Team:ETHZ Basel/Biology
From 2010.igem.org
Biology & Wet Laboratory Overview
The core idea of E. lemming is based on the spatial localization of one of the species of the chemotactic network: one of the Che proteins. Thus, 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. 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.
Inside the cell, the chemotactic proteins CheA, CheY and CheZ tend to co-localize with methyl accepting chemotaxis proteins (MCPs) at the membrane. Whereas CheA and CheZ almost exclusivly localize at the MCPs, CheY is also present in significant concentrations in the cytoplasm. Due to that we argue that the diffusion of a CheY-LSP-fusion protein to its anchor is less affected by the affinity to its natural interaction partner making its re-localization more straightforward [1].
For spatial localization of the Che-protein, three different anchor-proteins have been considered:
- The tetracyclin repressor tetR anchoring the Che-protein to the DNA by binding to its operator site tetO that has been inserted into a high copy plasmid [2].
- The ribosome binding domain of triggor factor TrigA anchoring the Che protein to the ribosome by binding to the large ribosomal subunit [3].
- The prokaryotic actin homologue MreB which assembles into helical filaments underneath the cytoplasmic membrane [4].