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Protein degradation in a TX-TL cell-free expression system using ClpXP protease
Abstract We explored the possibility of supplementi
We explored the possibility of supplementing an in vitro S30-based Escherichia coli expression system (or “TX-TL”) with ClpXP, an AAA+ protease pair that selectively degrades tagged proteins, to provide finely-tuned degradation. The mechanism of ClpXP degradation has been extensively studied both in vitro and in vivo. However, it has not been characterized for use in synthetic circuits -- metrics such as toxicity, ATP usage, degradation variation over time, and cellular loading need to be determined. In particular, TX-TL in batch mode is known to be resource limited, and ClpXP is known to require significant amounts of ATP to unfold different protein targets. We find that ClpXP’s protein degradation dynamics is dependent on protein identity, but can be determined experimentally. Degradation follows Michaels- Menten kinetics, and can be fine tuned by ClpX or ClpP concentration. Added purified ClpX is also not toxic to TX-TL reactions. Therefore, ClpXP provides a controllable way to introduce protein degradation and dynamics into synthetic circuits in TX-TL. <p> NOTE: This is a technical report for future inclusion in work pending submission, review, and publication. Therefore, this work has not been peer-reviewed and is presented as-is.
been peer-reviewed and is presented as-is.  +
Authors Zachary Z. Sun, Jongmin Kim, Vipul Singhal, Richard M. Murray  +
Funding Biomolecular Breadboards for Prototyping and Debugging Synthetic Biocircuits +
ID 2014g  +
Source Technical Report, 7 July 2014  +
Tag sksm14-clpxp  +
Title Protein degradation in a TX-TL cell-free expression system using ClpXP protease +
Type Technical Report  +
Categories Papers
Modification date
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15 May 2016 06:14:45  +
URL
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http://www.cds.caltech.edu/~murray/preprints/sksm14-clpxp.pdf  +
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Protein degradation in a TX-TL cell-free expression system using ClpXP protease + Title
 

 

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