Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems
Abstract Cell‐free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell‐free measurements is limited by the use of channel‐limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell‐Free Transcription and Sequencin...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Springer Nature
2019-08-01
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| Series: | Molecular Systems Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.15252/msb.20198875 |
| _version_ | 1797283850958667776 |
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| author | Sung Sun Yim Nathan I Johns Jimin Park Antonio LC Gomes Ross M McBee Miles Richardson Carlotta Ronda Sway P Chen David Garenne Vincent Noireaux Harris H Wang |
| author_facet | Sung Sun Yim Nathan I Johns Jimin Park Antonio LC Gomes Ross M McBee Miles Richardson Carlotta Ronda Sway P Chen David Garenne Vincent Noireaux Harris H Wang |
| author_sort | Sung Sun Yim |
| collection | DOAJ |
| description | Abstract Cell‐free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell‐free measurements is limited by the use of channel‐limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell‐Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual‐species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell‐free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology. |
| first_indexed | 2024-03-07T17:37:47Z |
| format | Article |
| id | doaj.art-2b873f070b894259b22ee0164734d743 |
| institution | Directory Open Access Journal |
| issn | 1744-4292 |
| language | English |
| last_indexed | 2024-03-07T17:37:47Z |
| publishDate | 2019-08-01 |
| publisher | Springer Nature |
| record_format | Article |
| series | Molecular Systems Biology |
| spelling | doaj.art-2b873f070b894259b22ee0164734d7432024-03-02T16:31:38ZengSpringer NatureMolecular Systems Biology1744-42922019-08-01158n/an/a10.15252/msb.20198875Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systemsSung Sun Yim0Nathan I Johns1Jimin Park2Antonio LC Gomes3Ross M McBee4Miles Richardson5Carlotta Ronda6Sway P Chen7David Garenne8Vincent Noireaux9Harris H Wang10Department of Systems Biology Columbia University New York NY USADepartment of Systems Biology Columbia University New York NY USADepartment of Systems Biology Columbia University New York NY USADepartment of Immunology Memorial Sloan Kettering Cancer Center New York NY USADepartment of Systems Biology Columbia University New York NY USADepartment of Systems Biology Columbia University New York NY USADepartment of Systems Biology Columbia University New York NY USADepartment of Systems Biology Columbia University New York NY USASchool of Physics and Astronomy University of Minnesota Minneapolis MN USASchool of Physics and Astronomy University of Minnesota Minneapolis MN USADepartment of Systems Biology Columbia University New York NY USAAbstract Cell‐free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell‐free measurements is limited by the use of channel‐limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell‐Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual‐species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell‐free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.https://doi.org/10.15252/msb.20198875cell‐free expression systemsgene expressionmassively parallel reporter assaysynthetic biologytranscription |
| spellingShingle | Sung Sun Yim Nathan I Johns Jimin Park Antonio LC Gomes Ross M McBee Miles Richardson Carlotta Ronda Sway P Chen David Garenne Vincent Noireaux Harris H Wang Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems Molecular Systems Biology cell‐free expression systems gene expression massively parallel reporter assay synthetic biology transcription |
| title | Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems |
| title_full | Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems |
| title_fullStr | Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems |
| title_full_unstemmed | Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems |
| title_short | Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems |
| title_sort | multiplex transcriptional characterizations across diverse bacterial species using cell free systems |
| topic | cell‐free expression systems gene expression massively parallel reporter assay synthetic biology transcription |
| url | https://doi.org/10.15252/msb.20198875 |
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