulose; however, its substrate recognition mechanism is unknown. In this study, the crystal structures of AgLRE and its complex with d-allulose and d-fructose were determined. Upon substrate binding, the hydrophobic residues around the active-site entrance move toward the bound substrate. A comparison of AgLRE and other KEase structures revealed that the substrate-binding residues are not the main factors responsible for its marked specificity for d-allulose and d-fructose, but the hydrophobicity of the active site pocket influences substrate recognition. Particularly, the two hydrophobic regions at the active site entrance are the regulatory elements that modulate substrate recognition by AgLRE. This study provides useful information for designing AgLRE to increase its affinity for d-allulose and d-fructose.
{"title":"Elucidation of d-allulose recognition mechanism in ketose 3-epimerase.","authors":"Masahiro Watanabe, Yusuke Nakamichi, Shohei Mine","doi":"10.1016/j.jbiosc.2024.08.010","DOIUrl":"https://doi.org/10.1016/j.jbiosc.2024.08.010","url":null,"abstract":"<p><p>d-Allulose is a low-calorie sweetener with multiple nutritional functions that can be produced through d-fructose isomerization by ketose 3-epimerase (KEase). l-Ribulose 3-epimerase from Arthrobacterglobiformis (AgLRE) is one of the most important enzymes that produce d-allulose; however, its substrate recognition mechanism is unknown. In this study, the crystal structures of AgLRE and its complex with d-allulose and d-fructose were determined. Upon substrate binding, the hydrophobic residues around the active-site entrance move toward the bound substrate. A comparison of AgLRE and other KEase structures revealed that the substrate-binding residues are not the main factors responsible for its marked specificity for d-allulose and d-fructose, but the hydrophobicity of the active site pocket influences substrate recognition. Particularly, the two hydrophobic regions at the active site entrance are the regulatory elements that modulate substrate recognition by AgLRE. This study provides useful information for designing AgLRE to increase its affinity for d-allulose and d-fructose.</p>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347275","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-19DOI: 10.1016/j.jbiosc.2024.08.003
Rui Lei, Xi-Peng Liu
Single nucleotide polymorphism (SNP), as one of the key components of the genetic factors, is important for disease detection and early screening of hereditary diseases. Current SNP genotyping methods require laboratory instruments or long operating times. To facilitate the diagnosis of hereditary diseases, we developed a new method referred to as the LwaCas13a-based SNP genotyping platform (Cas13a platform), which is useful for detecting disease-related SNPs. We report a CRISPR/Cas13a-based SNP genotyping platform that couples recombinase-aided amplification (RAA), T7 transcription, and Leptotrichia wadei Cas13a (LwaCas13a) detection for simple and fast genotyping of human disease-related SNPs. We used this Cas13a platform to identify 17 disease-related SNPs, demonstrating that position 2 in gRNA is suitable for the introduction of additional mismatches to achieve high discrimination in genotyping across a wide range of SNP targets. The discrimination specificity of 17 SNPs was improved 3.0-35.1-fold after introducing additional mismatches at position 2 from the 5'-end. We developed a method, which has a lower risk of cross-contamination and operational complexity, for genotyping SNPs using human saliva samples in an one-pot testing that delivers results within 60 min. Compared to TaqMan probe qPCR, RFLP, AS-PCR and other SNP genotyping methods, the Cas13a platform is simple, rapid and reliable, expanding the applications of the CRISPR/Cas system in nucleic acid detection and SNP genotyping.
