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

 
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<strong>[Expression of Dronpa 145K and Dronpa 145N]</strong><br>
 
<strong>[Expression of Dronpa 145K and Dronpa 145N]</strong><br>
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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).<br>
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We inserted our parts，Dronpa 145N(BBa_K1740000) and Dronpa 145K(BBa_K1740001) into pcold vector. And we expressed Dronpa 145N and Dronpa 145K in Ecoli(DH5α).<br>
 
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<br>
 
<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>
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We irradiated the Ecoli with 400nm light. We confirmed that Dronpa 145N and Dronpa 145K were bright green fluorescence under 400nm light.  <br>
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Figure1 shows the Ecoli that expressed was bright with 400nm light. We confirmed that Dronpa 145N and Dronpa 145K were bright green fluorescence under 400nm light.  <br>
 
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<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>
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We irradiated Dronpa 145N and Dronpa145K with 400nm light.Both of them were bright green fluorescence.<br>
+
We irradiated Dronpa 145N and Dronpa145K with 400nm light.Both of them were bright green fluorescence.Figure2 show that Dronpa 145N was bright with 400nm light.<br>
 
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<br>
 
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<br>
 
<strong>[SDS-PAGE]</strong><br>
 
<strong>[SDS-PAGE]</strong><br>
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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>
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After we purified Dronpa145N and Dronpa 145K by using His-tag purification,we ran these proteins on SDS-PAGE.We analyzed the bands.<br>
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<br>
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<img src="https://static.igem.org/mediawiki/2015/6/6f/Saegusaaaaaaaaaaaaaaaa12.PNG"><br>
 
<img src="https://static.igem.org/mediawiki/2015/6/6f/Saegusaaaaaaaaaaaaaaaa12.PNG"><br>
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<br>Figure 3 shows the result of the SDS-PAGE.The 1 lane was the molecular maker. 2 lane and 3 lane were respectively Dronpa 145K and 145N.<br>
 +
The molecure weight of Dronpa is 28.8 kDa. We were able to see the bands that was estimated about 30.0kDa. 
 +
We　concluded that we succeeded expressing and purifying Dronpa 145N and 145K.<br>
 
<br>
 
<br>
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Dronpa is about 28.8 kDa. The 1 lane is the molecular maker. 2 lane and 3 lane are respectively Dronpa 145K and 145N.<br>
 
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We were obtained Dronpa 145N and 145K.<br>
 
 
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<br>
 
<br>
 
<br>
 +
<strong>[Fluorescence intensity]</strong><br>We irradiated 400 nm (5 hours) and 500 nm (5 hours) collectively for Dronpa 145K and 145N., we analyzed for fluorescence intensity of Dronpa 145N and Dronpa 145K. Figure4 shows the result. Initial state Dronpa 145N and 145K had middle fluorescence intensity, irradiated with 500nm light, the fluorescent intensity was decreasing and Dronpa 145N and 145K were not bright green fluorescence. After irradiated with 400nm light, the fluorescent intensity was increasing, Dronpa 145N and 145K were bright green fluorescence.
 +
 
<br>
 
<br>
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<strong>[Fluorescence intensity]</strong><br>
 
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After we irradiated each Dronpa with 400 nm or 500 nm, we analyzed for fluorescence intensity of Dronpa 145N and Dronpa 145K.
 
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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.
 
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(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.)
 
 
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<br><img src="https://static.igem.org/mediawiki/2015/9/92/444444444444444444444444444444444444444444444444.PNG"><br>
 
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<img src="https://static.igem.org/mediawiki/2015/8/8c/Saegusaaaaaaaa2.PNG"><br>
 
 
<br>
 
<br>
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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/2015/9/9a/Saegusaaaaaaaaaaaaaaaaaa3.PNG"><br>
+
Fluorescence intensity of Dronpa 145K was bigger than that of Dronpa 145N in each state. 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>
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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>
+
 
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<br>
 
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<br>
 
<br>
 
<strong>[Native- PAGE]</strong><br>
 
<strong>[Native- PAGE]</strong><br>
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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>
+
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.Figure8 shows the Native-PAGE. We obtained a large molecular weight band in lane Dronpa 145N (-,- ),Dronpa 145N(-,+),Dronpa145N(+,+). This result showed that Dronpa 145N formed tetramer by 400nm light. 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)). <br>
 
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<img src="https://static.igem.org/mediawiki/2015/7/7c/Saegusaaaaaaaaaaaaaaaaaaa4.PNG"><br>
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<img src="https://static.igem.org/mediawiki/2015/0/04/5555555555555555555555555555555555555.PNG"><br>
 
<br>
 
<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 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>
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<strong>[Confocal laser scanning microscope (CLSM)]</strong><br>
 
<strong>[Confocal laser scanning microscope (CLSM)]</strong><br>
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We observed E.coli which has Dronpa 145N or 145K using CLSM.<br>
+
We observed E.coli which had Dronpa 145N or 145K using CLSM.Figure9,and Figure10 show the result of CLSM.<br>
 
