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ESBS - Strasbourg





The aim of our project is to engineer a new fundamental component that could be universally used to build more complex or more controllable biological circuits inside chassis organisms. This new component consists of the E.coli protease ClpXP to which the phytochrome B of arabidopsis thaliana is fused. Any given protein can be degraded as long as it is tagged with our especially designed Biobrick containing a PIF sequence. This Biobrick can be added to the C-terminal end by standard assembly methods. The activity of this system is tightly controlled and reversible by light inducement.

The system can be constitutively expressed in the chassis but remains inactive for the tagged protein. However it is expected to stay active for the background of naturally SsrA tagged proteins and so it will not interfere with the metabolism of E.coli. Instantly after the light inducement the system is turned on, due to the lack of transcriptional or translational delay and is expected to remain active until another light signal turns it off.

In our modeling part we will used this system for a genetic oscillator which could be used for multi step synthesis.

Theoretical Background:

Our system combines two main components: the ClpXP protease and the phytochrome B/ PIF system The ClpXP component is important for the specificity and the phytochrome B/ PIF system is important for the light sensitivity.

-Specificity : We based our system on the ClpXP protease, from E. coli. In wild type E. coli, ClpX forms a hexametric ring 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 down peptide bonds.

Note : In the native organism, 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). This system has two configurations. The Pf configuration which describes the inactive state of the PhyB and the Pfr configuration which describes the active state. The PCB of the 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 the native ClpX system 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.

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

How did we proceed?

For the protease, we fused either the 642 or 908 first amino acids of PhyB to a trimer of the last 364 amino acids 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 amino acids 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 further tagged proteins.