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Azobenzene Photo-induced Magical Molecule
From NPs Aggregation to Synthetic Biofilm
(1) Our bacterium has two main features - one is a sensor and the second is azobenzene attached to the LPS. When the bacterium detects a substance it changes color by producing green luciferase
(2) The light emitted from the bacterium causes the azobenzene molecules to change conformation to a "sticky" form
(3) The azobenzene molecules cause the bacteria to aggregate by forming bonds through azobenzene, allowing fast diffusion of quorum sensing molecules and the rest of the bacteria turn green as well
Azobenzene is an organic molecule which responds to light and selectively photoswitches from an extended Trans conformation to a more compact Cis conformation.
Background
In the dark, at equilibrium, azobenzene is >99% trans. Irradiation at specific short wavelength causes ~90% to switch to the cis isomer. Irradiation at longer wavelengths and/or thermal relaxation causes the molecule to return to the trans isomer.
Figure1: Photochromism of azobenzene derivatives and energetic profile for the switching process.
iGEM new azobenzene synthesis breakthrough
Most azobenzene-based photoswitches use UV light for photoisomerization. This limits their application in biological systems, where UV light be harmful to the cell. Substitution of all four ortho positions with methoxy groups in an amidoazobenzene derivative leads to a substantial (~35 nm) red shift. This red shift makes trans-to-cis photoswitching possible using green light (530-560 nm). The cis state is thermally stable with a half-life of ~2.4 days in the dark in aqueous solution. Reverse (cis-to-trans) photoswitching can be accomplished with blue light (460 nm), so bidirectional photoswitching between thermally stable isomers is possible without using UV light at all. Our team synthesized this azobenzene molecule by ourselves.
Bacteria-azobenzene connection
We attached the azobenzene molecules to the bacteria's modified lipopolysaccharide (LPS) on the outer-membrane. Azobenzene reacts with the Kdo sugar of the LPS giving stable amide bonds of azo-bacteria connections. Amide bonds cannot form at room temperature. To overcome this we used an EDC catalyst. EDC is used to form amide bonds between peptides. We could not find any information about how much EDC molecules are safe for E.coli so we performed a series of experiments to check the concentration range of EDC that is safe for a bacterial culture and used it to attach the azobenzene to the E.coli's LPS.
Diagram A: azobenzene attaches to KDO sugers
We run a concentration gradient of EDC along with Top10 bacteria, 1 is the highest concentration of EDC, 2 is the lowest concentration of EDC. As you can see in the tubes 1 and 2 we did not get a bacterial growth, but in 3-10 tubes we did. Therefore, we chose to work with the concentration of tube 4 for the rest of the experiment where we use EDC with azobenzene and bacteria.
Synthetic biofilm formation
After mixing azobenzene with bacteria, we exposed them to 530-560 nm wavelength and we saw the “sticky” bacteria aggregate to form a biofilm. The quorum sensing molecules, AHL can diffuse faster between them leading to a faster information exchange.
left: E.coli sample with azobenzene, right: E.coli sample with-out azobenzene
The dipole force between azobenzene rings are more powerful than the natural negative repulsion of bacteria.
We are the first people ever to demonstrate this technique. Our vision can help many researchers study the kinetics of biofilm formation in real time.
Signal focusing by azobenzene
Quorum sensing bacteria release chemical signal molecules called autoinducers (AHL molecules) that increase in concentration as a function of cell density.
By making the bacteria aggregate, they exchange AHL molecules faster, which speeds up the detection process.
We engineered E. coli bacteria to emit light at a wavelength range of 530-560nm (green) when they detect the desired substance. The green light is absorbed by azobenzene, photo-switches to its “sticky” form causing the bacteria clump together forming a reversible synthetic biofilm. The AHL molecules diffuse quickly to all the other bacteria and they all produce green light, which can be seen by the naked eye.
Azobenzene aggregate Nano-Particles (NPs)
We established our iGEM azobenzene biological conceptions based on the Nano-word. We collaborated with Weizmann institute to test azobenzene molecules. We have seen that azobenzene can aggregate various NPs like iron oxide, gold and big particles like silica (see reference and TEM figures), and based on this behaviors we established our vision to use azobenzene as a photo-induced molecule to aggregate bacteria forming a synthetic biofilm.
