Peking 2018 joined the fifth Interlab measurement study. This year we helped to answer this question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
The introduction of this year Interlab: https://2018.igem.org/Measurement/InterLab
Plate Reader: Perkin Elemer EnSpire TM Multilabel Reader 2300
Flow Cytometry: BD LSRFortessa TM Cell Analyzer
96 - Well Pate: Corning Incorporated Costar®️ 3603
 |
 |
|---|---|
| (a) Particle Standard Curve - Linear | (b) Particle Standard Curve - Log Scale |
Figure. 3 The result of particle standard curve
| |
 |
 |
|---|---|
| (a) Fluorescence Standard Curve - Linear | (b) Fluorescence Standard Curve - Log Scale |
Figure. 5 The result of fluorescence standard curve
| |
Now, we have artificially designed phase separation in cells and synthesized membraneless organelles. But how can we fulfill intended functions with synthetic organelles? Here, we propose two ideas. We reserve two sites to implement functions, which means function modules, such as enzymes in metabolism, proteins in signaling pathway, transcription factors in transcription and so on, have two sites in our designs.
Just as we characterize synthetic organelles with fluorescent proteins, we can fuse function modules to the C-terminus of interaction modules and to the N-terminus of HOTags. Then, the function modules can be “kidnapped” into the synthetic organelles to fulfill intended functions.
We introduce a magic protein, anti-GFP nanobody, which is very small (only 13-kDa, 1.5nm 2.5nm) and high-affinity (0.59nM) camelid antibody to GFP[8]. So we can use its characteristic to improve our designs. We can fuse GFP to the C-terminus of interaction modules and to the N-terminus of HOTags, and fuse function modules to the C-terminus of anti-GFP nanobodies. Then, with the help of interaction between anti-GFP nanobodies and GFP, synthetic organelles will “welcome” function modules, expected functions can be realized. You may ask: How does anti-GFP nanobody improve the design? Firstly, it will not make the protein extremely large and will reduce the effect on the structure of function modules, which can ensure the quality of functions. Secondly, it can bring components not belonging to the original structure to synthetic organelles, which can enlarge the enrichment range of synthetic organelles. Thirdly, it is easy to regulate the expression of target proteins. So you can see, nanobodies may do better and give you a surprise!
We artificially designed phase separation in cells and synthesized membraneless organelles. And the main work to synthesize an organelle is to fulfill phase separation in a cell, so we stress the importance of interactions and multivalency. For these two aspects, we gave our ideas and the feasibility was analyzed. At last, we proposed two ideas to implement functions. We believe that in the near future, “millions of dollars” will no longer be a dream!
[1] Zhang, Q., Huang, H., Zhang, L., Wu, R., Chung, C. I., Zhang, S. Q., ... & Shu, X. (2018). Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter. Molecular cell, 69(2), 334-346.
[2] Woolfson, D. N., Bartlett, G. J., Burton, A. J., Heal, J. W., Niitsu, A., Thomson, A. R., & Wood, C. W. (2015). De novo protein design: how do we expand into the universe of possible protein structures?. Current opinion in structural biology, 33, 16-26.
[3] Banani, S. F., Rice, A. M., Peeples, W. B., Lin, Y., Jain, S., Parker, R., & Rosen, M. K. (2016). Compositional control of phase-separated cellular bodies. Cell, 166(3), 651-663.
[4] Husnjak, K., Keiten-Schmitz, J., & Müller, S. (2016). Identification and characterization of SUMO-SIM interactions. In SUMO (pp. 79-98). Humana Press, New York, NY.
[5] Banaszynski, L. A., Liu, C. W., & Wandless, T. J. (2005). Characterization of the FKBP Rapamycin FRB Ternary Complex. Journal of the American Chemical Society, 127(13), 4715-4721.
[6] Park, S. Y., Fung, P., Nishimura, N., Jensen, D. R., Fujii, H., Zhao, Y., ... & Alfred, S. E. (2009). Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. science, 324(5930), 1068-1071.
[7] Yin, P., Fan, H., Hao, Q., Yuan, X., Wu, D., Pang, Y., ... & Yan, N. (2009). Structural insights into the mechanism of abscisic acid signaling by PYL proteins. Nature structural & molecular biology, 16(12), 1230.
[8] Ries, J., Kaplan, C., Platonova, E., Eghlidi, H., & Ewers, H. (2012). A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nature methods, 9(6), 582.
.