Team:ETHZ Basel/Biology/Implementation

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Biological Implementation

Experimental design

Considering the tremendously hight amount of 81 fusion proteins, we had to assign priorities to the different possibilities. This was made with the various models the dry-lab team implemented in order to help us prioritizing. We had to consider the following parameters. The detailed explanations for our choices you find by clicking on the question itself which is internally linked to the appropriate wiki page:

Experimental realization

Now that we got the input from the experimental design we had to realize or verify the stated parameters in our biological implementation.

Fusion proteins: First at all, accoring to the given input, we decided to assemble the following fusion proteins: CheY fused to Pif3, TetR fused to PhyB and Trigger factor fused to PhyB.


Ratio between anchor and binding partner: For the implementaion of E. lemming we need to express two fusion proteins simultaneously in one cell -

Plasmid copy number estimation.

Anchor to As the ratio between anchor protein and its binding partner has been proven to be essential, according to the experimental design evaluation, the plasmid copy number was determined by the normalization of cell number via optical density measurement followed by plasmid concentration measurements (using a commercial Miniprep kit).


From the modeling perspective, working vector 1 (BBR1 ori) should have a higher copy number than working vector 2 (RK2 ori). Our results showed that working vector 1 has a 1.1x higher frequency in the cell that working vector 2. This is acceptable, as the molecular models suggest an optimal ratio of 1.5x.

In view of the proportion of anchor to anchor binding protein, the aim of an intracellular tetO7 concentration of 50 µM can't be achieved, even not by ligation of the tet07 construct into a high copy number plasmid such as pUC19. The measured amount of 266 vectors per cell results in the exposure of approximately 5 µM of tetO binding sites. Therefore, we decided to integrate a second anchor binding protein into the plasmid, which is also fused to the light sensitive protein PhyB in one operon.

Functionality assays

The constructs are tested for the following properties:

  • Che protein fusion: Using the chemotaxis assay described by Mazumder et al. [2], the functionality of Che protein fusions can be tested.
  • Localizer fusion: The spatial localization of the anchor protein can be investigated by fusing it to a fluorescent protein (fluorescent GFP-tag). The anchor protein can fuse to the plasmid (tetR-tetO), the cell membrane (MreB) or the ribosome (TrigA).
  • PhyB-Pif3 system: Fusing a second fluorescent protein to Pif3 would enable us to visualize of the light-dimerization (photodimerization).

References

[1] [http://dspace.mit.edu/handle/1721.1/46721 BBF RFC 28: A method for combinatorial multi-part assembly based on the Type IIs restriction enzyme AarI. Peisajovich et al. (2009)]
[2] [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-3X3BN58-6&_user=10&_coverDate=09%2F30%2F1999&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1510762895&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=be555c903c4a328ea42a549fff7d9ac4&searchtype=a: Determining chemotactic responses by two subsurface microaerophiles using a simplified capillary assay method. Mazumder et al. (1999)]