Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements
Abstract To develop artificial cell models that mimic living cells, cell-sized lipid vesicles encapsulating cell-free protein synthesis (CFPS) systems are useful for protein expressions or artificial gene circuits for vesicle–vesicle communications. Therefore, investigating the transcriptional and t...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2024-02-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-024-53135-8 |
| _version_ | 1797274688872775680 |
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| author | Akari Miwa Masatoshi Wakamori Tetsuro Ariyoshi Yasushi Okada Mikako Shirouzu Takashi Umehara Koki Kamiya |
| author_facet | Akari Miwa Masatoshi Wakamori Tetsuro Ariyoshi Yasushi Okada Mikako Shirouzu Takashi Umehara Koki Kamiya |
| author_sort | Akari Miwa |
| collection | DOAJ |
| description | Abstract To develop artificial cell models that mimic living cells, cell-sized lipid vesicles encapsulating cell-free protein synthesis (CFPS) systems are useful for protein expressions or artificial gene circuits for vesicle–vesicle communications. Therefore, investigating the transcriptional and translational properties of CFPS systems in lipid vesicles is important for maximizing the synthesis and functions of proteins. Although transcription and translation using CFPS systems inside lipid vesicles are more important than that outside lipid vesicles, the former processes are not investigated by changing the lipid composition of lipid vesicles. Herein, we investigated changes in transcription and translation using CFPS systems inside giant lipid vesicles (approximately 5–20 μm in diameter) caused by changing the lipid composition of lipid vesicles containing neutral, positively, and negatively charged lipids. After incubating for 30 min, 1 h, 2 h, and 4 h, the transcriptional and translational activities in these lipid vesicles were determined by detecting the fluorescence intensities of the fluorogenic RNA aptamer on the 3′-untranslated region of mRNA (transcription) and the fluorescent protein sfCherry (translation), respectively. The results revealed that transcriptional and translational activities in a lipid vesicle containing positively charged lipids were high when the protein was synthesized using the CFPS system inside the lipid vesicle. Thus, the present study provides an experimental basis for constructing complex artificial cell models using bottom-up approaches. |
| first_indexed | 2024-03-07T15:01:51Z |
| format | Article |
| id | doaj.art-e556d5036f8846bba93418939d59267e |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-07T15:01:51Z |
| publishDate | 2024-02-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-e556d5036f8846bba93418939d59267e2024-03-05T19:06:56ZengNature PortfolioScientific Reports2045-23222024-02-0114111010.1038/s41598-024-53135-8Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurementsAkari Miwa0Masatoshi Wakamori1Tetsuro Ariyoshi2Yasushi Okada3Mikako Shirouzu4Takashi Umehara5Koki Kamiya6Division of Molecular Science, Graduate School of Science and Technology, Gunma UniversityLaboratory for Epigenetics Drug Discovery, RIKEN Center for Biosystems Dynamics ResearchLaboratory for Cell Polarity Regulation, RIKEN Center for Biosystems Dynamics ResearchLaboratory for Cell Polarity Regulation, RIKEN Center for Biosystems Dynamics ResearchLaboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics ResearchLaboratory for Epigenetics Drug Discovery, RIKEN Center for Biosystems Dynamics ResearchDivision of Molecular Science, Graduate School of Science and Technology, Gunma UniversityAbstract To develop artificial cell models that mimic living cells, cell-sized lipid vesicles encapsulating cell-free protein synthesis (CFPS) systems are useful for protein expressions or artificial gene circuits for vesicle–vesicle communications. Therefore, investigating the transcriptional and translational properties of CFPS systems in lipid vesicles is important for maximizing the synthesis and functions of proteins. Although transcription and translation using CFPS systems inside lipid vesicles are more important than that outside lipid vesicles, the former processes are not investigated by changing the lipid composition of lipid vesicles. Herein, we investigated changes in transcription and translation using CFPS systems inside giant lipid vesicles (approximately 5–20 μm in diameter) caused by changing the lipid composition of lipid vesicles containing neutral, positively, and negatively charged lipids. After incubating for 30 min, 1 h, 2 h, and 4 h, the transcriptional and translational activities in these lipid vesicles were determined by detecting the fluorescence intensities of the fluorogenic RNA aptamer on the 3′-untranslated region of mRNA (transcription) and the fluorescent protein sfCherry (translation), respectively. The results revealed that transcriptional and translational activities in a lipid vesicle containing positively charged lipids were high when the protein was synthesized using the CFPS system inside the lipid vesicle. Thus, the present study provides an experimental basis for constructing complex artificial cell models using bottom-up approaches.https://doi.org/10.1038/s41598-024-53135-8 |
| spellingShingle | Akari Miwa Masatoshi Wakamori Tetsuro Ariyoshi Yasushi Okada Mikako Shirouzu Takashi Umehara Koki Kamiya Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements Scientific Reports |
| title | Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements |
| title_full | Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements |
| title_fullStr | Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements |
| title_full_unstemmed | Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements |
| title_short | Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements |
| title_sort | efficiency of transcription and translation of cell free protein synthesis systems in cell sized lipid vesicles with changing lipid composition determined by fluorescence measurements |
| url | https://doi.org/10.1038/s41598-024-53135-8 |
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