دورية أكاديمية
Cell-Free Translation Quantification via a Fluorescent Minihelix.
| العنوان: | Cell-Free Translation Quantification via a Fluorescent Minihelix. |
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| المؤلفون: | Willi JA; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States., Karim AS; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States., Jewett MC; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States.; Department of Bioengineering, Stanford University, Stanford, California 94305, United States. |
| المصدر: | ACS synthetic biology [ACS Synth Biol] 2024 Jul 19; Vol. 13 (7), pp. 2253-2259. Date of Electronic Publication: 2024 Jul 09. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: American Chemical Society Country of Publication: United States NLM ID: 101575075 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2161-5063 (Electronic) Linking ISSN: 21615063 NLM ISO Abbreviation: ACS Synth Biol Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Washington, D.C. : American Chemical Society, c2012- |
| مواضيع طبية MeSH: | Cell-Free System* , Protein Biosynthesis* , Fluorescent Dyes*/chemistry, Cysteine/metabolism ; Cysteine/genetics ; Ribosomes/metabolism ; Ribosomes/genetics |
| مستخلص: | Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems. |
| فهرسة مساهمة: | Keywords: cell-free gene expression; fluorescent reporter; high-throughput; in vitro transcription and translation; protein quantification; synthetic biology |
| المشرفين على المادة: | 0 (Fluorescent Dyes) K848JZ4886 (Cysteine) |
| تواريخ الأحداث: | Date Created: 20240709 Date Completed: 20240719 Latest Revision: 20240719 |
| رمز التحديث: | 20240719 |
| DOI: | 10.1021/acssynbio.4c00266 |
| PMID: | 38979618 |
| قاعدة البيانات: | MEDLINE |
Find this issue from the American Chemical Society | تدمد: | 2161-5063 |
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| DOI: | 10.1021/acssynbio.4c00266 |