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

Line 123: Line 123:
 
<img src="https://static.igem.org/mediawiki/2015/3/37/Kekka1.PNG"><br>
 
<img src="https://static.igem.org/mediawiki/2015/3/37/Kekka1.PNG"><br>
 
<br>
 
<br>
−
E.coli fluorescence when we irradiated about 400 nm.<br>
+
Dronpa 145N in E.coli irradiated with 400nm light. We confirmed that there were bright green fluorescence. <br>
 
<br>
 
<br>
 
<br>
 
<br>
Line 132: Line 132:
 
<img src="https://static.igem.org/mediawiki/2015/3/3c/Kekka2.PNG"><br>
 
<img src="https://static.igem.org/mediawiki/2015/3/3c/Kekka2.PNG"><br>
 
<br>
 
<br>
−
We irradiated about 400 nm at purification of Dronpa 145N. It fluorescence green.<br>
+
Dronpa 145N irradiated with 400 nm. It was bright green fluorescence .<br>
 
<br>
 
<br>
 
<br>
 
<br>
 
<br>
 
<br>
 
<strong>[SDS-PAGE]</strong><br>
 
<strong>[SDS-PAGE]</strong><br>
−
We expressed Dronpa 145N, 145K and luciferase fusion protein at pcold vector in E.coli and obtained these proteins at His-tag purification. <br>
+
We expressed Dronpa 145N, 145K and Dronpa-Luciferase fusion protein at pcold vector in E.coli. After that, we obtained these proteins by using His-tag purification. <br>
 
<br>
 
<br>
 
<img src="https://static.igem.org/mediawiki/2015/3/3a/Kekka3.PNG"><br>
 
<img src="https://static.igem.org/mediawiki/2015/3/3a/Kekka3.PNG"><br>
Line 147: Line 147:
 
<br>
 
<br>
 
<strong>[Fluorescence intensity]</strong><br>
 
<strong>[Fluorescence intensity]</strong><br>
−
We analyzed for fluorescence intensity of Dronpa 145N and Dronpa 145K when we irradiated 400 nm or 500 nm.<br>
+
After we irradiated each Dronpa with 400 nm or 500 nm, we analyzed for fluorescence intensity of Dronpa 145N and Dronpa 145K.
−
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.<br>
+
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 wavelengths. Spectrum of this device shows it 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 is including some wavelength. However, 400 nm of energy was stronger than 500 nm of energy. So, Dronpa fluorescent by fluorescent lamp.)<br>
+
(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.)
 +
<br>
 
<br>
 
<br>
 
<img src="https://static.igem.org/mediawiki/2015/a/af/%E3%82%AD%E3%83%A3%E3%83%97kekka4.PNG"><br>
 
<img src="https://static.igem.org/mediawiki/2015/a/af/%E3%82%AD%E3%83%A3%E3%83%97kekka4.PNG"><br>
Line 155: Line 156:
 
We irradiated 400 nm (5 hours) and 500 nm (5 hours) collectively for Dronpa 145K and 145N.<br>
 
We irradiated 400 nm (5 hours) and 500 nm (5 hours) collectively for Dronpa 145K and 145N.<br>
 
<img src="https://static.igem.org/mediawiki/2015/7/72/Kekka5.PNG"><br>
 
<img src="https://static.igem.org/mediawiki/2015/7/72/Kekka5.PNG"><br>
−
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.<br>
+
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.<br>
 
<br>
 
<br>
 
<br>
 
<br>
 
<br>
 
<br>
 
<strong>[Native- PAGE]</strong><br>
 
<strong>[Native- PAGE]</strong><br>
−
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.<br>
+
We irradiated Dronpa 145N with 400 nm (5 hours), 500 nm (5 hours) and 500 nm (5 hours) + 400 nm (10 hours). Structure of Dronpa 145N changed by light wavelength (400 nm, 500 nm). This structure change is reversible reaction. We confirmed it. On the other hand, Dronpa 145K isn’t changed the structure.<br>
 
<br>
 
<br>
 
<img src="https://static.igem.org/mediawiki/2015/6/6b/Kekka6.PNG"><br>
 
<img src="https://static.igem.org/mediawiki/2015/6/6b/Kekka6.PNG"><br>
 
<br>
 
<br>
−
The 500 nm irradiated tetramer band was paler than 400 nm irradiated tetramer band. <br>
+
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.<br>
−
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.<br>
+
 
<br>
 
<br>
 
<br>
 
<br>
Line 174: Line 174:
 
<img src="https://static.igem.org/mediawiki/2015/e/e8/Kekka7.PNG"><br>
 
<img src="https://static.igem.org/mediawiki/2015/e/e8/Kekka7.PNG"><br>
 
<br>
 
<br>
−
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.<br>
+
Dronpa 145N and 145K were bright green fluorescence when we irradiate each Dronpa with 400 nm. Also, these fluorescence were extinguished when we irradiate each Dronpa with 500 nm. This change time was fast. We were able to control in vivo.<br>
 
<br>
 
<br>
 
<br>
 
<br>

Revision as of 14:34, 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.



Dronpa 145N in E.coli irradiated with 400nm light. We confirmed that there were bright green fluorescence.



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



Dronpa 145N irradiated with 400 nm. It was bright green fluorescence .



[SDS-PAGE]
We expressed Dronpa 145N, 145K and Dronpa-Luciferase fusion protein at pcold vector in E.coli. After that, we obtained these proteins by using 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]
After we irradiated each Dronpa 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 wavelengths. Spectrum of this device shows it 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). Structure of Dronpa 145N changed by light wavelength (400 nm, 500 nm). This structure change is reversible reaction. We confirmed it. On the other hand, Dronpa 145K isn’t changed the structure.



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.



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


-----------------------------------------------------------------------------------------------------------------

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


-----------------------------------------------------------------------------------------------------------------
"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"