Under construction

Motile cells, such as Escherichia Coli and Salmonella typhimurium swim by means of flagella. So, we can regard cells as biological paints, we named ColorColi, that can move automatically. Although we can create a lot of artworks (you can see our works at WORKS) by using innate motility of cells, engineering cells motility should expand the application of ColorColi in Art. This year, we aimed to stop the swarming motility of cell for out-put of arithmetic processing by cell-cell communication.(see SIGNAL).

In past iGEM projects, several teams tried to control cell motility (iHKU2008, Imperial2008, Penn State Uni2006). However, their approaches require modified or specified chassis. To control the cell motility more conveniently, more general methods will be needed.

So we made a new part to inhibit flagellar assembly and as a result stop the motility. And in addition, we tested the compatibility of EpsE that work as molecular clutch and stop the motor rotation in B. subtilis for further work.

Bacterial flagellar assembly is proceed by highly sophisticated manner in which gene regulation coordinates with self assembly of motor proteins[1].To form the flagellar axial structure, "molecular propeller", at the cell exterior, these protein subunits must be translocated across the cell membrane. And this work is carried out by flagellar type III secretion system [2]. By a lot of study, the molecular mechanism of this systems is being elucidated recently.Currently, the following model for flagellar protein export is suggested (Fig. 1). N-terminal of segment of a substrate is initially docked with by formation of the FliH_x-FliI₆ complex. And then, ATP hydrolysis induces dissociation of the FliH_x-FliI₆ and successive unfolding and translocation of the substrates is driven by the PMF.

FliH, the regulator of ATPase FliI, is one of soluble components of export apparatus. In the absence of FliI, FliH inhibits the protein translocation [3]. Further more, even in the presence of FliI, FliH expression from pTrc99A vector caused pronounced inhibition of swariming motility of the cell [4].

Considering above mentioned molecular mechanism, we aimed to control flagellar protein transloaction by expressing FliH. Because the molecular propeller is required for motility, the inhibition of flagellar assembly should lead the inhibition of cell motility. Furthermore, the components of flagellar export apparatus is highly conserved among a number of bacteria, this method can be potentially applied for various bacteria.

We successfully constructed BioBrick fliH.（パーツのリンク）To characterize the effect of FliH on the swarming motility and protein secretion level, we used p_Tet and p_T7 promoters.

[1] Fabienne F. V. Chevance and Kelly T. Hughes, “Coordinating assembly of a bacterial macromolecular machine,” Nat Rev Micro 6, no. 6 (June 2008): 455-465.

[2] Tohru Minamino, Katsumi Imada, and Keiichi Namba, “Molecular motors of the bacterial flagella,” Current Opinion in Structural Biology 18, no. 6 (December 2008): 693-701.

[3] Minamino, T. & Namba, K. Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export. Nature 451, 485-488(2008).

[4] Minamino, T. & Macnab, R.M. Interactions among components of the Salmonella flagellar export apparatus and its substrates. Molecular Microbiology 35, 1052-1064(2000).