Team:ETHZ Basel/Biology

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(Biology & Wet Laboratory: Overview)
 
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= Biology & Wet Laboratory Overview =
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= Biology & Wet Laboratory: Overview =
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<iframe title="YouTube video player" class="youtube-player" type="text/html" width="425" height="349" src="http://www.youtube.com/embed/yif8KekgVY8?hd=1" frameborder="0"></iframe>
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<iframe title="YouTube video player" class="youtube-player" type="text/html" width="400" height="325" src="http://www.youtube.com/embed/yQdX8o8i_uc?hd=1" frameborder="0"></iframe>
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<div class="thumbcaption"><div class="magnify"><a href="http://www.youtube.com/watch?v=yif8KekgVY8?hd=1" class="external" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Molecular mechanism of E. lemming.</b> 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.</div></div></div></div>  
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<div class="thumbcaption"><div class="magnify"><a href="http://www.youtube.com/watch?v=yQdX8o8i_uc?hd=1" class="external" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Molecular mechanism of E. lemming.</b> 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.</div></div></div>
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The core element of E. lemming is '''spatial localization''' of certain elements of the chemotactic network (Che proteins) and thus affecting the activity of their downstream partners. This anchoring is achieved with the help of '''light-sensitive proteins LSP's''' that dimerize upon a light signal. Dimerization results in spatial relocalization of the Che-protein and therefore altering the 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 chemotaxis network, so called '''Che proteins'''. Phosphorylated CheY (further referred to as CheYp) binds to the flagellar motor protein FliM, where it induces tumbling. Our research aimed at gaining control over this molecular switch and thus over the [https://2010.igem.org/Team:ETHZ_Basel/Modeling/Movement flagellar machine]. Through localizing (intracellular anchoring), the effective concentration of the free cytosolic CheY protein is decreased at its site of action, greatly affecting the activity on its downstream partners. Anchoring is achieved with the help of '''light-sensitive proteins (LSPs)''' that dimerize upon a light signal (photodimerization). The Che protein is fused to LSP1, while its binding partner LSP2 is itself fused to a so called '''anchor protein'''. Dimerization of the two LSPs into an LSP1/LSP2 complex, where LSP1 is still bound to CheY, results in spatial re-localization of the Che protein, which, as a final measurable output, induces a change in the ratio between tumbling and directed flagellar movement. The general idea is nicely represented by the video on the right side. Read more about the [[Team:ETHZ_Basel/Biology/Molecular_Mechanism|'''Molecular mechanism''']].
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Inside the cell, the chemotactic proteins CheA, CheY and CheZ tend to co-localize with methyl accepting chemotaxis protein MCPs at the membrane. But whereas CheA and CheZ nearly only localize at the MCPs, CheY is also present in significant concentrations in the cytoplasm making it's localization more straightforward [1].
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For spatial localization of the Che-protein complex, three different '''anchor-proteins''' will be utilized:
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<br> 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 plasmid [2].
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<br> 2. The '''triggor factor TrigA''' binding to the large ribosomal subunit [3].
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<br> 3. The '''prokaryotic actin homologue MreB''' which assembles into helical filaments underneath the cytoplasmic membrane [4].
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These anchors should enable to control the tumbling frequency by localizing a Che-protein and therefore interfering with its activity.
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Remove all the rest?
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== generation of BioBricks ==
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All utilized parts will be generated by PCR and subcloned into the storage vector pSEVA132 (Victor de Lorenzo's lab, KanR, BBR1 ori) allowing blue white screening. The working process for the generation of the subparts is as follows:
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<br>1. Ordering of primers (if template is available)
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<br>2. [[Team:ETHZ_Basel/Lab/protocols#PCR|PCR]]
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<br>3. [[Team:ETHZ_Basel/Lab/protocols#PCR_clean-up|clean-up of PCR product]]
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<br>4. Ligation into storage vector
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<br>5. Transformation
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<br>6. Blue-white screening
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<br>7. Sequencing
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Due to the presence of rare codons in the sequence of PhyB and Pif3, these two genes will be ordered from GenArt. However, as synthesizing takes several weeks, expression of the wild-type gene of these two proteins will be tested and if satisfying proceeded with these constructs.
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In this picture all the details (origin of DNA, length of gene, restriction sites, sources) about the BioBricks generated are displayed.
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[[Image:BioBricksI.png|200px|thumb|none|BioBricksI: light system, linkers]]
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[[Image:BioBricksII.png|200px|thumb|none|BioBricksII: Che proteins, localizer]]
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== generation of fusion proteins ==
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The image shows all the constructs we plan to clone.[[Image:Constructs.jpg|200px|thumb|none|fusion proteins]]
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Once the BioBricks in the storage vectors are available, the fusion proteins in the acceoptor vector will be generated according to the cloning strategy BBF RFC 28.
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== Functionality assays ==
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The constructs will have to be tested for the following properties:
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*Che protein fusion: Influencing of chemotactic network
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*Localizer fusion:  Spatial localization
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*PhyB-Pif system: Activation by light
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==== Chemotactic Functionality ====
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A second approach for the design of E. lemming is the usage of a photoreceptor connected to the bacterial chemotaxis system. Find out more about the [[Team:ETHZ_Basel/Biology/Archeal_Light_Receptor|'''Archeal Light Receptor''']] that enabled us to '''successfully''' implement the light-inducible synthetic network via the fusion of archeal and eubactarial parts.
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Is the Che fusion protein is still functional? <br> Possible assays to investigate the functionality:
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==== Localization ====
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The fusion proteins were constructed according to the [[Team:ETHZ_Basel/Biology/Cloning|'''Cloning Strategy BBF RFC28''']], a method for the combinatorial multi-part assembly based on the type II restriction enzmye AarI.
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Is the localizer spatially separating PIF3 or PhyB to a certain area within the cell? <br> Fluorescence microscopy  will be utilized to answer this question. Therefore, fusions of the anchor to fluorescent proteins (cyfp, gfp) have to be generated.
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==== PhyB-PIF-system ====
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In the section [[Team:ETHZ_Basel/Biology/Implementation|'''Implementation''']], you find details on the experimental design such as the ideal conditions for the observation of chemotaxis behavior (strain, media, growth temperature, growth phase etc.) and the functionality and expression level assays of the fusion proteins. We also provide you with some lab impressions.
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== Bringing ''E. lemming'' to life ==
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Of course, we also reflected a lot about [[Team:ETHZ_Basel/Biology/Safety|'''Human Practices and Safety''']] during our project, because knowledge also means responsibility. This section summarizes our findings on potential risks and safety issues and the measures we have taken in order to work as safely as possible.

