Background

In fields such as nano-scale robotics and manufacturing researchers have encountered problems in generating motion and force reliably. In recent years many attempts have been made at using micro-organisms to create useable mechanical force. Since microorganisms have adapted ways of efficiently creating movement in nanoscale environments, they pose an interesting alternative to conventional mechanical devices as means of driving nanoscale machines, much in the same way that animals have been used in agriculture and production in the past. Different approaches have been taken including using swimming bacteria to drive microgears(1), move objects(2) and to generate organised flow on surfaces and in pump-like systems(3),(4). Different attempts at introducing remote control in such systems have also been made using magnetism(5), chemical stimuli(4) and light(6). It seems obvious how synthetic biology might contribute by creating systems for these purposes.

The Idea

Inspired by an article on flow generated in a micro-capillary tube by a bacterial "pump" [http://microfluids.engin.brown.edu/Breuer_Papers/Journals/Small2008_Bacterial_Pump.pdf (4)] we have decided to attempt construction of a similar system. In the article pump was constructed by coating the inside of a tube with Serratia Marcesens. This was done by washing a bacterial suspension through the tube at a speed, that allowed the cells to adhere to the surface. The flow also had the effect of alligning most of the bacteria, so their flagellae were facing downstream. When the induction of flow was stopped, the bacteria kept the solution flowing with their flagellae, in essens acting as a pump. Control was lent by altering the glucose concentration of the buffer solution. They were able to show a measurable force for several hours, before the cells became de-energized and died.

Our Approach

Our approach centers on E. coli that will be modified to overexpress flagella, in an attempt to increase force generation potential. We also want to be able to regulate the flow that is generated with a light sensor, that integrates into the chemotaxis pathway, giving us very fast response times. Finally for the photosensor to function propperly we will need to introduce retinal biosynthesis to the system. In this way we can avoid altering the buffer solution flowing through the system, appart from the trace amounts of waste products from the cells metabolism.

Hyperflagellation

To achieve hyperflagellation we have decided to focus mainly on increasing the expression of the flhD and flhC transcriptional regulators, also know as the master regulon of flagella synthesis(?). In normal E. coli the flhDC operon is tightly regulated by numerous factors(), resulting in average expression of 3-7 flagellae(). In some hyperflagellated strains mutations have been found upstream of the regulon that increase expression(), making the cells hypermotile. We have decided to take a down-and-dirty approach to increasing flagella expression, overriding the regulation all together by putting the two genes on a constitutive promotor. We hereby hope to increase the pumping power of our system.

Phototaxis

Retinal Biosynthesis

Regulation of the pump will be introduced through a photo-sensing chimeric fusion protein that has recently been shown to integrate with the E. coli chemotaxis system [http://pubs.acs.org/doi/abs/10.1021/bi034399q], to control tumbling frequencies in our bacteria, thereby giving us control over the amount of turbulence disrupting our pump via a blue light source. This will result in an off-switch, since the blue light increases the tumbling frequency, which disturbs the flow. The more bacteria tumble instead of following the "run" pattern, the weaker (or nonexistent) the flow woll be. The parts contributed will in effect introduce phototaxic ability to E. coli.

For the photoreceptor to work, we will need to supply it with retinal. Therefor we will be building on work done in the Cambridge 2009 project, that introduced beta-carotene synthesis in E. coli, and contribute an enzyme that will catalyze cleavage of beta-carotene into retinal. By this our biological machine can supply it self with everything it needs to function, so that once the system has been established, there is need to intervene (except for the light regulation).

On top of creating a microfluidic flow generator, we will hopefully at the same time create a system that can mix fluids in microtubes. It is often a problem when working with tubes of this dimension, that if you let two liquids flow into them, they will not mix. The turbulence created by the bacteria's flagella will make both liquids move around randomly in the tube, so that they will become one in the end.

The plan is that these three subprojects will result in a biobrick each:
- A constitutively active operon with the master regulator of flagella synthesis.
- A bluelight photosensor coupled to the chemotaxis pathway.
- A generator for the enyzme that cleaves beta-carotene to retinal.

References

[http://prl.aps.org/abstract/PRL/v102/i4/e048104 (1)] Angelani L, Di Leonardo R, Ruocco G, Self-starting micromotors in a bacterial bath. Phys Rev Lett (2009) 102:048104.
[http://apl.aip.org/resource/1/applab/v90/i26/p263901_s1 (2)] Steager E, Kim CB, Patel J, Bith S, Naik C, Reber L, Kim MJ, Control of microfabricated structures powered by flagellated bacteria using phototaxis, Appl. Phys. Lett. 90, 263901 (2007), DOI:10.1063/1.2752721
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1304020/ (3)] Darnton N, Turner L, Breuer KS, Berg HC, Moving Fluid with Bacterial Carpets, Biophys J. 2004 March; 86(3): 1863–1870.
[http://onlinelibrary.wiley.com/doi/10.1002/smll.200700641/abstract (4)] Kim MJ, Breuer KS,Microfluidic pump powered by self-organizing bacteria. Small 4, 111 (2008).
[http://apl.aip.org/resource/1/applab/v89/i23/p233904_s1?isAuthorized=no (5)] Martel S, Tremblay CC, Ngakeng S, Langlois G, (2006) Controlled manipulation and actuation of micro-objects with magnetotactic bacteria, Appl. Phys. Lett. 89, 233904 (2006); doi:10.1063/1.2402221
[http://apl.aip.org/resource/1/applab/v90/i26/p263901_s1?isAuthorized=no (6)] Steager E, Kim CB, Patel J, Bith S, Naik C, Reber L, Kim MJ, (2007) Control of microfabricated structures powered by flagellated bacteria using phototaxis, Appl. Phys. Lett. 90, 263901 (2007); doi:10.1063/1.2752721

For further details and closer descriptions, please visit the "Theory" section.

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