Part:BBa_K2980009
CIB1-GCN(4)-mEGFP-FUSLCD
The fusion protein can induce consistant phase-separation in cell once well expressed. Under blue light stimulation, cry2-mcherry (Part:BBa K2980006) will be recruited into its phase.
Light stimulation
To achieve the response to light stimulation, light-sensitive proteins, CIB1(Part:BBa K2980002) and cry2(Part:BBa K2980000), are fused to our phase separation elements and application elements, respectively. When exposed to 488nm laser, CIB1 and cry2 will bind to each other. Since CIB1 is primarily amplified in the compartment formed by phase separation elements (BBa K2980009), cry2 would be recruited to phase, as the switch turns on. Therefore, the distribution of enzyme or other proteins fused to cry2 would be altered by light stimulation.
E. coli transformed with CIB1-GCN(4)-GFP-FUS(BBa K2980009) and cry2-mcherry(BBa K2980006) are placed under confocal microscope. Only mcherry channel and TD channel are shown in the video below (figure 2A), 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. (Figure 2B) Yet, after stimulation, it is recruited to the ends of the cell and form two sphere-like droplets. The screen shots below show the distribution of mcherry before and after 488 nm laser stimulation. (Figure 2C) In order to reflect the recruitment of cry2-mcherry into phase, we use the ratio of light intensity in phase to the rest of the cell as a standard. (Figure 2D) As presented in the plot, this ratio quickly increases after stimulation and can stay at a rather static level for a long time. (Figure 2E)
Usage and Biology
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 112
- 1000COMPATIBLE WITH RFC[1000]
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