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<img src="https://static.igem.org/mediawiki/2015/4/43/Saegusaaaaaaaaaaaaaaaaaaa5.PNG"><br>
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<img src="https://static.igem.org/mediawiki/2015/d/dc/66666666666666666666666666666666666666666666.PNG"><br>
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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>
+
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. <br>
 
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<br><br>
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<br>We confirmed our parts sequence(BBa_K170000(coding Dronpa 145N),BBa_K170003(coding Dronpa 145K)). See our result below
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<br>We confirmed our parts sequence(BBa_K1740000(coding Dronpa 145N),BBa_K1740003(coding Dronpa 145K)). See our result below
 
<img src="https://static.igem.org/mediawiki/2015/e/e2/Mozidarake.PNG"><br>
 
<img src="https://static.igem.org/mediawiki/2015/e/e2/Mozidarake.PNG"><br>
 
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<a href="https://static.igem.org/mediawiki/2015/1/13/20150817_DronpaN_sequence_confirmed_Fw_%281%29.pdf">"BBa_170000(Dronpa 145N) sequence confirmed Fw"</a><br>
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<a href="https://static.igem.org/mediawiki/2015/1/13/20150817_DronpaN_sequence_confirmed_Fw_%281%29.pdf">"BBa_1740000(Dronpa 145N) sequence confirmed Fw"</a><br>
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<a href="https://static.igem.org/mediawiki/2015/0/02/20150817_DronpaN_sequence_confirmed_Rv.pdf">"BBa_170000(Dronpa 145N) sequence confirmed Rv"</a><br>
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<a href="https://static.igem.org/mediawiki/2015/0/02/20150817_DronpaN_sequence_confirmed_Rv.pdf">"BBa_1740000(Dronpa 145N) sequence confirmed Rv"</a><br>
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<a href="https://static.igem.org/mediawiki/2015/c/cc/20150907_DronpaK_sequence_confirmed_Fw.pdf">"BBa_170003 (Dronpa 145K)sequence confirmed Fw"</a><br>
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<a href="https://static.igem.org/mediawiki/2015/c/cc/20150907_DronpaK_sequence_confirmed_Fw.pdf">"BBa_1740003 (Dronpa 145K)sequence confirmed Fw"</a><br>
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<a href="https://static.igem.org/mediawiki/2015/d/dd/20150907_DronpaK_sequence_confirmed_Rv.pdf">"BBa_170003 (Dronpa 145K) sequence confirmed Rv"</a><br>
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<a href="https://static.igem.org/mediawiki/2015/d/dd/20150907_DronpaK_sequence_confirmed_Rv.pdf">"BBa_1740003 (Dronpa 145K) sequence confirmed Rv"</a><br>
 
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Latest revision as of 01:32, 19 September 2015

Results


[Expression of Dronpa 145K and Dronpa 145N]
We inserted our parts，Dronpa 145N(BBa_K1740000) and Dronpa 145K(BBa_K1740001) into pcold vector. And we expressed Dronpa 145N and Dronpa 145K in Ecoli(DH5α).



Figure1 shows the Ecoli that expressed was bright 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.Figure2 show that Dronpa 145N was bright with 400nm light.



[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 shows the result of the 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 28.8 kDa. We were able to see the bands that was estimated about 30.0kDa. We　concluded that we succeeded expressing and purifying Dronpa 145N and 145K.



[Fluorescence intensity]
We irradiated 400 nm (5 hours) and 500 nm (5 hours) collectively for Dronpa 145K and 145N., we analyzed for fluorescence intensity of Dronpa 145N and Dronpa 145K. Figure4 shows the result. Initial state Dronpa 145N and 145K had middle fluorescence intensity, irradiated with 500nm light, the fluorescent intensity was decreasing and Dronpa 145N and 145K were not bright green fluorescence. After irradiated with 400nm light, the fluorescent intensity was increasing, Dronpa 145N and 145K were bright green fluorescence.





Fluorescence intensity of Dronpa 145K was bigger than that of Dronpa 145N in each state. 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.Figure8 shows the Native-PAGE. We obtained a large molecular weight band in lane Dronpa 145N (-,- ),Dronpa 145N(-,+),Dronpa145N(+,+). This result showed that Dronpa 145N formed tetramer by 400nm light. 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 had Dronpa 145N or 145K using CLSM.Figure9,and Figure10 show the result of 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_K1740000(coding Dronpa 145N),BBa_K1740003(coding Dronpa 145K)). See our result below


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"BBa_1740000(Dronpa 145N) sequence confirmed Fw"
"BBa_1740000(Dronpa 145N) sequence confirmed Rv"
"BBa_1740003 (Dronpa 145K)sequence confirmed Fw"
"BBa_1740003 (Dronpa 145K) sequence confirmed Rv"