Team:Osaka/MOTILITY

MOTILITY

Overview 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 on soft agar. 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 control the swarming motility of cell, and in the future, integrate it with 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, all their approaches focused on the motor rotation itself. Bacteria flagella, one of nano machines, has a noteworthy characteristic except motor mechanism, which is "protein translocation" from the cytoplasm to the external environment. This protein translocation mechanism might be broadly applicable to problems in biotechnology if it is possible to control protein translocation. So we made a new part to inhibit flagellar protein translocation and as a result stop the motility as a result of failure of flagellar assemebly. And in addition, we tested the compatibility of EpsE that work as molecular clutch and stop the motor rotation in B. subtilis. Design Bacterial flagellar assembly is proceed by highly sophisticated manner in which gene regulation coordinates with self-assembly of motor proteins[1]</a>.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 that is evolutionary related with pathogenic type III secretion system <a href="#2">[2]</a>. By a lot of study, the molecular mechanism of this systems is being elucidated. 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 FliHx-FliI6 complex. And then, ATP hydrolysis induces dissociation of the FliHx-FliI6 and successive unfolding and translocation of the substrates is driven by the PMF. <img src="http://2009.igem.org/wiki/images/3/36/Export_model.jpg" width="622" height="214" > FliH, the regulator of ATPase FliI, is one of soluble components of export apparatus. In the absence of FliI, FliH inhibits the protein translocation <a href="#3">[3]</a>. Further more, even in the presence of FliI, FliH expression from pTrc99A vector caused pronounced inhibition of swariming motility of the cell <a href="#4">[4]</a>. 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.

Results We successfully constructed BioBrick fliH (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K204500">K204500</a>).To characterize the effect of FliH on the swarming motility and protein secretion level, we used pTet and pT7 promoters. Salmonella typhimurium</I> (MMHI0117) was transformed with pTet FliH (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K204502">K204502</a>), pT7_FliH (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K204503">K204503</a>), and pUC19 for vector control. In this experiment, we used MMHI0117, which is the fliH fliI null mutant. So it was speculated that less FliH expression will be required for wild type cell to control protein translocation <a href="#3">[3]</a>. So as first step, we tested our parts in this strain. FliH expression from both parts decreased the swarming motility of the cell (Fig. 1). Because T7 promoter is leaky in the absence of T7 RNAP in Salmonella typhimurium</I>, the swarming motility was decreased transforming pT7_FliH (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K204503">K204503</a>). <img src="http://2009.igem.org/wiki/images/9/91/P1010002.png" width="270" height="272" > And the secretion level of flagellar protein is also decreased (Fig. 2). <img src= "http://2009.igem.org/wiki/images/f/f4/Export_level.jpg" border="1" width="213" height="135" align="middle"> So, these results showed that FliH can be used to control the swarming motility and the secretion level of flagellar protein as we expected. For application for other strain, the check of compatibility and improving FliH expression will be needed. We also tried to stop the flagellar rotation by using <a href="http://partsregistry.org/Part:BBa_K143032">EpsE</a>. We used pTet promotor to express EpsE. However, EpsE had no effect on swarming motility of the cell (Data not shown). Although FliG which EpsE interacts with is highly conserved between Salmonella typhimurium</I> and B. subtilis, EpsE was no functional in Salmonella typhimurium</I>. Furthermore, other B. subtilis's intrinsic protein might be required for mediating the interaction between EpsE and FliG (personal communication with Dr. Phil Aldridge).Taken all together, using EpsE in any host other than B. subtilis to control the flagellar rotation and cell motility is ill-advised action. Discussion We showed that FliH is useful parts to control the swarming motility and protein secretion level. This method is relatively easy because it require only expressing FliH. However, the level of expression of FliH is important to control the protein secretion. Now we are integrating this parts with cell-cell communication systems. Although we used FliH to control the swarm size, there are potentially several applications of this part. Flagellar type III protein export is notably fast and the protein export rate is 55kDa subunits persec. Therefore, this system can be applied to drug delivery system or material production in the future<a href="#5">[5]</a>. And engineering these molecular machine to exert its ability and integrating it with other module should be crucial work for creating useful bio robot.

Method 1. Swarming motility assay Fresh colonies were inoculated on soft tryptone agar plates and incubated at 30℃. 2. Export assay Cells were grown in 5 ml LB medium at 37℃ with shaking until the cell density had reached at OV600 of 0.8-1.0. 1.5 ml of culture were centrifuge to obtain the cell pellets and culture supernatants. Cell pellets were re-suspended in the SDS-loading buffer, and normalized to a cell density give a constant number of cells. Protein in the culture supernatants were precipitated by 10% trichlorocetic acid, suspended in the 0.1M Tris/SDS loading buffer and heated at 95℃ for 5 min. After SDS-PAGE, proteins were stained by CBB.

Reference <a name="1" id="1" class="internal">[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.</a> <a name="2" id="2" class="internal">[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. </a> <a name="3" id="3" class="internal">[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).</a> <a name="4" id="4" class="internal">[4] Minamino, T. & Macnab, R.M. Interactions among components of the Salmonella flagellar export apparatus and its substrates. Molecular Microbiology 35, 1052-1064(2000).</a> <a name="5" id="5" class="internal">[5] Galan, J.E. Energizing type III secretion machines: what is the fuel? Nat Struct Mol Biol 15, 127-128(2008).</a>

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