Part:BBa_K2980014
(SIM-SUMO)3-SUMO3-mEGFP-Cry2
This part is the fusion of a photo-regulated protein Cry2, a green fluorescence protein mEGFP, and tandem repeats of SIM-SUMO as well as SIM. This part undergoes liquid-liquid phase separation in E. coli, forming green spherical liquid-like puncta. It can be used for light-induced recruitment of other clients into phase. The clients should be connected to the other photo-regulated CIB1 in another vector. Under 488nm laser stimulation, Cry2 and CIB1 will interact with each other, so that the clients will be recruited and enriched in the phase-separated droplets, and further regulation can be performed.
Phase separation
We first expressed the (SIM-SUMO)3-SUMO3-GFP-Cry2 in E. coli and observe it using confocal microscopy. Spherical green droplets indicating phase separation were observed (Figure 1) in E.coli.
Light stimulation
3-SUMO3-GFP-cry2.png)
E. coli transformed with (SIM-SUMO)3-SUMO3-GFP-Cry2 (Part:BBa K2980014) and CIB1-mCherry (Part:BBa_K2980015) are placed under confocal microscope. Since 488 nm laser, which is used to stimulate GFP, can also lead to bound of CIB1 and Cry2. At 0 second, Cry2-mCherry is almost smear in the cell. Yet, after stimulation, it is recruited to the spherical droplets formed by (SIM-SUMO)3-SUMO3-GFP-Cry2. The screen shots below show the distribution of mCherry before and after 488 nm laser stimulation as well as the location of GFP signal on the right. (Figure 2B, C)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 393
Illegal BglII site found at 852
Illegal BamHI site found at 1331 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 277
Illegal AgeI site found at 1006 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 629
Illegal BsaI.rc site found at 38
Illegal SapI.rc site found at 146
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