Team:ETHZ Basel/Biology

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== Overview ==
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= Biology & Wet Laboratory: Overview =
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The goal of the iGEM project 2010 of ETH Basel "E.lemming" is to control the tumbling frequency of ''E. coli''. This is achieved by spatially localizing certain elements of the chemotactic network (Che proteins) and thus affecting the activity of their downstream partners.
 
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Reversible localization is achieved by the light-inducible PhyB-PIF3 system detected in plants. PIF3 will be fused to a Che protein and PhyB to a localized anchor within the cell. Red light illumination converts PhyB Pr to Pfr facilitating PhyB-Pif3 interaction and therefore spatially separating the Che protein from its binding partner. Upon far red light stimulus, Pfr converts back to Pr resulting in the dissociation of PhyB-Pif3 and free diffusion of the Che protein.
 
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<br>As modeling of the chemotactic network did not give a clear answer which Che protein should be attacked, several combinations will be investigated.
 
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In view of the chemotactic proteins this includes CheY, CheB and CheR and concerning the localizer MreB (localization to the membrane), tetR (localization to the plasmid via tetO) and trigger factor (localization to the ribosome).
 
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The goal of the wet lab team is to implement this localization system into ''E. coli''.
 
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== Cloning Strategy ==
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<html>
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As we plan to generate several fusion proteins with different linkers, we decided to use the cloning strategy BBF RFC 28: A method for combinatorial multi-part assembly based on the Type IIs restriction enzyme AarI (http://dspace.mit.edu/handle/1721.1/46721).  
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<div class="thumb tright"><div class="thumbinner" style="width:402px;">
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The advantage of this strategy is that we can clone up to 3 different inserts into one vector simultaneously in a 96 well format.
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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=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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</html>
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== generation of BioBricks ==
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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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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.  
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We use the vector pSEVA 132 of Victor de Lorenzo's lab. It has a kanamycin resistance and a BBR1 origin.
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The working process for the generation of the storage vectors is as follows:
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<br>Ordering of primers (if template is available) -> [[Team:ETHZ_Basel/Lab/protocols#PCR|PCR]] -> [[Team:ETHZ_Basel/Lab/protocols#PCR_clean-up|clean-up of PCR product]] -> ligation into storage vector -> transformation of competent cells -> plating of cells -> selection of clones (blue-white-screening) -> sequencing
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<br>For proteins for which no template is available we let the let the genes synthesize directly.
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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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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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== generation of biobricks ==
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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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<br>Currently we are working on putting all the subparts into a storage vector. The image shows all the constructs we plan to clone.[[Image:Constructs.jpg|200px|thumb|none|fusion proteins]]
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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.
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Once the storage vectors are created the acceptor vectors can be generated according to the cloning strategy BBF RFC 28.
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== testing of biobricks ==
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We will have to test our constructs for the following properties:
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*Fusion of che-protein and PhyB or PIF3 for chemotactic functionality
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*Fusion of PIF3/PhyB and localizer for proper localization
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==== Chemotactic Functionality ====
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Is our fusion protein still able to maintain its function in the chemotactic pathway? To answer this question we plan to use a swarm test.
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==== Localization ====
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Is our localizer directing PIF3 or PhyB sufficiently to a certain area within the cell?
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We will use fluorescence microscopy to test for this. To do so we will need an additional fusion protein containing a fluorescent protein. We decided to use GFP and alternatively cyFP because they shouldn’t interfere with our coupling system of PhyB and PIF3 which uses red and far red light. If the fusion between the localizer the fluorescent protein is attracted to a specific site chances are high that a fusion of localizer and PIF3/PhyB also will.
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==== PhyB-PIF-system ====
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== Bringing ''E. lemming'' to life ==
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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.

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