Difference between revisions of "Part:BBa K2980014"

Latest revision as of 11:07, 20 October 2019

(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.

Figure_1

Light stimulation

Figure_2A

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)

Figure_2B
Figure_2C

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 393
Illegal BglII site found at 852
Illegal BamHI site found at 1331
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 277
Illegal AgeI site found at 1006
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 629
Illegal BsaI.rc site found at 38
Illegal SapI.rc site found at 146

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2980014 short</partinfo>
-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.
+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 Cry2-(SIM-SUMO)3-SIM3-GFP in E. coli and observe it using confocal microscopy. Spherical green droplets indicating phase separation were observed ('''Figure 1''') in E.coli.
+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''.
-[[File:PhASE#2-sti-2-GFP_crop_RGB_EGFP.png|thumb|none|200px|Figure_1]]
+[[File:Screen Shot 2019-10-20 at 16.50.57.png|thumb|none|200px|Figure_1]]
 ===Light stimulation===
-[[File:Figure_1A.png|200px|thumb|right|Figure_2A]]
+[[File:(SIMSUMO)3-SUMO3-GFP-cry2.png|200px|thumb|right|Figure_2A]]
-E. coli transformed with (SIM-SUMO)3-SUMO3-GFP-Cry2(BBa K2980014) and CIB1-mcherry(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(BBa K2980014). 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''')
+''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''')
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