Junzhu Yang , Yuntao Cui , Zhou Cao , Shengnan Ma, Yuan Lu
{"title":"Strategy exploration for developing robust lyophilized cell-free systems","authors":"Junzhu Yang , Yuntao Cui , Zhou Cao , Shengnan Ma, Yuan Lu","doi":"10.1016/j.biotno.2021.08.004","DOIUrl":"10.1016/j.biotno.2021.08.004","url":null,"abstract":"<div><p>Cell-free protein synthesis system is emerging as a powerful tool for rapid expression of therapeutic proteins. However, one drawback of cell-free technology is the necessity to store the major components below freezing in bulky aqueous solutions. To preserve the cell-free synthesis system for a longer time, the lyophilized cell-free system was developed, and its stability under freeze-drying conditions was explored. The novelty and the difference of this study from the others lie in determining more detailed storage conditions, which could provide a more accurate storage strategy. The effects of the treatment time, the storage temperature, and the component-mixing mode on the freeze-dried cell-free system were investigated. This study explored a robust storage strategy for freeze-dried cell-free systems and could further open up a better opportunity for on-demand synthesis of therapeutic proteins.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"2 ","pages":"Pages 44-50"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.biotno.2021.08.004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84815577","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Vasil D'Ambrosio , Subrata Pramanik , Kati Goroncy , Tadas Jakočiūnas , David Schönauer , Mehdi D. Davari , Ulrich Schwaneberg , Jay D. Keasling , Michael K. Jensen
{"title":"Directed evolution of VanR biosensor specificity in yeast","authors":"Vasil D'Ambrosio , Subrata Pramanik , Kati Goroncy , Tadas Jakočiūnas , David Schönauer , Mehdi D. Davari , Ulrich Schwaneberg , Jay D. Keasling , Michael K. Jensen","doi":"10.1016/j.biotno.2020.01.002","DOIUrl":"https://doi.org/10.1016/j.biotno.2020.01.002","url":null,"abstract":"<div><p>Allosterically regulated transcription factors (aTFs) based biosensors from prokaryotes have been widely used to screen large gene libraries, stabilize engineered microbes from evolutionary drifting, and for detection of soil pollutants, among many other applications. However, even though aTF-based biosensors have been established as successful tools for bioengineering and remediation, rational engineering of aTF small molecule-specificity have so far not been demonstrated, highlighting the need for a deeper understanding of the sequence-function relationships that govern aTF allostery. Here, by combining directed evolution of a naïve library of VanR, a vanillic acid transcriptional regulator from <em>Caulobacter crescentus</em> in yeast, followed by saturation mutagenesis of selected positions we identify residues required for vanillic acid responsiveness, while at the same time maintaining responsiveness to vanillin. Selected single-position VanR mutants show both complete repression of transcription in the absence of any ligand, complete loss of vanillic acid responsiveness, while still maintaining high derepression in the presence of vanillin. By computational ligand docking analyses we also discuss the structure-function relationship single mutations can have on aTF specificity, an attribute potentially accounting for the wide-spread arise of aTF members belonging to the GntR superfamily of transcriptional regulators.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"1 ","pages":"Pages 9-15"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.biotno.2020.01.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91684845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Copper-tripeptide complexes for rapid inactivation of Bacillus subtilis endospores","authors":"Yonghao Cui, Kang Zhou, Kun-Lin Yang","doi":"10.1016/j.biotno.2020.03.002","DOIUrl":"https://doi.org/10.1016/j.biotno.2020.03.002","url":null,"abstract":"<div><p>Bacterial endospores can be pathogenic to human beings. However, they are robust and thus difficult to kill due to their rigid structure. Conventional methods such as autoclaving for inactivating endospores are energy-intensive and time-consuming. In this study, we developed a copper-tripeptide complexes reagent composed of copper-tripeptide complexes, hydrogen peroxide, and cetyl trimethylammonium bromide (CTAB), to kill endospores. This copper-tripeptide complexes reagent can achieve a 10<sup>7.8</sup>-fold reduction in viable endospore count after 60-min treatment at ambient conditions. As a cost-effective and stable sporicidal agent, this reagent may be applied as a general-purpose disinfectant and a replacement of standard sterilization procedures in the future.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"1 ","pages":"Pages 16-19"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.biotno.2020.03.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91684846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Improving Spinach2-and Broccoli-based biosensors for single and double analytes","authors":"Shuo-Fu Yuan , Hal S. Alper","doi":"10.1016/j.biotno.2020.01.001","DOIUrl":"https://doi.org/10.1016/j.biotno.2020.01.001","url":null,"abstract":"<div><p>The use of “Spinach”-based RNA sensors for the detection of small metabolites and proteins has received growing interest in the recent years. While this approach can be used <em>in vivo</em> for cell sensing and <em>in vitro</em> for microfluidic assays, their overall utility is limited as a result of a high magnesium ion dependence. Here, we alleviate this limitation through incorporating a human tRNA<sup>Lys3</sup> or an engineered viral F30 (a three-way junction RNA motif) scaffold to facilitate aptamer folding <em>in vitro</em> and improve the performance of selected streptavidin, tyrosine, and thrombin aptamers as exemplary cases. Furthermore, we demonstrate the use of a Broccoli aptamer scaffold in conjunction with the viral F30 to enable simultaneous sensing of two molecules in a logic gate type fashion. Our proof-of-concept results demonstrate the ability to redesign these aptamer sensors for improved brightness as well as signal stability without the need for high magnesium—both traits that can further enhance downstream screening applications.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"1 ","pages":"Pages 2-8"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.biotno.2020.01.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91684844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Vasil D'ambrosio, S. Pramanik, Kati Goroncy, Tadas Jakočiūnas, David Schönauer, M. Davari, U. Schwaneberg, J. Keasling, M. K. Jensen
{"title":"Directed evolution of VanR biosensor specificity in yeast","authors":"Vasil D'ambrosio, S. Pramanik, Kati Goroncy, Tadas Jakočiūnas, David Schönauer, M. Davari, U. Schwaneberg, J. Keasling, M. K. Jensen","doi":"10.1016/j.biotno.2020.01.002","DOIUrl":"https://doi.org/10.1016/j.biotno.2020.01.002","url":null,"abstract":"","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83494182","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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