In our design, the interaction module is replaceable. We have used SUMO-SIM modules and FKBP-Frb modules to build spontaneous and induced synthetic organelles. To enrich our platform, more interaction modules are being considered, such as the GA-induced heterodimer system and ABA-induced heterodimer systems.
In addition to the use of small chemical molecules as SPOT inducer, light-induced interaction may be another promising strategy with several advantages. Firstly, it is very fast for the light-induced module to response and dimerize. Secondly, light-induced dimerization is reversible, make it much more flexible than chemical-induced. Another advantage for light control is that it can achieve high spatial and temporal specificity. Last but not least, light induced system contains high orthogonality, which is very important in human-design system.
In the near future, we can try phyB / PIF6 dimerization system. With red light to induce dimerization to test the feasibility of the light-induced organelle. (Figure.1A) With red light, dimerization happens, while with far-red light, the two components will disassociate. (Figure.1B) The feasibility, orthogonality and spatial and temporal specificity of the light-induced organelle may be a useful tool in synthetic biology
We have observed the formation process of synthetic organelles. However, in our previous work, the inducer rapamycin is difficult to be removed from the system, so we have not seen the disassociation process of organelles. We think that the formation and disassociation of synthetic organelles can be both controlled by using light-induced interaction module or chemical-induced module with weaker strength. We believe that our synthetic organelles can perform more functions with this ability.
Another assumption is that two orthogonal synthetic organelles can co-exist in cells. Two organelles can be induced and perform functions independently.
When we try to use synthetic organelles to accelerated reaction, we found the enzyme fused to the system cannot perform well as free enzyme. So it inspires us to develop a new method to load function modules to the whole systems, where the organelle acts as an organization hub.
In the design, the function modules will not be fused into the components, and be recruited into the organelles by protein-protein interactions. We have tested the feasibility of the design by using anti-GFP nanobody. Anti-GFP nanobody is a protein that can bind GFP. We can fuse the protein to be controlled with anti-GFP nanobody to aggregate it at the synthetic organelles.
We verified the function by fusing CFP with nanobody, and we found the co-localization of the blue and green fluorescence.
This system is modular and flexible. We can fuse almost any protein with nanobody and then it can aggregate in the synthetic organelles. What’s more, this strategy avoids fusing protein in the large system, which might result in the loss of functions because of structure change. These effects will be tested in the future, especially in the metabolism regulation protein.
This system also has the potential to aggregate the endogenous protein and even macromolecules by fusing the ligand of the substance with nanobody as medium.
Another assumption is that two orthogonal synthetic organelles can co-exist in cells. Two organelles can be induced and perform functions independently.
We have tested several functions of synthetic organelles platform. But more functions have not been tested owing to the time limit. Here we'll show some expectations of potential applications:
As we demonstrate before, SPOT can act as a sensor as they response to the environment rapidly and sensitively, so we wonder that they can be used to sense small molecules semi quantitatively in real-time scale in living cells. Our plan includes an NAD+ sensor in the future, which plays an important role in the study of cell's growth and metabolism. By using interaction modules that could be induced by NAD+, our synthetic organelles can work well.
SPOT also have the potential to detect the posttranslational modification of protein. There are some modification such as ubiquitination and SUMOylation. Measure the ubiquitination and SUMOylation of a protein can be a hard work including protein extract, western blot etc. Using the protein target to the substrate and ubiquitin as interaction modules, we might have the chance the catching the dynamic change of ubiquitination in the cell.
Now we can indicate that enzymatic reactions can happen normally in synthetic organelles. So what's the fittest situation of such a reaction hub? Beyond just accelerating the reaction rates, there are much more functions it can perform. By accelerating part of the reaction pathway, we can change the final product of the engineered cells. Besides, some intermediates in metabolism pathways are toxic to cells, which limits the application in engineered cells. If the enzymes of the reaction are recruited into the organelles, the toxicity problem may be solved just as what happens in lysosome.
Phase separation process shows sensitive dynamics so that we think our synthetic organelles can be introduced into artificial signal pathway as a signal amplifier.