From 2010.igem.org

Biology & Wet Laboratory: Overview

Molecular mechanism of E. lemming. A light-sensitive dimerizing complex fused to proteins of the chemotaxis pathway at a spatially fixed location is induced by light pulses and therefore localization of the two molecules can be manipulated.

The core idea of E. lemming is based on the spatial localization of one of the species of the chemotaxis network, so called Che proteins. Phosphorylated CheY (further referred to as CheYp) binds to the flagellar motor protein FliM, where it induces tumbling. Our research aimed at gaining control over this molecular switch and thus over the flagellar machine. Through localizing (intracellular anchoring), the effective concentration of the free cytosolic CheY protein is decreased at its site of action, greatly affecting the activity on its downstream partners. Anchoring is achieved with the help of light-sensitive proteins (LSPs) that dimerize upon a light signal (photodimerization). The Che protein is fused to LSP1, while its binding partner LSP2 is itself fused to a so called anchor protein. Dimerization of the two LSPs into an LSP1/LSP2 complex, where LSP1 is still bound to CheY, results in spatial re-localization of the Che protein, which, as a final measurable output, induces a change in the ratio between tumbling and directed flagellar movement. The general idea is nicely represented by the video on the right side. Read more about the Molecular mechanism.

A second approach for the design of E. lemming is the usage of a photoreceptor connected to the bacterial chemotaxis system. Find out more about the Archeal Light Receptor that enabled us to successfully implement the light-inducible synthetic network via the fusion of archeal and eubactarial parts.

The fusion proteins were constructed according to the Cloning Strategy BBF RFC28, a method for the combinatorial multi-part assembly based on the type II restriction enzmye AarI.

In the section Implementation, you find details on the experimental design such as the ideal conditions for the observation of chemotaxis behavior (strain, media, growth temperature, growth phase etc.) and the functionality and expression level assays of the fusion proteins.

Of course, we also reflected a lot about Human Practices and Safety during our project, because knowledge also means responsibility. This section summarizes our findings on potential risks and safety issues and the measures we have taken in order to work as safely as possible..