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Revision as of 04:11, 24 October 2010

Implementation

Generation of fusion proteins

Schematic overview of the planned constructs. In the planning phase, 81 constructs were considered.

The picture graphically represents all the fusion proteins we have in our wetlab production pipeline. The BioBricks for all those constructs were generated either via PCR or synthesized from GeneArt. For sequencing, the bricks were then ligated into the storage vector. Cutting and pasting of constructs into working vectors should be feasible, applying the cloning strategy BBF RFC28 [1].

Experimental Design

Considering the tremendously hight amount of 81 fusion proteins, we had to assign priorities to the different genes. This was made with the various models the dry-lab team implemented in order to help us prioritizing.

• Chemotaxis protein: We chose CheY as the first target.
• Anchor: TetR was the first choice due to its wide application in synthetic biology and extensive characterization.
• Pif3 linked to Che-Protein: Because PhyB has been reportet to have a sequestering tendency in plants, we chose not to link it to the Che protein.
• Ratio anchor to binding partner: The simulations favored a ratio of 50 µM anchor to 40 µM of anchor binding partner.

Plasmid copy number

Plasmid copy number estimation.

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 tetO7 = 50 µM in one cell can't even be achieved by ligation into a high copy number plasmid such as pUC19. The measured amount of 266 vectors in one cell gives approximately 5 µM of tetO binding sites. Therefore, the decision was made to integrate a second anchor binding protein 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)]