Difference between revisions of "Team:KAIT Japan/Results"

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Revision as of 10:48, 18 September 2015

Results


[Expression of Dronpa 145K and Dronpa 145N]
We were cloning Dronpa 145K and Dronpa 145N in E.coli. And we expressed these parts using pcold vector.



E.coli fluorescence when we irradiated about 400 nm.



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



We irradiated about 400 nm at purification of Dronpa 145N. It fluorescence green.



[SDS-PAGE]
We expressed Dronpa 145N, 145K and luciferase fusion protein at pcold vector in E.coli and obtained these proteins at His-tag purification.



Dronpa is about 28.8 kDa. The 1 lane is the molecular maker. 2 lane and 3 lane are respectively Dronpa 145K and 145N.
We were obtained Dronpa 145N and 145K.



[Fluorescence intensity]
We analyzed for fluorescence intensity of Dronpa 145N and Dronpa 145K when we irradiated 400 nm or 500 nm.
We were irradiated 500 nm by Figure4 of machine. This is 68mW and it can irradiate LED light. This machine can choose wavelength by combining wavelength of 3 kinds. This machine spectrum is Figure5. However, we don’t have machine which isn't able to irradiate 400 nm. So we irradiated 400 nm using light of the fluorescent lamp. This is 32W. Spectrum of fluorescent lamp is Figure6.
(The fluorescent lamp is including some wavelength. However, 400 nm of energy was stronger than 500 nm of energy. So, Dronpa fluorescent by fluorescent lamp.)



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

The Dronpa 145K was bigger change of fluorescence intensity than Dronpa 145N. We confirmed becoming stronger when we irradiated 400 nm also, becoming weaker when we irradiated 500 nm. We proved by specific wavelength.



[Native- PAGE]
We irradiated 400 nm (5 hours), 500 nm (5 hours) and 500 nm (5 hours) + 400 nm (10 hours) at Dronpa 145N. This structure changed by light wavelength (400 nm, 500 nm). It is reversible reaction. We confirmed it. The 145K isn’t changed structure.



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



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



The Dronpa 145N and Dronpa 145K were fluorescence when we irradiate 400 nm. Also, these were fluorescence extinguished when we irradiate 500 nm. This change time was quickly. We were able to control in vivo.