Team:ETHZ Basel/Biology

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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.
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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 [https://2010.igem.org/Team:ETHZ_Basel/Biology/Molecular_Mechanism].
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Inside the cell, the chemotaxis proteins ([[Team:ETHZ_Basel/Biology/Molecular_Mechanism|Che 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 [[Team:ETHZ_Basel/Biology#References|[1]]].
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The fusion proteins were constructed according to the '''cloning strategy BBF RFC28''' [https://2010.igem.org/Team:ETHZ_Basel/Biology/Cloning], a method for the combinatorial multi-part assembly based on the type II restriction enzmye AarI.
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For spatial localization of the Che-protein, three different '''anchor-proteins''' have been considered:
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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 [https://2010.igem.org/Team:ETHZ_Basel/Biology/Implementation].
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# The '''[https://2010.igem.org/Team:ETHZ_Basel/Biology/Molecular_Mechanism#Spatial_localization_in_a_bacterial_cell:_Anchor_proteins: 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 [[Team:ETHZ_Basel/Biology#References|[2]]].
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# The ''' [https://2010.igem.org/Team:ETHZ_Basel/Biology/Molecular_Mechanism#Spatial_localization_in_a_bacterial_cell:_Anchor_proteins: ribosome binding domain of trigger factor TrigA]''' anchoring the Che protein to the ribosome by binding to the large ribosomal subunit [[Team:ETHZ_Basel/Biology#References|[3]]].
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# The '''[https://2010.igem.org/Team:ETHZ_Basel/Biology/Molecular_Mechanism#Spatial_localization_in_a_bacterial_cell:_Anchor_proteins: prokaryotic actin homologue MreB]''' which assembles into helical filaments underneath the cytoplasmic membrane [[Team:ETHZ_Basel/Biology#References|[4]]].
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Another possibility for the generation of our our E. lemming is the fusion of an '''archean photoreceptor to a bacterial chemotactic transducer'''. Detailed information can be found here [https://2010.igem.org/Team:ETHZ_Basel/Biology/Archeal_Light_Receptor].
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== References ==
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For sure, we thought about human practices and safety during our project [https://2010.igem.org/Team:ETHZ_Basel/Biology/Safety].
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[1] [http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.2000.02044.x/pdf Sourjik and Berg: Localization of components of the chemotaxis machinery of Escheria coli using fluorescent protein fusions. Molecular Biology. 2000; 37:4.]
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[2] [http://onlinelibrary.wiley.com/doi/10.1111/j.1751-7915.2007.00001.x/pdf Bertram and Hillen: The application of Tet repressor in prokaryotic gene regulation and expression. Microbial Biotechnology. 2008; 1:1.]
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[3] [http://www.jbc.org/content/272/35/21865.full.pdf+html Hesterkamp, Deuerling and Bukau: The Amino-terminal 118 amino acids of Escherichia coli Trigger factor constitute a domain that is necessary and sufficient for binding to ribosomes. The Journal of Biologcial Chemistry. 1997; 272:35.]
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[4] [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2004.04367.x/pdf Kruse, Bork-Jensen and Gerdes: The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex. Molecular Microbiology. 2005;55:1.]
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Revision as of 17:15, 26 October 2010

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 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 archean 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].

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