Team:KAIT Japan/Results

Results


[Expression of Dronpa 145K and Dronpa 145N]
We inserted our parts，Dronpa 145N(BBa_K170000) and Dronpa 145K(BBa_K170001) into pcold vector. And we expressed Dronpa 145N and Dronpa 145K in Ecoli(DH5α).



We irradiated the Ecoli with 400nm light. We confirmed that Dronpa 145N and Dronpa 145K were bright green fluorescence under 400nm light.



[Purification of Dronpa 145N and 145K]
We purified Dronpa 145N and Dronpa 145K by His-tag purification.



We irradiated Dronpa 145N and Dronpa145K with 400nm light.Both of them were bright green fluorescence.



[SDS-PAGE]
After we purified Dronpa145N and Dronpa 145K by using His-tag purification,we ran these proteins on SDS-PAGE.

We analyzed the bands.

Figure 3 shoes the result of SDS-PAGE.The 1 lane was the molecular maker. 2 lane and 3 lane were respectively Dronpa 145K and 145N.
The molecure weight of Dronpa is about 28.8 kDa. We were able to see the bands that was estimated about We　concluded that we succeeded expressing and purifying Dronpa 145N and 145K.



[Fluorescence intensity]
After we irradiated Dronpa145N and 145K with 400 nm or 500 nm, we analyzed for fluorescence intensity of Dronpa 145N and Dronpa 145K. We irradiated each Dronpa with 500 nm using figure4 of device. This device can irradiate LED light at the 68mW. This device can choose wavelength by the combination of three colors of LED light.We showed the spectrum that we used to irradiate 500nm in figure5. However, this device didn’t use because it couldn’t choose 400 nm wavelength. So we irradiated 400 nm using light of the fluorescent lamp. The fluorescent lamp is at the 32W. Spectrum of fluorescent lamp shows it in figure6. (The fluorescent lamp which we usually use contains some wavelength. 400 nm of energy is stronger than 500 nm. So, Dronpa fluoresces by fluorescent lamp.)



We irradiated 400 nm (5 hours) and 500 nm (5 hours) collectively for Dronpa 145K and 145N.

Dronpa 145K was bigger change of fluorescence intensity than Dronpa 145N. We confirmed that Dronpa’s fluorescence intensity was becoming stronger when we irradiated Dronpa with 400 nm. And it was becoming weaker when we irradiated Dronpa with 500 nm. We proved by specific wavelength.



[Native- PAGE]
We irradiated Dronpa 145N with 400 nm (5 hours), 500 nm (5 hours) and 500 nm (5 hours) + 400 nm (10 hours). And we ran these samples on Native-PAGE.We obtained a large molecular weight band in lane Dronpa 145N (-,- ),Dronpa 145N(-,+),Dronpa145N(+,+). iit was estimated that protein formed tetramer from this result.We determined that Dronpa 145N formed teteramer, when it was exposed by 400nm light. We also determined Dronpa 145K did not form tetramer. We concluded that Dronpa 145N reversibly changed their olligomerization by light (400 nm(tetramer), 500 nm(monomer)).



The tetramer band irradiated with 500nm was paler than the tetramer band irradiated with 400nm. The tetramer band irradiated with 500nm+400nm is deeper than the tetramer band irradiated with 500nm. Therefore, Dronpa 145N changed structure by specific light.



[Confocal laser scanning microscope (CLSM)]
We observed E.coli which has Dronpa 145N or 145K using CLSM.



Ecoli that expressed Dronpa 145N and 145K were bright green fluorescence when we irradiate Ecoli with 400 nm. These fluorescence were extinguished when we irradiate each ecoi with 500 nm.We concluded Dronpa 145N and 145K worked in our Ecoli.


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We confirmed our parts sequence(BBa_K170000(coding Dronpa 145N),BBa_K170003(coding Dronpa 145K)). See our result below


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"BBa_170000(Dronpa 145N) sequence confirmed Fw"
"BBa_170000(Dronpa 145N) sequence confirmed Rv"
"BBa_170003 (Dronpa 145K)sequence confirmed Fw"
"BBa_170003 (Dronpa 145K) sequence confirmed Rv"