Construction a RBS library with different translational activity
The first step in programming and controlling cell behavior is to establish a library of well-defined components, a.k.a. "biobricks", that serve as the building blocks of artificial gene networks. The main challenge in genetic circuit design lies in selecting well-matched genetic component...
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| Format: | Technical Report |
| Language: | English |
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2010
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| Online Access: | http://hdl.handle.net/1721.1/60088 |
| _version_ | 1811078105605865472 |
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| author | Lee, Hsiao-Ching |
| author_facet | Lee, Hsiao-Ching |
| author_sort | Lee, Hsiao-Ching |
| collection | MIT |
| description | The first step in programming and controlling cell behavior is to establish a library of well-defined components, a.k.a. "biobricks", that serve as the building blocks of artificial gene networks. The main challenge in genetic circuit design lies in selecting well-matched genetic components that when coupled, reliably and consistently act in a desired behavior. Although the parameter values are calculated by model equations, it is hard to select the biobricks that reliably implements a desired cellular function with quantitative values. To overcome this problem, the RBSs were designed to control the expression of downstream genes when necessary. This protocol will describe how to generate a RBS library with different ribosome binding affinities. The degenerated primers designed for PCR are used to generate mutations in RBS regions, while the mutated RBS activity are assayed using green fluorescence protein on a low copy number plasmid. In addition, a library of RBS's with different transcriptional strength can be built to fit the specific parameter values derived from model equations. Because the reporter protein activity has a positive correlation to RBS translational activity, we can design customizable RBS translational strengths for application. |
| first_indexed | 2024-09-23T10:53:29Z |
| format | Technical Report |
| id | mit-1721.1/60088 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T10:53:29Z |
| publishDate | 2010 |
| record_format | dspace |
| spelling | mit-1721.1/600882019-04-12T11:36:35Z Construction a RBS library with different translational activity Lee, Hsiao-Ching RBS The first step in programming and controlling cell behavior is to establish a library of well-defined components, a.k.a. "biobricks", that serve as the building blocks of artificial gene networks. The main challenge in genetic circuit design lies in selecting well-matched genetic components that when coupled, reliably and consistently act in a desired behavior. Although the parameter values are calculated by model equations, it is hard to select the biobricks that reliably implements a desired cellular function with quantitative values. To overcome this problem, the RBSs were designed to control the expression of downstream genes when necessary. This protocol will describe how to generate a RBS library with different ribosome binding affinities. The degenerated primers designed for PCR are used to generate mutations in RBS regions, while the mutated RBS activity are assayed using green fluorescence protein on a low copy number plasmid. In addition, a library of RBS's with different transcriptional strength can be built to fit the specific parameter values derived from model equations. Because the reporter protein activity has a positive correlation to RBS translational activity, we can design customizable RBS translational strengths for application. 2010-12-05T20:59:58Z 2010-12-05T20:59:58Z 2010-12-05 Technical Report http://hdl.handle.net/1721.1/60088 en BBF RFC;79 Attribution-No Derivative Works 3.0 United States http://creativecommons.org/licenses/by-nd/3.0/us/ application/pdf |
| spellingShingle | RBS Lee, Hsiao-Ching Construction a RBS library with different translational activity |
| title | Construction a RBS library with different translational activity |
| title_full | Construction a RBS library with different translational activity |
| title_fullStr | Construction a RBS library with different translational activity |
| title_full_unstemmed | Construction a RBS library with different translational activity |
| title_short | Construction a RBS library with different translational activity |
| title_sort | construction a rbs library with different translational activity |
| topic | RBS |
| url | http://hdl.handle.net/1721.1/60088 |
| work_keys_str_mv | AT leehsiaoching constructionarbslibrarywithdifferenttranslationalactivity |