Difference between revisions of "Part:BBa K2980014"

 
 
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<partinfo>BBa_K2980014 short</partinfo>
 
<partinfo>BBa_K2980014 short</partinfo>
  
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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.
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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.
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===Phase separation===
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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''.
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[[File:Screen Shot 2019-10-20 at 16.50.57.png|thumb|none|200px|Figure_1]]
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===Light stimulation===
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[[File:(SIMSUMO)3-SUMO3-GFP-cry2.png|200px|thumb|right|Figure_2A]]
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''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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<div><ul>
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<li style="display: inline-block;"> [[File:Screen Shot 2019-10-20 at 16.39.00.png|200px|thumb|left|Figure_2B]] </li>
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<li style="display: inline-block;"> [[File:Screen Shot 2019-10-20 at 16.38.52.png|200px|thumb|left|Figure_2C]] </li>
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</ul></div>
  
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===Usage and Biology===
 
  
 
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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