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                                    <p>As mentioned above, interactions can be formed not only by inducers such as rapamycin and gibberellin, but also spontaneously, just as SUMO and SIM. So can we combine these two ways of interactions? To solve this problem, we did further studies about phytohormone and found ABA.
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Abscisic acid (ABA) is an important phytohormone that regulates plant stress responses. Proteins from the PYR-PYL-PCAR family were identified as ABA receptors<sup>[6]</sup>. Upon binding to ABA, a PYL protein associates with type 2C protein phosphatases (PP2Cs) such as ABI1 and ABI2, inhibiting their activity<sup>[7]</sup>. Previous structural and biochemical observations have provided insight into PYL-mediated ABA signaling and given rise to a working model. In the absence of ABA signaling, PP2Cs are fully active and PYLs exist as inactive homodimers in cells, unable to bind or inhibit PP2Cs, mainly due to the incompatible conformation of CL2loop<sup>[7]</sup>. In response to ABA binding, the CL2 loop undergoes a conformational rearrangement to close onto the ABA-bound pocket, then, the interaction between PYLs and PP2Cs can be formed.
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Here we chose PYL1 and ABI1 as a pair of interaction modules. Then, we assembled them on to yeast plasmid as the same as the construction of FKBP and FRB and transformed them into yeast. Based on the interaction of PYL1 and ABI1, we can get a wonderful scene: In the absence of ABA, the synthetic organelles composed only of PYL1 appear, because of the homodimers of PYL1. And after we add ABA into yeast, ABI1 can enter the organelles with the interaction of ABI1 and PYL1, and we can see red droplets colocalize with green droplets in cells through fluorescence microscope. In this way, new components can enter the original organelles and the time of occurrence can be regulated as it is inducer-mediated regulation. So it give our designs and functions more possibilities. 
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/3/35/T--Peking--project_design6.jpeg" width="700px" height="410 px" ></div>
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                                  Figure. 5 Interaction of PYL and PP2C<sup>[7]</sup>
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<div class="texttitle">Two function sites
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<div class="texttitle">Cell Measurement
  
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                                     <p>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.</p>
 
                                     <p>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.</p>
 
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Materials:
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Competent cells (Escherichia coli strain DH5α),
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LB (Luria Bertani) media,
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Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH),
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50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light) Incubator at 37°C,
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1.5 ml eppendorf tubes for sample storage,
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Ice bucket with ice,
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Micropipettes and tips,
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96 well plate, black with clear flat bottom preferred.
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/8a/T--Peking--interlabdevice.png" ></div>
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                                  Figure. 6 The required test devices.                             
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                                     <h3> Direct integration into the skeleton</h3>
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                                     <h3>Plate Reader</h3>
 
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Revision as of 19:22, 16 October 2018

Interlab

Introduction

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

 

Equipment Information

Plate Reader: Perkin Elemer EnSpire TM Multilabel Reader 2300

Flow Cytometry: BD LSRFortessa TM Cell Analyzer

96 - Well Pate: Corning Incorporated Costar®️ 3603

 

Results

 

1.

Calibration 1: OD600 Reference point - LUDOX

 

Figure. 1 The result of LUDOX calibration. The correction factor of our plate reader is 3.316


 

2.

Calibration 2: Particle Standard Curve – Microsphere

 

Figure. 2 The result of particle calibration.


(a) Particle Standard Curve - Linear (b) Particle Standard Curve - Log Scale
Figure. 3 The result of particle standard curve

 

3.

Calibration 3: Fluorescence standard curve – Fluorescein

 

Figure. 4 The result of fluorescein calibration.


(a) Fluorescence Standard Curve - Linear (b) Fluorescence Standard Curve - Log Scale
Figure. 5 The result of fluorescence standard curve

 

Cell Measurement

 

Materials:

Competent cells (Escherichia coli strain DH5α), LB (Luria Bertani) media, Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH), 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light) Incubator at 37°C, 1.5 ml eppendorf tubes for sample storage, Ice bucket with ice, Micropipettes and tips, 96 well plate, black with clear flat bottom preferred.

Figure. 6 The required test devices.


1.

Plate Reader

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.

Figure. 6 Integrate function modules to the skeleton
2.

Targeting GFP with nanobody

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!

Figure. 7 Interaction of anti-GFP nanobody and GFP
Conclusion

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!

References

[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.
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