From 2010.igem.org

Biology & Wet Lab: 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. Through localizing, the effective concentration of the Che protein is decreased at its site of action, affecting its activity on its downstream partners. Anchoring is achieved with the help of light-sensitive proteins (LSPs) that dimerize upon a light signal. The Che protein is fused to one of the LSPs, while the other LSP is fused to a so called anchor protein. Dimerization of the LSPs results in spatial re-localization of the Che protein, which, as a final measurable output, induces an altered ratio between tumbling and directed flagellar movement. Read more about the Molecular Mechanism.

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 we represent our reflections on the experimental design and how we intend to tackle the related challenges, such as the ideal conditions (strain, media, growth temperature, growth phase etc.) for the observation of chemotaxis behavior had to be investigated. Moreover, the fusion proteins have to be tested for functionality and expression levels. Read more about all the wet lab experiments here [1].

The Archeal Light Receptor is another approach to implement the light-inducible synthetic network via the fusion of archeal and eubactarial parts. You can find detailed information in this part.

For sure, we thought about Human Practices and Safety during our project [2]. This section summarizes our findings on potential risk and safety issues.