单核苷酸多态性(SNP)作为遗传因子的重要组成部分之一,对于疾病检测和遗传性疾病的早期筛查具有重要意义。目前的 SNP 基因分型方法需要实验室仪器或较长的操作时间。为了促进遗传性疾病的诊断,我们开发了一种新方法,即基于LwaCas13a的SNP基因分型平台(Cas13a平台),它可用于检测与疾病相关的SNP。我们报告了一种基于CRISPR/Cas13a的SNP基因分型平台,它将重组酶辅助扩增(RAA)、T7转录和Leptotrichia wadei Cas13a(LwaCas13a)检测结合在一起,用于简单快速地对人类疾病相关SNP进行基因分型。我们利用这一 Cas13a 平台鉴定了 17 个疾病相关 SNP,证明 gRNA 中的第 2 位适合引入额外的错配,从而在广泛的 SNP 目标基因分型中实现高分辨。在 5'-end 的第 2 位引入额外的错配后,17 个 SNP 的鉴别特异性提高了 3.0-35.1 倍。我们开发了一种利用人体唾液样本进行 SNP 基因分型的方法,该方法具有较低的交叉污染风险和操作复杂性,可在 60 分钟内完成一次检测。与 TaqMan 探针 qPCR、RFLP、AS-PCR 和其他 SNP 基因分型方法相比,Cas13a 平台简单、快速、可靠,拓展了 CRISPR/Cas 系统在核酸检测和 SNP 基因分型中的应用。
{"title":"Rapid one-pot human single nucleotide polymorphism genotyping platform with Cas13a nuclease.","authors":"Rui Lei, Xi-Peng Liu","doi":"10.1016/j.jbiosc.2024.08.003","DOIUrl":"https://doi.org/10.1016/j.jbiosc.2024.08.003","url":null,"abstract":"<p><p>Single nucleotide polymorphism (SNP), as one of the key components of the genetic factors, is important for disease detection and early screening of hereditary diseases. Current SNP genotyping methods require laboratory instruments or long operating times. To facilitate the diagnosis of hereditary diseases, we developed a new method referred to as the LwaCas13a-based SNP genotyping platform (Cas13a platform), which is useful for detecting disease-related SNPs. We report a CRISPR/Cas13a-based SNP genotyping platform that couples recombinase-aided amplification (RAA), T7 transcription, and Leptotrichia wadei Cas13a (LwaCas13a) detection for simple and fast genotyping of human disease-related SNPs. We used this Cas13a platform to identify 17 disease-related SNPs, demonstrating that position 2 in gRNA is suitable for the introduction of additional mismatches to achieve high discrimination in genotyping across a wide range of SNP targets. The discrimination specificity of 17 SNPs was improved 3.0-35.1-fold after introducing additional mismatches at position 2 from the 5'-end. We developed a method, which has a lower risk of cross-contamination and operational complexity, for genotyping SNPs using human saliva samples in an one-pot testing that delivers results within 60 min. Compared to TaqMan probe qPCR, RFLP, AS-PCR and other SNP genotyping methods, the Cas13a platform is simple, rapid and reliable, expanding the applications of the CRISPR/Cas system in nucleic acid detection and SNP genotyping.</p>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142288070","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A comprehensive analysis of the microbiome and volatile organic compounds (VOC) in the moromi of soy sauce during fermentation and aging was conducted under industrial production. Microbiome analysis using next-generation sequencing revealed the presence and dynamics of microorganisms other than Aspergillus, Tetragenococcus, Zygosaccharomyces, and Wickerhamiella, which were used as starters. The bacterial community of the moromi on the first day of this process was rich in diversity. Staphylococcus, Bacillus, Kurthia, Acinetobacter, Enterococcus, and Macrococcus that grew during koji making were relatively dominant. However, as the fermentation progressed, only Tetragenococcus became dominant in the bacterial communities. In contrast, the fungal community was simple at the beginning of fermentation and aging, with Aspergillus present almost exclusively. After adding Zygosaccharomyces rouxii on day 42, the fungal community changed significantly. At the end of fermentation and aging, the fungal community diversified, with Millerozyma, Wickerhamiella, Yamadazyma, and Saccharomycopsis becoming dominant. The analysis of VOC showed that the VOC profile changed during fermentation and aging, and that the VOC profile changed significantly after adding Z. rouxii. The correlation analysis between the microbiome and VOC showed that Wickerhamiella, Millerozyma, Debaryomyces, Yamadazyma, and Candida had a significant positive correlation with alcohols, esters, and phenols produced in the later stage of fermentation and aging, indicating that not only Z. rouxii but also various fungi may contribute to the formation of the complex aroma profile of soy sauce.
