Team:Kyoto/Project





Raise the Titanic!

Project Description In many biotechnological contexts, bacterial cells are considered as "chemical facilities." A number of studies have genetically engineered cells to produce various desired compounds. They further aim at accurate and precise regulation of material production. Cells are also power suppliers in terms of their motility. This aspect, however, has been much less featured. Here comes our project, which started with the gigantic goals of lifting up the Titanic from the deep-sea with bacterial power. Toward our general goal – to engineer cells to carry larger order of objects – we have been designing and constructing cells so that these micro-order entities can move a centimeter or larger objects. We have equipped E. coli with the functions of attachment to an object surface, cell density dependent buoyancy production, and regulatable flagella and examined by quantitating the parameters to what extent our goal is achieved. Our study presents the possibility of bacterial physical power.

  

Functions: Bind, Buoy, and Push!

Our E. coli machine has these 3 functions.

Function A: Binding to Ti and polystyrene (Modified genes: luxI, luxR, gvpA, gvpB, gvpC) Fisrt, cells must bind to the surface of the Titanic. For this purpose, we employed the cell surface display method with Lpp-OmpA-fusion protein. This method enables to display particular peptides on the surface of the gram negative bacteria. In this project, we fused two kinds of binding peptides with OmpA; one is Titanium-binding peptide (TBP)3 and the other is polystyrene binding peptide (PBP)4. Both of which were obtained by PCR cloning.

Function B: Cell density-dependent buoyancy (Modified genes: luxI, luxR, gvpA, gvpB, gvpC) We designed the cell density dependent buoyancy system by which cells produce buoyancy only when they have sufficiently increased. This aims at both fast growth rate and enough buoyancy. E. coli gains buoyancy from intracellular hollow organelles called gas vesicles. The expression of this gene is regulated by quorum sensing (QS) mechanism. Not activating gas vesicle genes during, for eample, an exponential growth phase enhances their grow rate. For QS, E. coli was transformed with two genes luxI and luxR. LuxI is an autoinducer synthase, which produces acyl-homoserin lactone (AHL). LuxR is a protein that binds to AHL. Since AHL freely diffuses though cell membrane, its concentration reflects density of the cells. When bound to AHL, LuxR stimulates Lux promoter of the gas vesicle gene. For gas vesicle proteins, only gvpA – C among at least 15 related genes are used to minimize the cost of the protein production.

Function C: Light-dependent flagella rotation Our engineered E. coli is designed more likely to continue counter clockwise (CCW) flagella rotatation in the darkness due to cheZ. The mechanism consists of following four points:

 CCW is promoted in the presence of CheZ. In our E.coli, the synthesis of CheZ is accelerated in the darkness and is repressed in the light. Then, the number of E.coli rotating flagella CCW on the darker side of the object on which E.coli is binding exceeds that on the lighter side. The object on which E.coli is binding can gain force in the direction of lighter place.</li> </ol>

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Our E. coli is designed to sense light and then generate thrust as following.

<ul class="functionC"> CCW rotation of flagella produces thrust</li> CCW is promoted in the presence of CheZ.</li> In our E.coli, cheZ expression is activated by light.</li> </ul>

For the production of light activated protein. three genes, ho1, pcyA, and cph8 of BioBrick parts were introduced. This protein is phosphorylated in light exposure and activates a specific promoter and expression of its downstream gene cheZ.