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| - | {{ETHZ_Basel10}}
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| - | = Animations =
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| - | == Elsa ==
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| - | For the animations: would be nice to have titles with questions, that structure the videos (not yet added in my suggestions).
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| - | ANIMATION:
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| - | molecular mechanism
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| - | the synthetic molecular chemotaxis signal transduction pathway of the ETH E. lemming enables us to switch the flagellar rotary machine between its cw and ccw states, in response to an extracellular input-which is in our case red and far-red light. in wild-type bacteria, chemoattractants and chemorepellants are the stimuli-generating compounds which produce a chemotaxis response: consequently, the prokaryotes, moves away or towards the compound by altering its flagellar tumbling frequency via thenaturally occuring signaling cascade. the E. lemming is addidtionally equipped with a synthetic set of light-inducible proteins and anchor proteins, which add a novel tool to the chemotaxis machinery.
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| - | What are the key players in the synthetic cascade and how do they act?
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| - | The attractant or repellent compound binds to the methylaccepting proteins (MCP), embedded in the (inner) bacterial membrane. Through conformational change, this input signal is conveyed via an elaborate regulatory pathway, including CheW and CheA, to CheY, by tightly adjusting it's level of phosphorylation. CheY in its phoshorylated form can bind to the flagellar motor complex and induce a clockwise rotation of the apparatus, which results in tumbling.
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| - | unphosphorylated Chy, in contrast, canot bind to the motor complex. if chey is not phosphorylated, the motor remains rotating in its default ccw direction and the bacterium moves continuously forward. Although constant phases of ccw rotation are time-limited and not long-lasting, the phosphorylation state and frequency of chey has great influence on the tumbling frequency and therefore, chemotaxis as an entire system.
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| - | our research therefore aims at manipulating the concntration of free Cheyp available for via synthetic anchor proteins. as phosphorylated chey is then “anchored” to those localizers, the cheyp-concentration is decreased and and the flagellal switch can be inhibited. Via light-sensitive proteins (LSP's), cheyp is reversibly anchored to lsp1, itself bound to lsp2. the “kidnapping” of cheyp this has drastical consequences for its network of downstream partners-and on the tumbling requency of the motor.
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| - | When come our light-pulses into play? Well, it's right at this stage! the far-red light induces dissociation of the LSP proteins and Chey is “freed” and released to the cytosol, where it acts as a “tumbling agent” for the prokaryotic motor complex. this results in an increased tumbling frequency.
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| - | A pulse of red light induces the fusion of lsp1 and lsp2. again, cheyp is anchored to the lsp1/Lsp2-complex. as a consequence, the motor rotation remains in its default ccw direction and the bacterium moves forward. our research efforts focuse on resolving the questions related to the signaling protein chey
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| - | A pulse of red lights removes cheyp from the cytosol(by anchoring it to the lsp1/lsp2-complex) and the bacterium moves forward.
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| - | Far-red-light, kidnaps relaeses cheyp to the plasma, as the lsp1/lsp2-complex dissociates and the bacterium tumbles.
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| - | A challenging issue was how to automatize the process of assessing the movement of the E. lemming and applying corresponding light pulses. We build an information processing pipeline, consisting of different modules. The imaging module, the controller and the E. lemming itself. The imaging module (consisting of a microscope connected to a controller, detects movement. By comparing the input of the imaging module with the desired direction, the controller determines which light-pulse it has to send. This enables the “user” of the E. lemming to watch its movement on a screen and direct it at the same time via the joystick.
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| - | The fusion of all those components into exchangeable standard biobricks, the stimulating interworking between molecular biologists and computer scientists as well as the in vivo and in silico research on the E. lemming brought our bacterial robot alive!
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