From 2010.igem.org

Biology & Wet Lab: Overview

In this section, we are giving an overview on the wet lab work.

Molecular Mechanism

Cloning Strategy

Implementation

Archeal Light Receptor

Human Practices and Safety

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 chemotactic 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 [1].

The fusion proteins were constructed according to the cloning strategy BBF RFC28 [2], a method for the combinatorial multi-part assembly based on the type II restriction enzmye AarI.

For the implementation of the system, the ideal conditions (strain, media, growth temperature, growth phase etc.) for the observation of chemotactic behaviour 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 [3].

Another possibility for the generation of our our E. lemming is the fusion of an archeal photoreceptor to a bacterial chemotactic transducer. Detailed information can be found here [4].

For sure, we thought about human practices and safety during our project [5].