Part:BBa_K2601008

FKBP-yEGFP

Introduction

A widespread use of rapamycin, an anti-fungal antibiotic, has been developed to take advantage of the small molecule’s ability to heterodimerize proteins. Rapamycin binds to the FK506 binding protein (FKBP) as well as to a 100-amino acid domain of the mammalian target of rapamycin (mTOR), known as the FKBP-rapamycin binding domain (Frb). Proteins of interest can be expressed as fusions to FKBP or Frb, and then conditionally dimerized by adding rapamycin.

Figure 1. Structure of FKBP-Frb interaction induced by rapamycin.

Design

Some membrane-less organelles, such as stress granules and P bodies, have been discovered in recent years. Proteins condense into droplets and assemble these organelles through a process called phase separation. Physically, phase separation is the transformation of a one-phase thermodynamic system to a multi phase system, much like how oil and water demix from each other. According to thermodynamics, molecules will diffuse down the gradient of chemical potential instead of concentration. This is exactly why proteins will self organize into granules, diffusing from regions of low concentration to regions of high concentration. Here is an illustration of phase separation in cells.

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In order to rationally design a synthetic organelle based on protein phase separation, we needed a multivalent module and a protein-protein interaction module. The paired FKBP and Frb was one of the bioparts that we chose to introduce protein-protein interaction. FKBP and Frb could dimerize upon adding rapamycin. As for the multivalent module, we turned to de novo-designed homo-oligomeric short peptides. These short peptides are called HOTags (homo-oligomeric tags). HOTags contain approximately 30 amino acids. They have high stoichiometry, forming hexamer or tetrameric spontaneously. The hexameric HOTag3, together with the tetrameric HOTag6, could robustly drive protein phase separation upon protein interaction (achieved by FKBP-Frb module). To verify the feasibility of the system, we fused two fluorescence proteins with the two components of synthetic organelles. Thus, we could observe the self-organization of components and the formation of organelles under fluorescence microscope. We named our system SPOT(Synthetic Phase separation-based Organelle Platform) because it could form granules (fluorescent spots) in yeast. Here is a demonstration of our overall design.

Figure 2. The overall design of the synthetic organelle with florescence reporters. Based on the principle of multivalence and interaction, we fused FKBP-Frb module with homo-oligomeric tags (HOTags) to form synthetic organelle.

Properties

The interaction between FKBP and rapamycin has been well characterized (Kd ≈ 0.2 nM), and early experiments suggest that formation of a ternary complex including FRB is quite favorable (Kd ≈ 2.5 nM). FKBP and FRB do not interact in the absence of rapamycin.

Our results confirm that FKBP/Frb module fused with HOTags can drive phase separation in yeast.

Figure 3.

Figure 4.

We set up a thermodynamic model characterizing our system. Phase separation is easy to take place in the green area, where the system is unstable, while it can also happen in the blue area, where the system is metastable. The parameter chi represents the interaction energy. Whether chi one three is equal to chi two three decides whether the diagram is symmetric. This is a useful instruction for our experiments. To some extends it can save us from some unnecessary trials.

Figure 5. The strength of three different yeast promoters tested by flow cytometry.

‎ Figure 6. A screenshot taken from the GIFs. FKBP-HOTag3 and Frb-HOTag6 were under the control of promoters with different strength.

‎ Figure 7. The thermodynamic model of our system.

Here are the kinetic simulation results. We can see the larger chi is, the sooner the system relaxes into equilibrium state, which means the faster the phase separation appears.

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Figure 4.

Figure 4.

Sequence and Features

Assembly Compatibility:

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