{"title":"Dynamics of the microbiome and volatile organic compounds during fermentation and aging of soy sauce.","authors":"Yuichi Mizuno, Takashi Yoshimura, Kazutaka Sawada, Keisuke Tsuge, Yukio Nagano, Yumiko Yoshizaki, Masatoshi Goto, Genta Kobayashi","doi":"10.1016/j.jbiosc.2024.08.009","DOIUrl":"https://doi.org/10.1016/j.jbiosc.2024.08.009","url":null,"abstract":"<p><p>A comprehensive analysis of the microbiome and volatile organic compounds (VOC) in the moromi of soy sauce during fermentation and aging was conducted under industrial production. Microbiome analysis using next-generation sequencing revealed the presence and dynamics of microorganisms other than Aspergillus, Tetragenococcus, Zygosaccharomyces, and Wickerhamiella, which were used as starters. The bacterial community of the moromi on the first day of this process was rich in diversity. Staphylococcus, Bacillus, Kurthia, Acinetobacter, Enterococcus, and Macrococcus that grew during koji making were relatively dominant. However, as the fermentation progressed, only Tetragenococcus became dominant in the bacterial communities. In contrast, the fungal community was simple at the beginning of fermentation and aging, with Aspergillus present almost exclusively. After adding Zygosaccharomyces rouxii on day 42, the fungal community changed significantly. At the end of fermentation and aging, the fungal community diversified, with Millerozyma, Wickerhamiella, Yamadazyma, and Saccharomycopsis becoming dominant. The analysis of VOC showed that the VOC profile changed during fermentation and aging, and that the VOC profile changed significantly after adding Z. rouxii. The correlation analysis between the microbiome and VOC showed that Wickerhamiella, Millerozyma, Debaryomyces, Yamadazyma, and Candida had a significant positive correlation with alcohols, esters, and phenols produced in the later stage of fermentation and aging, indicating that not only Z. rouxii but also various fungi may contribute to the formation of the complex aroma profile of soy sauce.</p>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142288069","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
To plant crops (especially dry crops such as water spinach) with concomitant electricity recovery, a hanging-submerged-plant-pot system (HSPP) is developed. The HSPP consists of a soil pot (anodic) partially submerged under the water surface of a cathode tank. The microbial communities changed with conditions were also investigated. It was found that with chemical fertilizers the closed-circuit voltage (CCV, with 1 kΩ) was stable (approximately 250 mV) within 28 d; however, without fertilizer, the water spinach could adjust to the environment to obtain a better power output (approximately 3 mW m-2) at day 28. The microbial-community analyses revealed that the Pseudomonas sp. was the only exoeletrogens found in the anode pots. Using a secondary design of HSPP, for a better water-level adjustment, the maximum power output of each plant was found to be approximately 27.1 mW m-2. During operation, high temperature resulted in low oxygen solubility, and low CCV as well. At this time, it is yet to be concluded whether the submerged water level significantly affects electricity generation.
{"title":"Developing a plant microbial fuel cell by planting water spinach in a hanging-submerged plant pot system.","authors":"Yi-Hsuan Chen,Shiue-Lin Li,Ching-Ya Hung,Pei-Ching Wu,Yue-Xiang Hong,Wen-Jing Chen,Shu-Yi Chang,Yu-Ya Hsu,Wei-Yi Chao,Kai-Jhih Tsai,You-Chen Chen,Ji-Teng Chen,Chia-Le Hsu,Yun-Ju Lu,Li-Ming Fang,Ming-Han Yang,I-Ting Tan,Ying-Chuan Hsu,Hong-Yu Yang,Rui-Hong Jiang","doi":"10.1016/j.jbiosc.2024.08.007","DOIUrl":"https://doi.org/10.1016/j.jbiosc.2024.08.007","url":null,"abstract":"To plant crops (especially dry crops such as water spinach) with concomitant electricity recovery, a hanging-submerged-plant-pot system (HSPP) is developed. The HSPP consists of a soil pot (anodic) partially submerged under