Latest revision as of 16:39, 2 March 2011

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 chemotaxis network, so called Che proteins. Phosphorylated CheY (further referred to as CheYp) binds to the flagellar motor protein FliM, where it induces tumbling. Our research aimed at gaining control over this molecular switch and thus over the flagellar machine. Through localizing (intracellular anchoring), the effective concentration of the free cytosolic CheY protein is decreased at its site of action, greatly affecting the activity on its downstream partners. Anchoring is achieved with the help of light-sensitive proteins (LSPs) that dimerize upon a light signal (photodimerization). The Che protein is fused to LSP1, while its binding partner LSP2 is itself fused to a so called anchor protein. Dimerization of the two LSPs into an LSP1/LSP2 complex, where LSP1 is still bound to CheY, results in spatial re-localization of the Che protein, which, as a final measurable output, induces a change in the ratio between tumbling and directed flagellar movement. The general idea is nicely represented by the video on the right side. Read more about the Molecular mechanism.

A second approach for the design of E. lemming is the usage of a photoreceptor connected to the bacterial chemotaxis system. Find out more about the Archeal Light Receptor that enabled us to successfully implement the light-inducible synthetic network via the fusion of archeal and eubactarial parts.

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, you find details on the experimental design such as the ideal conditions for the observation of chemotaxis behavior (strain, media, growth temperature, growth phase etc.) and the functionality and expression level assays of the fusion proteins. We also provide you with some lab impressions.

Of course, we also reflected a lot about Human Practices and Safety during our project, because knowledge also means responsibility. This section summarizes our findings on potential risks and safety issues and the measures we have taken in order to work as safely as possible.