Team:ESBS-Strasbourg/Project/Strategy

Our device construction is divided into 4 components :

1. Degradation system
2. Light detection system
3. Protein Tagging
4. Light controllable protease

Notebook

The work of the former IGEM Team had a great influence on our choice, particularly those of The ESBS 2008 Team. Their goal was to control cells’ state by linking the natural variability of life to the binary system of computing. In the project of The ESBS 2008 Team, the incrementing from one bit to another required a protease which was expressed during the mitosis in yeast. Renaud Renault of the actual iGEM team had thus the idea of inventing a degradation system which would be not only temporally controllable but also specific.

Based on this idea we chose to create a light-controllable specific protein degradation system. The system contains several parts: degradation system with the bacterial ClpXP protease from Escherichia coli (E.Coli), the light detection system with the photoreceptor protein Phytochrome B and the Phytochrome Interacting Factor (PIF 3 or 6) from Arabidopsis thaliana (A. thaliana) and the protein tagging with the DAS/LAA recognition sequences for ClpX represent the main parts of our system.

The different parts, their basic ideas and their strategic development will be discussed in detail in the following separated parts. The choice of the host organism will also be explained, followed by the final structure of the light controllable protease.

The ClpXP from E.coli protease rapidly established itself as an evident choice. Indeed, this protease has been very well studied, notably by the Tania Baker Team from Berkeley.

Description

Basically, ClpXP is an AAA protease present in bacteria, consisting of two main components, ClpX and ClpP. The ClpX is a hexamer consisting of six identical subunits. It recognizes specific degradation tags of target substrate proteins, unfolds them in an ATP-consuming hydrolysis reaction, and uses additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place. ClpP is a multi-subunit serine peptidase, in which the proteolytic active sites reside within a barrel-shaped structure.

                       The figure on the left shows the two heptamers forming ClpP in our light controllable protease
The figure on the right shows the hexamers of ClpX in our light controllable protease

How using the ClpXP protease ? : The question of the use of an adaptator

This question was really critical. We focused on different strategies before making a definitive choice.
The publication of Tania Baker (Baker and Sauer 2006) based on the ClpXP protease of E. Coli which degrades substrates bearing the specific SsrA recognition sequence, has been the starting point of our reflection. In this work Baker and colleagues designed a series of modified ssrA tags which have weakened interactions with ClpXP to engineer controlled degradation. In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. In the absence of SspB , substrates bearing the artificially altered DAS-tag were stable, in contrast the degradation of substrates bearing these engineered peptide tags was 100-fold more efficiently when SspB was present.

Upon these findings our first idea consisted in using the native ClpX with the mutated tag and a modified SspB whose binding to ClpX should be controlled by light in order to control protein degradation. Forcing cells to produce an inactive form of an adaptator seem to be a good solution to be able to stop the degradation at a certain point. This could be realized by producing two parts of the adaptator which could interconnect them after light-induction by fusing them to proteins which had this capacity, for instance the couple Phytochrome/PIF.

However, the use of the adaptor-based system posed some major problems concerning the complexity. Subsequent to another finding of Baker et. al., we decided to fuse our phytochrome directly to the N-terminal of ClpX, as it is not required for the basic enzymatic functions of ClpX (Baker and Sauer 2006).

Further, we decided to fuse the target protein, additionally to the specific degradation tag, with PIF which will assume the role of the adaptor protein SspB .

Phytochrome - PIF

bla bla bla

Tagging

bla bla bla

Light Inducible Degradation

bla bla bla