دورية أكاديمية
أعرض في EDS

Thermostable in vitro transcription-translation compatible with microfluidic droplets.

التفاصيل البيبلوغرافية
العنوان: Thermostable in vitro transcription-translation compatible with microfluidic droplets.
المؤلفون: Ribeiro ALJL; Centro de Biología Molecular 'Severo Ochoa' (UAM-CSIC), Nicolás Cabrera 1, 28049, Madrid, Spain.; Instituto de Biología Molecular, Universidad Autónoma de Madrid, Nicolás Cabrera 1, 28049, Madrid, Spain.; Departamento de Biología Molecular, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049, Madrid, Spain., Pérez-Arnaiz P; Centro de Biología Molecular 'Severo Ochoa' (UAM-CSIC), Nicolás Cabrera 1, 28049, Madrid, Spain.; Instituto de Biología Molecular, Universidad Autónoma de Madrid, Nicolás Cabrera 1, 28049, Madrid, Spain.; Departamento de Biología Molecular, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049, Madrid, Spain., Sánchez-Costa M; Centro de Biología Molecular 'Severo Ochoa' (UAM-CSIC), Nicolás Cabrera 1, 28049, Madrid, Spain.; Departamento de Biología Molecular, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049, Madrid, Spain., Pérez L; Centro de Biología Molecular 'Severo Ochoa' (UAM-CSIC), Nicolás Cabrera 1, 28049, Madrid, Spain.; Departamento de Biología Molecular, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049, Madrid, Spain., Almendros M; Centro de Biología Molecular 'Severo Ochoa' (UAM-CSIC), Nicolás Cabrera 1, 28049, Madrid, Spain.; Departamento de Biología Molecular, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049, Madrid, Spain., van Vliet L; Departament of Biochemistry, Cambridge University, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.; DropTech Ltd, 91 Canterbury Court, Cambridge, CB4 3QU, UK., Gielen F; DropTech Ltd, 91 Canterbury Court, Cambridge, CB4 3QU, UK.; Living Systems Institute, Faculty of Health and Life Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.; Department of Physics and Astronomy, Faculty of Environment, Science and Economy, University of Exeter, Stocker Road, Exeter, EX4 4QL, UK., Lim J; Prozomix Ltd, Building 4, West End Ind. Estate, Haltwhistle, Northumberland, NE49 9HA, UK., Charnock S; Prozomix Ltd, Building 4, West End Ind. Estate, Haltwhistle, Northumberland, NE49 9HA, UK., Hollfelder F; Departament of Biochemistry, Cambridge University, 80 Tennis Court Road, Cambridge, CB2 1GA, UK., González-Pastor JE; Centro de Astrobiología (CAB), CSIC-INTA, Ctra de Torrejón a Ajalvir, Km 4, 28850, Torrejón de Ardoz, Spain., Berenguer J; Centro de Biología Molecular 'Severo Ochoa' (UAM-CSIC), Nicolás Cabrera 1, 28049, Madrid, Spain.; Instituto de Biología Molecular, Universidad Autónoma de Madrid, Nicolás Cabrera 1, 28049, Madrid, Spain.; Departamento de Biología Molecular, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049, Madrid, Spain., Hidalgo A; Centro de Biología Molecular 'Severo Ochoa' (UAM-CSIC), Nicolás Cabrera 1, 28049, Madrid, Spain. ahidalgo@cbm.csic.es.; Instituto de Biología Molecular, Universidad Autónoma de Madrid, Nicolás Cabrera 1, 28049, Madrid, Spain. ahidalgo@cbm.csic.es.; Departamento de Biología Molecular, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049, Madrid, Spain. ahidalgo@cbm.csic.es.
المصدر: Microbial cell factories [Microb Cell Fact] 2024 Jun 10; Vol. 23 (1), pp. 169. Date of Electronic Publication: 2024 Jun 10.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: BioMed Central Country of Publication: England NLM ID: 101139812 Publication Model: Electronic Cited Medium: Internet ISSN: 1475-2859 (Electronic) Linking ISSN: 14752859 NLM ISO Abbreviation: Microb Cell Fact Subsets: MEDLINE
أسماء مطبوعة: Original Publication: London : BioMed Central, [2002-
مواضيع طبية MeSH: Thermus thermophilus*/genetics , Thermus thermophilus*/metabolism , Thermus thermophilus*/enzymology , Transcription, Genetic* , Protein Biosynthesis*, Microfluidics/methods ; Cell-Free System ; DNA-Directed RNA Polymerases/metabolism ; DNA-Directed RNA Polymerases/genetics ; Temperature ; Hot Temperature ; Bacterial Proteins/metabolism ; Bacterial Proteins/genetics
مستخلص: Background: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures.
Objectives: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets.
Results: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time.
Conclusions: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.
(© 2024. The Author(s).)
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معلومات مُعتمدة: 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; 324439, 635595, 685474, 695669 and 10100560 European Commission; BIO-2013-44963-R, RED2022-134755-T, CEX2021-001154-S Ministerio de Ciencia e Innovación; BIO-2013-44963-R, RED2022-134755-T, CEX2021-001154-S Ministerio de Ciencia e Innovación
فهرسة مساهمة: Keywords: Thermus thermophiles; Cell-free protein expression; Droplet microfluidics; In vitro transcription and translation; Thermozymes
المشرفين على المادة: EC 2.7.7.6 (DNA-Directed RNA Polymerases)
0 (Bacterial Proteins)
تواريخ الأحداث: Date Created: 20240610 Date Completed: 20240611 Latest Revision: 20240613
رمز التحديث: 20240613
مُعرف محوري في PubMed: PMC11165818
DOI: 10.1186/s12934-024-02440-y
PMID: 38858677
قاعدة البيانات: MEDLINE
الوصف
تدمد:1475-2859
DOI:10.1186/s12934-024-02440-y