Team:ESBS-Strasbourg/Project

From 2010.igem.org

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Our device construction is divided into 4 components :<br><br>
Our device construction is divided into 4 components :<br><br>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Protease">1. Degradation system</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project#degradation">1. Degradation system</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Light">2. Light detection system</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project#light">2. Light detection system</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Tagging">3. Protein Tagging</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project#tagging">3. Protein Tagging</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/System">4. Light controllable protease</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project#system">4. Light controllable protease</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/notebook">Notebook</a>
<a href="http://2010.igem.org/Team:ESBS-Strasbourg/notebook">Notebook</a>
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Revision as of 21:50, 12 September 2010

ESBS - Strasbourg

ESBS - Strasbourg



PROJECT INDEX

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Abstract


The aim of our project is to engineer a new component that could be universally used to build more complex or more controllable biological circuits inside chassis organisms. This new component consists in a protease that can be reversibly induced by light, after which it can specifically degrade any given target protein as long as it is properly tagged.
This system is to be constitutively expressed in the chassis, but will remain as an inactive state until light application. The protease activity is thus expected to be instantly turned on upon application of light since there is neither transcriptional nor translationnal delay.

Background:


The system combines two important different features : specificity, and light sensitivity.

-Specificity : We based our system on the ClpXP protease, from E. coli. In wild type E. coli, ClpX forms a ring of hexamer and binds to a double heptamer of ClpP. ClpX recognizes a specific C-terminal degradation tag called SsrA and starts to unfold the tagged protein. The denatured polypeptide is translocated into the ClpP subunit, which breaks peptide bonds.

Note : In vivo, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell.

-Light sensitivity : We used PhyB (phytochrome B) from A. thaliana and its natural chromophore PCB (phycocyanobilline). Holo phytochrome can absorb red light (pick at 660 nm) and through a structural transition it reaches its activated Pfr state. Under far red light (730 nm) the holo phytochrome goes back to its fundamental inactive Pr state. PIF-3 and PIF-6 (Phytochrome Interacting Factor 3 and 6) are natural partners of PhyB in A. thaliana and bind to PhyB in the Pfr state but not in the Pr state. This is the basis of the controllable feature of our system.


How does it work?



There are different variant of SsrA tags which are more or less efficiently recognized by ClpX. The DAS tag (T.Baker et al.) has a higher Kd value than wild type SsrA tag, and thus degradation of DAS-tagged proteins is not significant.

In vivo though, DAS-tagged protein can still be significantly degraded within the range of physiological concentrations under the action of an adaptator protein (SspB) which help tethering the tagged protein to the protease.

The idea is to replace this adaptator system with a light sensitive tethering system.


How did we proceed?


For the protease, we fused either the 650 or 900 first aminoacids of PhyB to a trimer of the last 364 aminoacids of ClpX. Indeed, the N terminal end of ClpX contains a domain which is responsible for interaction with its natural adaptator protein (SspB) that we would avoid. As this N-terminal domain is also partially responsible for ClpX subunits complexation into an hexamer, fusing three C-terminal end of ClpX together with appropriate linkers increases the stability of the system (T. Baker et al.) in the absence of this N-terminal domain.

The target protein will be tagged with both DAS at C-terminal end, and with the Active Phytochrome Binding (APB) motif (the first hundred aminoacids of PIF-3 or PIF-6), either at N terminal end or just upstream the DAS tag.

Under red light, the APB motif of the target protein binds to PhyB and facilitate recognition of DAS tag by ClpX, thus promoting degradation. Far red light instantly inhibits degradation of tagged proteins.