Effective Use of Linear DNA in Cell-Free Expression Systems
Cell-free expression systems (CFEs) are cutting-edge research tools used in the investigation of biological phenomena and the engineering of novel biotechnologies. While CFEs have many benefits over in vivo protein synthesis, one particularly significant advantage is that CFEs allow for gene express...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-07-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2021.715328/full |
| _version_ | 1818176686888321024 |
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| author | Megan A. McSweeney Mark P. Styczynski |
| author_facet | Megan A. McSweeney Mark P. Styczynski |
| author_sort | Megan A. McSweeney |
| collection | DOAJ |
| description | Cell-free expression systems (CFEs) are cutting-edge research tools used in the investigation of biological phenomena and the engineering of novel biotechnologies. While CFEs have many benefits over in vivo protein synthesis, one particularly significant advantage is that CFEs allow for gene expression from both plasmid DNA and linear expression templates (LETs). This is an important and impactful advantage because functional LETs can be efficiently synthesized in vitro in a few hours without transformation and cloning, thus expediting genetic circuit prototyping and allowing expression of toxic genes that would be difficult to clone through standard approaches. However, native nucleases present in the crude bacterial lysate (the basis for the most affordable form of CFEs) quickly degrade LETs and limit expression yield. Motivated by the significant benefits of using LETs in lieu of plasmid templates, numerous methods to enhance their stability in lysate-based CFEs have been developed. This review describes approaches to LET stabilization used in CFEs, summarizes the advancements that have come from using LETs with these methods, and identifies future applications and development goals that are likely to be impactful to the field. Collectively, continued improvement of LET-based expression and other linear DNA tools in CFEs will help drive scientific discovery and enable a wide range of applications, from diagnostics to synthetic biology research tools. |
| first_indexed | 2024-12-11T20:20:08Z |
| format | Article |
| id | doaj.art-bd67a931774a4ee7a96f3cdc5e21280d |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-12-11T20:20:08Z |
| publishDate | 2021-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-bd67a931774a4ee7a96f3cdc5e21280d2022-12-22T00:52:06ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852021-07-01910.3389/fbioe.2021.715328715328Effective Use of Linear DNA in Cell-Free Expression SystemsMegan A. McSweeneyMark P. StyczynskiCell-free expression systems (CFEs) are cutting-edge research tools used in the investigation of biological phenomena and the engineering of novel biotechnologies. While CFEs have many benefits over in vivo protein synthesis, one particularly significant advantage is that CFEs allow for gene expression from both plasmid DNA and linear expression templates (LETs). This is an important and impactful advantage because functional LETs can be efficiently synthesized in vitro in a few hours without transformation and cloning, thus expediting genetic circuit prototyping and allowing expression of toxic genes that would be difficult to clone through standard approaches. However, native nucleases present in the crude bacterial lysate (the basis for the most affordable form of CFEs) quickly degrade LETs and limit expression yield. Motivated by the significant benefits of using LETs in lieu of plasmid templates, numerous methods to enhance their stability in lysate-based CFEs have been developed. This review describes approaches to LET stabilization used in CFEs, summarizes the advancements that have come from using LETs with these methods, and identifies future applications and development goals that are likely to be impactful to the field. Collectively, continued improvement of LET-based expression and other linear DNA tools in CFEs will help drive scientific discovery and enable a wide range of applications, from diagnostics to synthetic biology research tools.https://www.frontiersin.org/articles/10.3389/fbioe.2021.715328/fullcell-free expressionlinear expression templatenuclease inhibitiongenetic circuitsrapid prototypingDNA aptamers |
| spellingShingle | Megan A. McSweeney Mark P. Styczynski Effective Use of Linear DNA in Cell-Free Expression Systems Frontiers in Bioengineering and Biotechnology cell-free expression linear expression template nuclease inhibition genetic circuits rapid prototyping DNA aptamers |
| title | Effective Use of Linear DNA in Cell-Free Expression Systems |
| title_full | Effective Use of Linear DNA in Cell-Free Expression Systems |
| title_fullStr | Effective Use of Linear DNA in Cell-Free Expression Systems |
| title_full_unstemmed | Effective Use of Linear DNA in Cell-Free Expression Systems |
| title_short | Effective Use of Linear DNA in Cell-Free Expression Systems |
| title_sort | effective use of linear dna in cell free expression systems |
| topic | cell-free expression linear expression template nuclease inhibition genetic circuits rapid prototyping DNA aptamers |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2021.715328/full |
| work_keys_str_mv | AT meganamcsweeney effectiveuseoflineardnaincellfreeexpressionsystems AT markpstyczynski effectiveuseoflineardnaincellfreeexpressionsystems |