Team:ETHZ Basel/Biology

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(Biology & Wet Laboratory Overview)
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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 -- 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.
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.
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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 it's re-localization more straightforward [1].
+
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 it's re-localization more straightforward [[Team:ETHZ_Basel/Biology#References|[1]]].
For spatial localization of the Che-protein, three different '''anchor-proteins''' have been considered:
For spatial localization of the Che-protein, three different '''anchor-proteins''' have been considered:
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# The '''tetracyclin repressor tetR''' anchoring the Che-protein to the DNA by binding to it's operator site tetO that has been inserted into a high copy plasmid [2].
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# The '''tetracyclin repressor tetR''' anchoring the Che-protein to the DNA by binding to it's operator site tetO that has been inserted into a high copy plasmid [[Team:ETHZ_Basel/Biology#References|[2]]].
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# The ''' ribosome binding domain of triggor factor TrigA''' anchoring the che protein to the ribosome by binding to the large ribosomal subunit[3].
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# The ''' ribosome binding domain of triggor 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 '''prokaryotic actin homologue MreB''' which assembles into helical filaments underneath the cytoplasmic membrane [4].
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# The '''prokaryotic actin homologue MreB''' which assembles into helical filaments underneath the cytoplasmic membrane [[Team:ETHZ_Basel/Biology#References|[4]]].
== References ==
== References ==
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[1] 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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[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] Bertram and Hillen: The application of Tet repressor in prokaryotic gene regulation and expression. Microbial Biotechnology. 2008; 1:1.
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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] 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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[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] 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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[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.]

Revision as of 23:52, 21 October 2010

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 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 it's re-localization more straightforward [1].

For spatial localization of the Che-protein, three different anchor-proteins have been considered:

  1. The tetracyclin repressor tetR anchoring the Che-protein to the DNA by binding to it's operator site tetO that has been inserted into a high copy plasmid [2].
  2. The ribosome binding domain of triggor factor TrigA anchoring the che protein to the ribosome by binding to the large ribosomal subunit[3].
  3. The prokaryotic actin homologue MreB which assembles into helical filaments underneath the cytoplasmic membrane [4].


References

[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.]

[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.]

[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.]

[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.]