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 (PTS1). 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 to create unique strains. Once expressed in the cytosol, the bait zipper will be recruited to the peroxisome due to the PTS1 targeting sequence. A range of possible phenotypes can be observed:
- 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.
- If the zipper pair has no binding interaction, then the prey zipper will remain in the cytosol. Effectively, in the event of no interaction, the yeast cell will have diffuse red fluorescent protein in the cytosol.
- In the event of a medium level interaction between the bait and prey zippers, both red punctate in the peroxisomes and diffuse red fluorescent protein in the cytosol should be observed. The proportion of localized red (peroxisome to cytosol) is dependent on the strength of interaction.
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, found in 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:
As it is shown in the figures above, the yeast strain with the strong zipper interaction showed red punctate in the peroxisomes of the yeast cells. The medium interaction strain gave diffuse RFP in the cytosol along with some concentrated red in the peroxisomes. The weak interaction strain gave only diffuse RFP in the cytosol.
Our computational team conducted an analysis of the zipper data from the images shown above. Our metric for gauging zipper-interaction was amount of protein in peroxisome divided by amount of protein in cytosol. This ratio was calculated for each cell in each population using the cell segmentation software described in the automation section and an additional program that the computational team developed for identifying and analyzing peroxisomes. Based on this analysis the interaction strengths in the table below were found.
The MATLAB analysis allows us to assign quantitative values to the qualitative
interactions strengths we observe in the microscopy images. We found that the
MATLAB analysis agreed well with the "eye-ball" interaction test shown above.
In order to test the MiCode application for the leucine zipper assay we created two MiCodes- one for the known strong binding pair (20+2) and one for the know weak binding pair (20+13). MiCode 1 was programmed to have a green nucleus, blue plasma membrane and strong leucine zipper binding. Micode 2 was programmed to have a blue nucleus, green plasma membrane and weak leucine zipper binding. Below are the resulting images from this proof of concept demonstration. Please click on the images for full resolution.
In order to generate a rich library of zippers to screen for orthogonality, we sought a collaboration with the lab of Amy Keating at MIT. The Keating Lab has developed powerful software for predicting leucine zipper interaction pairs de novo (SYNZIPs). We hope that by combining our assay with these computational techniques, we can generate and characterize a large set of orthogonal binding partners that can be used by the broader synthetic biology community.
Based on computational predictions from the Keating Lab, we synthesized 20 pairs of zippers (40 total), which we named 1A, 1B, 2A, 2B, etc. Zipper 1A was predicted to specifically bind to zipper 1B and not bind itself or any other members of the library. The affinities of predicted binders were calculated to be 1-10nM, and that of all other interactions to be greater than 1µM.
We are working on testing all 40 zippers as both bait and prey to generate a 1600-member orthogonality matrix. We have synthesized the zippers, and are currently finishing the final cloning steps - detailed on our construction page.