Team:Kyoto/Project

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.

Systems

Our E. coli machine has mainly these 3 functions.

Function A: Binding to Ti and polystyrene

(Modified genes: luxI, luxR, gvpA, gvpB, gvpC)

To make cells stiffly bind to the surface of the Titanic, we use a famous method of cell surface display; Lpp-OmpA-fusion protein used display1,2. We can apply this method to display particular peptide on the surface of the gram negative bacteria. In this case, we fused two affinity holding peptides; one is Titan binding peptide (TBP)3 and the other is polystyrene binding peptide (PBP)4. We cited the dominant sequence from following papers and applied it into PCR cloning.

Function B: Cell density-dependent buoyancy

(Modified genes: luxI, luxR, gvpA, gvpB, gvpC)

E. coli is given buoyancy by interacellular hollow organelles called gas vesicle. The expression of gas vesicle is regulated by increased cell population. This system uses Quorum sensing that is cell to cell communication system. We transferred two genes luxI and luxR from V. fischeri to E.coli (iGEM parts). LuxI is an autoinducer synthase, which produces the acyl-homoserin lactone(AHL). LuxR is protein that can bind AHL. The AHL diffuse out of the cell and increases its concentration with increasing cell population. When bound to AHL, lux promoter stimulates and Gas vesicle appears.

Function C: Light/dark-dependent flagella

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:

1. CCW is promoted in the presence of CheZ.
2. In our E.coli, the synthesis of CheZ is accelerated in the darkness and is repressed in the light.
3. 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.
4. The object on which E.coli is binding can gain force in the direction of lighter place.