IGEM:IMPERIAL/2008/Projects/motility

Production of materials is relatively easy - genetic engineering has been doing and refining this for a relatively long time. Producing materials in useful 'formats', however, is slightly harder. This page lists some possible methods for directing bacteria, allowing them to construct tertiary structures or lay down materials in a pattern.

This page includes an overview of ideas that were touched upon in our brainstorming sessions, and expands on them.

Motile Control Mechanisms

Directional

Magnetism

It could be possible to steer bacteria using a magnetic field, if they were first polarised somehow. A shifting magnetic field should allow fine control of the movement of bacteria - the field would need to shift in line with the bacteria's movement speed however, and to regulate it may require a reporter gene being present (so they can be directed as they go).

Electricity

Similarly to magnetism, using an electric field could allow control over the movement of the bacteria - this may be easier to regulate than magnetism too, as you can change the strength of the field with ease.

Chemotaxis

Chemotaxis is an interesting possibility for controlling direction. Erika's mini-iGEM project (presentation on Thursday!) involved a chemotactic "dot-to-dot" set up, whereby bacteria were directed to a point (where a nutrient source was placed that they were attracted to) up a gradient. Quorum sensing was then used to trigger both an excretion of a marker protein (to draw the dot) and a shift in the attractant - so they would start moving toward a second point, the position of a second attractant.

Distributed

Quorum Sensing

Quorum sensing is a very valuable tool for this situation. With it we can trigger clustering or dispersal via a link to the flagellar motion of the bacteria (see the flagellar clutch below), and theoretically we should be able to arrange our bacteria in a monospaced array... This could be very useful for producing e.g. collagen or a similar product.

The Flagellar Clutch

1. 2005 Penn State Uni team did a similar project, but they didn't use EpsE/SinR gene expression. Instead, they played around with the actual motor of the bacteria, motB gene which generates the torque required to give flagellum its rotating power. http://ieeexplore.ieee.org/xpls/abs_all.jsp?arnumber=4290622

2. Here is a simple explanation of how the clutch system works: http://www.sciencemag.org/cgi/reprint/320/5883/1599.pdf What happens is that the gene SinR upregulates the expression of flagellar genes i.e. MotA/MotB etc. and downregulates expression of biofilm forming genes. If SinR is absent, biofilm takes over and the bacteria loses its motility. It was found that SinR represses the epsE gene, thus the absence of SinR causes the derepression of EpsE.

It was later determined that EpsE is responsible for disengaging the clutch in the flagellar motor. See http://www.sciencemag.org/cgi/reprint/320/5883/1636.pdf for more. This means that instead of turning the motor on or off, bacteria's motor is continuously spinning, but disengaging or engaging the flagella to the motor is regulated by EpsE and thus the epsE gene. If we can characterise the epsE gene, we should be able to control whether the bacteria moves or not, irregardless of its orientation, which brings us to the next problem.

Light

If one begins with a regular array of bacteria on a plate, the triggering of production and secretion of the biomaterial required could be linked to light - this could, conceivably, allow us to produce materials in a very complex pattern, and also allow gradients of production (should that be required). Repeating this multiple times for different layers could, therefore, allow us to build up a construction (not unlike a 3D printer [1]) of our material.

In 2000 a light-activated proton pump called Proteorhodopsin was discovered. In the absence of cellular respiration, this pump is activated by illumination, generating a proton motive force which drives the flagellar motor. However, there is no effect of light when respiration is not impaired, and there are no direct links between this light sensitive protein and EpsE which we are trying to control.

Possibilities

Combinations of the above techniques allow for powerful possibilities for control.

Fibrous Construction

Production of fibres would be useful, as we use fibrous materials a lot (textiles/nylon for example). Spider silk is the most notable example of a fibrous material that would be incredibly useful to make. Production of fibres, however, would be very hard from a Synthetic Biology point of view... If one were to attempt to make a fibre by combination of fibrils, each being produced by a different bacteria, exceptionally fine directional control would be needed. In addition it would be very hard to produce a long fibre from a bacteria, considering their mode of expulsion of product (sort of like a reverse-phage, a needle of protein from within pierces the outer membrane and product exits through the needle but the needle can pierce anywhere on the bacteria. Additionally you'd need all the bacteria to swim the same way while spinning out the fibrils at an approximately constant rate and it's probably not feasible. It may be better for us to focus on other materials, therefore.

Laminar Construction

Construction of laminar patterns, designs or sheets of biomaterial would be very valuable indeed - particularly, perhaps, in the medical field (production of collagen for instance, skin grafts, and so on). This might be the most exciting application or area of production of biomaterials that is feasible, with the complexity of 3D construction being inhibitory! Having said that...

3-Dimensional Construction

Perhaps the most exciting idea is that of a biological 3D printer, as referred to above; building up a structure, layer by layer. With the addition of a number of different materials being produced, it may be possible to form complex structures with different components. Layer by layer construction would take a long time, however, and it's uncertain whether each excreted protein could be connected to the ones around it.

Approaching from a different angle, one might envision a block of agar laced with nutrients that trigger production of a biomaterial; and bacteria forming up via chemotaxis/quorum sensing and excreting their product in a shape as defined by the presence or absence of certain nutrients, perhaps.

Scaffold Construction

A key possibility for construction of a complex shape is the utilisation of a scaffold. It has been suggested that a scaffold of chitin would be the most likely possibility, as it is produced by fungi (as well as insects, crustaceans) and therefore probably does not require any complex post-translational modifications. Building a scaffold and then laying a more detailed product on top is a valuable idea.

Drawbacks/Issues

Many of the problems are similar across all methods. Most obvious, perhaps, is that bacteria divide and that this could mess up our steering. One solution might be to produce a two-state bacteria - in one state it can divide but not move, and in the other it can not divide but can move about. Thus you could start with a fixed colony of immobile bacteria, breed them up to the desired amount, then flip the bistable switch with a trigger and put them into the motile state. They would stop dividing, and would move as directed (well, hopefully). Peking's 2007 iGEM project's push-on push-off switch could be ideal for this.