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Revision as of 05:26, 3 October 2012
Protein interaction domain-peptide systems have been used by synthetic biologists to oligomerize or localize proteins. In particular, protein-protein interaction pairs have been applied to both scaffolding and signaling applications with impressive results. In such systems, orthogonality of protein pair interactions is desired to reduce cross-talk between system components and allow synthetic biologists to gain more precise control. However, the limited number and diversity of well-characterized, orthogonal protein interaction pairs restricts the complexity of systems that can be designed.
Current screening methods for protein-protein interactions (such as transcription-driven GFP expression in yeast 2-hybrid systems) do not have high enough throughput to analyze large library sizes of protein interaction pairs. However, with the utilization of MiCodes and microscopy’s ability to record spatial information, we were able to design an assay to directly observe and screen for orthogonal protein-protein interactions.
We adopted a bait-prey scheme in our experimental design. Two types of constructs were made using the golden gate cloning scheme detailed in our Construction page. The zipper parts were 3a parts, fluorescent proteins were 3b parts, and a peroxisome targeting tag-terminator sequence was a 4 part within our golden gate scheme. A diagram and description of these constructs is provided below:
Diagram of bait and prey leucine zipper constructs with fused fluorescent proteins and targeting tags.
"Prey": The prey construct has a leucine zipper fused to a monomeric red fluorescent protein (mKate)."Bait": The bait construct has a different leucine zipper fused to a photoactivatable green fluorescent protein (PAGFP) and a peroxisome targeting signal sequence. PAGFP is primarily used as a positive control to ensure the bait constructs are correctly targeted to the peroxisome.
These two constructs are combined into a plasmid and integrated into yeast.
Once expressed in the cytosol, the bait will be recruited to the peroxisome due to the PTS1 targeting sequence. If the binding interaction between the zipper pair is strong, then the prey zipper will be recruited to the peroxisome along with the bait. Effectively, in the event of a strong interaction, the yeast cell will have red fluorescent protein concentrated in the peroxisome when viewed under a microscope.
If the zipper pair has no binding interaction, then the prey zipper will remain in the cytosol. Effectively, in the event of weak to no interaction, the yeast cell will have diffuse red fluorescent protein in the cytosol when viewed under a microscope.
This assay was tested using zipper pairs of binding affinity characterized by yeast two hybrid systems courtesy of the Keating Lab at MIT (figure shown below - Thompson, et. al 2012). The following zipper pairs were used: 20+2 (strong), 20+6 (medium), 20+13 (weak).
Cell fluorescence as a measure of MAPK pathway modulation by SYNZIP parts.
Interaction pairs are
ordered, left to right, by the relative mean cell fluorescence induced.
The following microscope images were obtained using these protein interaction pairs in our leucine zipper assay:
Cloning for the Home Run 40 bait x 40 prey interaction library is detailed on our Construction page. This experiment is currently in progress and results are pending.