the water surface of a cathode tank. The microbial communities changed with conditions were also investigated. It was found that with chemical fertilizers the closed-circuit voltage (CCV, with 1 kΩ) was stable (approximately 250 mV) within 28 d; however, without fertilizer, the water spinach could adjust to the environment to obtain a better power output (approximately 3 mW m-2) at day 28. The microbial-community analyses revealed that the Pseudomonas sp. was the only exoeletrogens found in the anode pots. Using a secondary design of HSPP, for a better water-level adjustment, the maximum power output of each plant was found to be approximately 27.1 mW m-2. During operation, high temperature resulted in low oxygen solubility, and low CCV as well. At this time, it is yet to be concluded whether the submerged water level significantly affects electricity generation.","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142248091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1016/j.jbiosc.2024.08.008
Mikiko Nakamura,Rinji Akada
pET vectors allow inducible expression of recombinant proteins in Escherichia coli. In this system, isopropyl β-d-1-thiogalactopyranoside (IPTG) drives lacUV5 promoter to produce T7 RNA polymerase, simultaneously releasing the suppression of T7lac promoter. T7 RNA polymerase then strongly transcribes the target gene. A lac repressor encoded by lacI in the vector represses the promoters. Despite stringent repression and inducible expression achieved with the pET system, unexpected leaky expression can occur without IPTG induction. Here, by evaluating leaky expression in recombinant cells cultured in various Luria-Bertani (LB) media, prepared using yeast extract and peptone from different suppliers, as well as in five commercial premix-LB media, we confirmed the presence of unknown lac inducers in LB. To explore these inducers, we examined E. coli growth in media comprising yeast extract or peptone. At 4% concentration, five commercial yeast extract and six peptone samples individually allowed E. coli growth equivalent to that in LB medium. We determined the luciferase activity of the luxCDABE operon in the pET vector under these conditions. The presence of different concentrations of inducers was detected in both the yeast extract and peptone. Furthermore, we blended yeast extract and peptone with low or high concentrations of lac inducers. The low-expression blend, used as a basal medium before IPTG addition, allowed leak-free, tightly controlled expression. The high-expression blend was used for constitutive high-expression and pET induction with the basal medium, in lieu of IPTG. These blended media can be used for well-controlled inducible and constitutive expression using the pET system.
pET 载体可在大肠杆菌中诱导表达重组蛋白。在该系统中,异丙基β-d-1-硫代吡喃半乳糖苷(IPTG)驱动 lacUV5 启动子产生 T7 RNA 聚合酶,同时释放对 T7lac 启动子的抑制。然后,T7 RNA 聚合酶强烈转录目标基因。载体中由lacI编码的lac抑制因子抑制启动子。尽管 pET 系统实现了严格的抑制和诱导表达,但在没有 IPTG 诱导的情况下也会出现意外的泄漏表达。在这里,通过评估用不同供应商提供的酵母提取物和蛋白胨制备的各种 Luria-Bertani(LB)培养基以及五种商用预混合 LB 培养基培养的重组细胞的泄漏表达,我们证实了 LB 中存在未知的 lac 诱导因子。为了探究这些诱导剂,我们检测了大肠杆菌在含有酵母提取物或蛋白胨的培养基中的生长情况。在 4% 的浓度下,五种商用酵母提取物和六种蛋白胨样品分别允许大肠杆菌生长,与在 LB 培养基中的生长相当。在这些条件下,我们测定了 pET 载体中 luxCDABE 操作子的荧光素酶活性。在酵母提取物和蛋白胨中都检测到了不同浓度的诱导剂。此外,我们还将酵母提取物和蛋白胨与低浓度或高浓度的lac诱导剂混合。低表达混合培养基在添加 IPTG 前用作基础培养基,可实现无泄漏、严格控制的表达。高表达混合培养基用于组成型高表达,并用基础培养基诱导 pET,以代替 IPTG。这些混合培养基可用于使用 pET 系统进行良好的诱导型和组成型表达。
{"title":"Blending of selected yeast extract and peptone for inducible and constitutive protein production in Escherichia coli using the pET system.","authors":"Mikiko Nakamura,Rinji Akada","doi":"10.1016/j.jbiosc.2024.08.008","DOIUrl":"https://doi.org/10.1016/j.jbiosc.2024.08.008","url":null,"abstract":"pET vectors allow inducible expression of recombinant proteins in Escherichia coli. In this system, isopropyl β-d-1-thiogalactopyranoside (IPTG) drives lacUV5 promoter to produce T7 RNA polymerase, simultaneously releasing the suppression of T7lac promoter. T7 RNA polymerase then strongly transcribes the target gene. A