Project 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 microgears8[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" [4]
we have decided to attempt construction of a similar system. In the article a 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 aligning 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 focuses on E. coli strain MG1655 that will be modified to overexpress flagella, in an attempt to increase the 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, apart 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 known as the master regulon of flagella synthesis[7]. In normal E. coli the flhDC operon is tightly regulated by numerous factors[7], resulting in average expression of 4 flagellae per cell[8]. In some hyperflagellated strains, mutations have been found upstream of the regulon that increase expression[9], making the cells hypermotile. We have decided to take a down-and-dirty approach to increase flagella expression, overriding the regulation alltogether by putting the two genes on a constitutive promotor. We hereby hope to increase the pumping power of our system.
Phototaxis
Regulation of the pump will be introduced through a synthetic photo-sensing protein that has recently been shown to integrate with the E. coli chemotaxis system [10]. Since the chemotaxis system regulates flagellar behaviour, we hope to introduce control of the amount of flow generated with very fast response times since chemotaxis is controlled by phosphorylation cascades rather than transcriptional regulation. Although the cells will be held in place in our system, the part will in effect introduce a phototactic ability to free-moving E. coli.
Retinal biosynthesis
For the photoreceptor to work, we will need to supply it with enzymes for retinal biosynthesis. Retinal is formed by cleaving beta-carotene, a reaction that is catalyzed by beta-carotene-oxygenases [11]. We will be supplying a new biobrick that expresses a beta-carotene 15'15-monooxygenase from Drosophila melanogaster. Beta-carotene biosynthesis will be supplied by a part made by the [http://partsregistry.org/Part:BBa_K274210 2009 Cambrigde team ]. We will also do further characterization of the Cambridge part in new strains and with different analytical methods.
Prospects
On top of creating a microfluidic flow generator, we hope to simultaneously create a system that can mix fluids in microtubes. It is often a problem when working in nano-scale spaces 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, thus causing them to mix.
The plan is that these three subprojects will result in at least one biobrick each:
• A constitutively active operon encoding the master regulator of flagella synthesis.
• A photosensor, reacting to blue light, coupled to the chemotaxis pathway.
• A generator for the enzyme that cleaves beta-carotene to retinal.
References
(1) Angelani L, Di Leonardo R, Ruocco G, [http://prl.aps.org/abstract/PRL/v102/i4/e048104 Self-starting micromotors in a bacterial bath]. Phys Rev Lett (2009) 102:048104.
(2) Steager E, Kim CB, Patel J, Bith S, Naik C, Reber L, Kim MJ, [http://apl.aip.org/resource/1/applab/v90/i26/p263901_s1 Control of microfabricated structures powered by flagellated bacteria using phototaxis], Appl. Phys. Lett. 90, 263901 (2007), DOI:10.1063/1.2752721
(3) Darnton N, Turner L, Breuer KS, Berg HC, [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1304020 Moving Fluid with Bacterial Carpets), Biophys J. 2004 March; 86(3): 1863–1870.
(4) Kim MJ, Breuer KS,[http://onlinelibrary.wiley.com/doi/10.1002/smll.200700641/abstract Microfluidic pump powered by self-organizing bacteria.] Small 4, 111 (2008).
(5) Martel S, Tremblay CC, Ngakeng S, Langlois G, (2006) [http://apl.aip.org/resource/1/applab/v89/i23/p233904_s1?isAuthorized=no Controlled manipulation and actuation of micro-objects with magnetotactic bacteria], Appl. Phys. Lett. 89, 233904 (2006); doi:10.1063/1.2402221
(6) Steager E, Kim CB, Patel J, Bith S, Naik C, Reber L, Kim MJ, [http://apl.aip.org/resource/1/applab/v90/i26/p263901_s1?isAuthorized=no Control of microfabricated structures powered by flagellated bacteria using phototaxis], Appl. Phys. Lett. 90, 263901 (2007); doi:10.1063/1.2752721
(7) Chilcott GS, Hughes TK,[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC99010/?tool=pubmed Coupling of Flagellar Gene Expression to Flagellar Assembly in Salmonella enterica Serovar Typhimurium and Escherichia coli], Microbiol Mol Biol Rev. 2000 December; 64(4): 694–708.
(8) Berg HC, [http://www.annualreviews.org/eprint/cDJrS190m62mDRwHrlp9/full/10.1146/annurev.biochem.72.121801.161737 The rotary motor of bacterial flagella], Annual Review of Biochemistry Vol. 72: 19-54 (2003)
(9)Barker CS, Prüß BM,Matsumura P, [http://jb.asm.org/cgi/content/full/186/22/7529?view=long&pmid=15516564 Increased Motility of Escherichia coli by Insertion Sequence Element Integration into the Regulatory Region of the flhD Operon], Journal of Bacteriology, November 2004, p. 7529-7537, Vol. 186, No. 22
(10) Trivedi VD, Spudich JL, [http://pubs.acs.org/doi/abs/10.1021/bi034399q Photostimulation of a Sensory Rhodopsin II/HtrII/Tsr Fusion Chimera Activates CheA-Autophosphorylation and CheY-Phosphotransfer in Vitro], Biochemistry 42 (47), 13887-13892(2003)
(11) von Lintig J, Vogt K, [http://www.jbc.org/content/275/16/11915 Filling the Gap in Vitamin A Research: Molecular Identification of An Enzyme Cleaving Beta-carotene to Retinal] Journal of Biological Chemistry (ASBMB) 275 (16): 11915–11920 (2000)
For further details and closer descriptions, please visit the "Theory" section.
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