IGEM:IMPERIAL/2008/New/Chassis 1

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<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype><img width=50px src=http://openwetware.org/images/f/f2/Imperial_2008_Logo.png></img</a></html> Home The Project B.subtilis Chassis Wet Lab Dry Lab Notebook

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Chassis 1

This page offers a brief overview of how B. subtilis meets our main project specifications - to a much higher degree than E. coli! You'll find information on the main mechanisms behind our proposed system, and some very interesting biology too. If you would like even more depth than the outlines below, you are welcome to visit our OpenWetWare pages on those topics - but don't get to engrossed and forget to come back!

Motility

A schematic of the B. subtilis rotary flagellar motor is shown. Motile cells are powered by interactions of the FliG protein with the MotA/B complex (which generates torque). The protein EpsE acts as a molecular clutch to disengage the rotary flagellar motor, leaving the flagellum intact but unpowered. This shuts down motility and facilitates biofilm formation. Fluorescence microscopy photos of B. subtilis show bacterial membranes in red and flagella in green. FliM and FliF are motor proteins [1]

To achieve accurate distribution of our biomaterial microfactories (an affectionate term for our bacteria), we must first exert fine control over their motility. Bacteria's primary method of getting about is via flagellar locomotion. A ring of protein in the cell membrane rotates and is attached to a flagellum that extrudes, acting like a long corkscrew propeller to push the cell through its environment.

The difference between B. subtilis and other bacteria lies in our knowledge of its precise mechanism for flagellar locomotion. A recent paper describes an elegant clutch mechanism involved in the process. The flagella can be detached from the rotor by expression of a molecule that interacts with the flagella and distorts it so it is disengaged from the rotor protein.

Control over the expression of this protein should allow us to control of the bacteria very quickly. When we want the bacteria to stop, we trigger expression of the clutch molecule which halts movement.

To draw a parallel with a car, current synthetic methods of stopping bacteria are akin to destroying the engine (which can take time!) in order to stop it powering the vehicle where the method we hope to take advantage of would be like putting the car into neutral - disengaging the engine from the driveshaft. It's an elegant solution that offers us quick control and also the opportunity for quick reversal (putting the car back into "drive").

More: http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Motility

Light Sensing

A holographic image of a work by sculptor Eileen Borgeson [2]

With a very elegant motility-control strategy up on the drawing board, we need to look at how we are going to use that system to trigger production of biomaterial in a set pattern. We need a stimulus - something that the bacteria can detect and respond to - that we can control accurately (possibly even in 3D!).

The most obvious candidate for this is light. Light allows us to generate complex patterns with well defined edges, while gradients in wavelength and intensity will allow us to build up varying concentrations of biomaterial. After examining a number of light pathways present in subtilis, other bacteria and light-sensing bricks in the Registry we decided to make use of a native pathway involving YtvA. This protein is used by B. subtilis to detect blue light, whereupon it triggers a cascade of interactions. Importantly some way down the chain a molecule called sigma B (σB) is produced which can act to boost the expression of genes.

We plan to over-express YtvA, and use σB as a promoter for genes that stop movement and produce biomaterial. Thus when the bacteria detects blue light those genes will turn on, the bacteria will stop and biomaterial will be produced.

If we were really ambitious, or as a continuation of this project if it works well, one might try using holograms of blue light in semi-solid media to produce a 3D image that the microfactories could fill in with biomaterial!

Biomaterials

Scaffolding!

After our bacteria are positioned correctly, they need to be able to express a biomaterial. B. subtilis is Gram-positive, meaning it has only a single membrane as opposed to a double membrane like E. coli. This means that physical expression of a biomaterial is a lot more tractable; biomaterial can be produced and secreted more efficiently. With a double membrane, material may accumulate inside the cell and the efficiency of biomaterial production may be very low.

Another important aspect of our biomaterial specifications is what we want to secrete. We did a lot of research on this and decided to express elastin peptides and EAK16-II. Both are small peptides and their molecular structures favour self-assembly outside the B. subtilis cells to form 3D bio-scaffolds under appropriate conditions. 3D bio-scaffolds are very useful for tissue culture and regenerative medicine, as they offer a 3D enviroment for implanted cells to grow and proliferate.



So B. subtilis fulfils our main specifications perfectly, and can be made to meet our minor specifications with relatively little re-working. On top of this it does have other benefits (along with some challenges!) and these are listed on the next page - as well as an overview of our development of it as a chassis. > The Second Chassis Page >


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