lac repressor encoded by lacI in the vector represses the promoters. Despite stringent repression and inducible expression achieved with the pET system, unexpected leaky expression can occur without IPTG induction. Here, by evaluating leaky expression in recombinant cells cultured in various Luria-Bertani (LB) media, prepared using yeast extract and peptone from different suppliers, as well as in five commercial premix-LB media, we confirmed the presence of unknown lac inducers in LB. To explore these inducers, we examined E. coli growth in media comprising yeast extract or peptone. At 4% concentration, five commercial yeast extract and six peptone samples individually allowed E. coli growth equivalent to that in LB medium. We determined the luciferase activity of the luxCDABE operon in the pET vector under these conditions. The presence of different concentrations of inducers was detected in both the yeast extract and peptone. Furthermore, we blended yeast extract and peptone with low or high concentrations of lac inducers. The low-expression blend, used as a basal medium before IPTG addition, allowed leak-free, tightly controlled expression. The high-expression blend was used for constitutive high-expression and pET induction with the basal medium, in lieu of IPTG. These blended media can be used for well-controlled inducible and constitutive expression using the pET system.","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142217177","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Gamma-aminobutyric acid (GABA), which is synthesized from l-glutamic acid via glutamate decarboxylase (Gad), is used as food, supplements, and biodegradable plastics. Our previous study demonstrated an Escherichia coli mutant (ΔΔ) strain, lacking type I NADH dehydrogenase (NDH-I) and cytochrome bo3 oxidase (Cytbo3), produced 7 g/L glutamic acid on MS1 glucose-minimal medium. In this study, the ΔΔ strain was used for improving GABA production. A plasmid (pMBL19-gadB') expressing a mutated E. coli GadB (Glu89Gln/Δ452-466), retaining activity at neutral pH, was introduced into the ΔΔ strain and its parent strain (W1485). The ΔΔ strain carrying pMBL19-gadB' exhibited a twofold increase in GABA production compared to the W1485 strain carrying pMBL19-gadB'. Deleting the C-terminal (Δ471-511) of GadC antiporter in the ΔΔ strain further improved GABA yield by 1.5 g/L when cultured in MS1 glucose-minimal medium. On the other hand, a large amount of glutamic acid produced by the ΔΔ strain was not fully converted to GABA, likely due to the inhibition of GadB activity by the accumulation of acetic acid. Although there is room for improvement, these results indicate the efficacy of the ΔNDH-IΔCytbo3 double mutation in augmenting GABA production.
{"title":"Improved fermentative gamma-aminobutyric acid production from glucose by the inactivation of respiratory chain components NDH-I and Cytbo₃ in Escherichia coli.","authors":"Hiroki Wakahara, Takuya Mizokoshi, Kotaro Yamagami, Satoru Fukiya, Atsushi Yokota, Tomoya Maeda","doi":"10.1016/j.jbiosc.2024.08.004","DOIUrl":"https://doi.org/10.1016/j.jbiosc.2024.08.004","url":null,"abstract":"<p><p>Gamma-aminobutyric acid (GABA), which is synthesized from l-glutamic acid via glutamate decarboxylase (Gad), is used as food, supplements, and biodegradable plastics. Our previous study demonstrated an Escherichia coli mutant (ΔΔ) strain, lacking type I NADH dehydrogenase (NDH-I) and cytochrome bo<sub>3</sub> oxidase (Cytbo<sub>3</sub>), produced 7 g/L glutamic acid on MS1 glucose-minimal medium. In this study, the ΔΔ strain was used for improving GABA production. A plasmid (pMBL19-gadB') expressing a mutated E. coli GadB (Glu89Gln/Δ452-466), retaining activity at neutral pH, was introduced into the ΔΔ strain and its parent strain (W1485). The ΔΔ strain carrying pMBL19-gadB' exhibited a twofold increase in GABA production compared to the W1485 strain carrying pMBL19-gadB'. Deleting the C-terminal (Δ471-511) of GadC antiporter in the ΔΔ strain further improved GABA yield by 1.5 g/L when cultured in MS1 glucose-minimal medium. On the other hand, a large amount of glutamic acid produced by the ΔΔ strain was not fully converted to GABA, likely due to the inhibition of GadB activity by the accumulation of acetic acid. Although there is room for improvement, these results indicate the efficacy of the ΔNDH-IΔCytbo<sub>3</sub> double mutation in augmenting GABA production.