Team:ETHZ Basel/Biology

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Revision as of 10:17, 13 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 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.

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]. For spatial localization of the Che-protein complex, three different anchor-proteins will be utilized:
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].
2. The triggor factor TrigA binding to the large ribosomal subunit [3].
3. The prokaryotic actin homologue MreB which assembles into helical filaments underneath the cytoplasmic membrane [4].

These anchors should enable to control the tumbling frequency by localizing a Che-protein and therefore interfering with its activity.

Remove all the rest?

generation of BioBricks

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:
1. Ordering of primers (if template is available)
2. PCR
3. clean-up of PCR product
4. Ligation into storage vector
5. Transformation
6. Blue-white screening
7. Sequencing

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.

In this picture all the details (origin of DNA, length of gene, restriction sites, sources) about the BioBricks generated are displayed.

BioBricksI: light system, linkers

BioBricksII: Che proteins, localizer

generation of fusion proteins

The image shows all the constructs we plan to clone.

fusion proteins

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.

Functionality assays

The constructs will have to be tested for the following properties:

Che protein fusion: Influencing of chemotactic network
Localizer fusion: Spatial localization
PhyB-Pif system: Activation by light

Chemotactic Functionality

Is the Che fusion protein is still functional?
Possible assays to investigate the functionality:

Localization

Is the localizer spatially separating PIF3 or PhyB to a certain area within the cell?
Fluorescence microscopy will be utilized to answer this question. Therefore, fusions of the anchor to fluorescent proteins (cyfp, gfp) have to be generated.

PhyB-PIF-system

Bringing E. lemming to life

Retrieved from "http://2010.igem.org/Team:ETHZ_Basel/Biology"

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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.
+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.
-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.
-<br>As modeling of the chemotactic network did not give a clear answer which Che protein should be attacked, several combinations will be investigated.
-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).
-The goal of the wet lab team is to implement this localization system into ''E. coli''.
-== Cloning Strategy ==
+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].
-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).
+For spatial localization of the Che-protein complex, three different '''anchor-proteins''' will be utilized:
-The advantage of this strategy is that we can clone up to 3 different inserts into one vector simultaneously in a 96 well format.
+<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].
+<br> 2. The '''triggor factor TrigA''' binding to the large ribosomal subunit [3].
+<br> 3. The '''prokaryotic actin homologue MreB''' which assembles into helical filaments underneath the cytoplasmic membrane [4].
+These anchors should enable to control the tumbling frequency by localizing a Che-protein and therefore interfering with its activity.
+Remove all the rest?
 == generation of BioBricks ==