</p>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142154190","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Plasmids are molecular genetic tools used for trans-complementation and gene expression in bacteria. Challenges faced by researchers include limited repertoire of antibiotic resistance of plasmids, issues related to plasmid compatibility and restricted or incompatible multiple cloning sites when needing to change plasmid copy number to tune production of their protein of interest. In this study, a series of plasmids were generated with compatible multiple cloning sites and homologous DNA regions to allow for modular cloning for rapid exchange of antibiotic resistance and plasmid origin. Plasmids generated in this series have options for high, mid, and low plasmid copy number, and have either an integrated FLAG epitope in the multiple cloning site or possess an uninterrupted multiple cloning site with the option of using the common LacZ-based blue/white screening method. Low copy plasmids also have one of five antibiotic selection markers. To demonstrate functionality of these plasmids, a representative FLAG tagged protein and mCherry were cloned into the low copy plasmids and expressed in various bacteria belonging to the Enterobacteriaceae family. In conclusion, by creating a new plasmid series, we have expanded the toolkit of available molecular biology tools for bacterial work.
质粒是用于细菌转基因和基因表达的分子遗传工具。研究人员面临的挑战包括:质粒的抗生素抗性种类有限、质粒兼容性相关问题,以及当需要改变质粒拷贝数以调整所需蛋白质的生产时,多重克隆位点受限或不兼容。在这项研究中,生成了一系列具有兼容的多重克隆位点和同源 DNA 区域的质粒,以便进行模块化克隆,快速交换抗生素抗性和质粒来源。这一系列质粒有高、中、低三种质粒拷贝数可供选择,在多重克隆位点上有一个整合的 FLAG 表位,或拥有一个不间断的多重克隆位点,可选择使用常见的基于 LacZ 的蓝/白筛选方法。低拷贝质粒还具有五种抗生素选择标记中的一种。为了证明这些质粒的功能,我们在低拷贝质粒中克隆了具有代表性的 FLAG 标记蛋白和 mCherry,并在属于肠杆菌科的各种细菌中进行了表达。总之,通过创建新的质粒系列,我们扩展了用于细菌工作的分子生物学工具包。
{"title":"Generation of a plasmid series for rapid sub-cloning and use in various Enterobacteriaceae.","authors":"Hannah Gertrude Braun, Nabeela Kanwal, Luisa Fernanda Rivera Lopez, Jenny-Lee Thomassin","doi":"10.1016/j.jbiosc.2024.08.006","DOIUrl":"https://doi.org/10.1016/j.jbiosc.2024.08.006","url":null,"abstract":"<p><p>Plasmids are molecular genetic tools used for trans-complementation and gene expression in bacteria. Challenges faced by researchers include limited repertoire of antibiotic resistance of plasmids, issues related to plasmid compatibility and restricted or incompatible multiple cloning sites when needing to change plasmid copy number to tune production of their protein of interest. In this study, a series of plasmids were generated with compatible multiple cloning sites and homologous DNA regions to allow for modular cloning for rapid exchange of antibiotic resistance and plasmid origin. Plasmids generated in this series have options for high, mid, and low plasmid copy number, and have either an integrated FLAG epitope in the multiple cloning site or possess an uninterrupted multiple cloning site with the option of using the common LacZ-based blue/white screening method. Low copy plasmids also have one of five antibiotic selection markers. To demonstrate functionality of these plasmids, a representative FLAG tagged protein and mCherry were cloned into the low copy plasmids and expressed in various bacteria belonging to the Enterobacteriaceae family. In conclusion, by creating a new plasmid series, we have expanded the toolkit of available molecular biology tools for bacterial work.</p>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142145722","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}