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 were cloning Dronpa 145K and Dronpa 145N in E.coli. And we expressed these parts using pcold vector.<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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<img src="https://static.igem.org/mediawiki/2015/3/37/Kekka1.PNG"><br>
 
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E.coli fluorescence when we irradiated about 400 nm.<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>
 
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We irradiated about 400 nm at purification of Dronpa 145N. It fluorescence green.<br>
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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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<strong>[SDS-PAGE]</strong><br>
 
<strong>[SDS-PAGE]</strong><br>
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We expressed Dronpa 145N, 145K and luciferase fusion protein at pcold vector in E.coli and obtained these proteins at 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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<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>
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The molecure weight of Dronpa is 28.8 kDa. We were able to see the bands that was estimated about 30.0kDa. 
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We　concluded that we succeeded expressing and purifying Dronpa 145N and 145K.<br>
 
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<img src="https://static.igem.org/mediawiki/2015/3/3a/Kekka3.PNG"><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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<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.
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<strong>[Fluorescence intensity]</strong><br>
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<br><img src="https://static.igem.org/mediawiki/2015/9/92/444444444444444444444444444444444444444444444444.PNG"><br>
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We analyzed for fluorescence intensity of Dronpa 145N and Dronpa 145K when we irradiated 400 nm or 500 nm.<br>
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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>  
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(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>
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<img src="https://static.igem.org/mediawiki/2015/a/af/%E3%82%AD%E3%83%A3%E3%83%97kekka4.PNG"><br>
 
 
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We irradiated 400 nm (5 hours) and 500 nm (5 hours) collectively for Dronpa 145K and 145N.<br>
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<img src="https://static.igem.org/mediawiki/2015/7/72/Kekka5.PNG"><br>
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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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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>
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<strong>[Native- PAGE]</strong><br>
 
<strong>[Native- PAGE]</strong><br>
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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>
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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). 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/2015/6/6b/Kekka6.PNG"><br>
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<img src="https://static.igem.org/mediawiki/2015/0/04/5555555555555555555555555555555555555.PNG"><br>
 
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The 500 nm irradiated tetramer band was paler than 400 nm irradiated tetramer band. <br>
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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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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>
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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>
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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/e/e8/Kekka7.PNG"><br>
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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>
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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>We confirmed our parts sequence(BBa_K1740000(coding Dronpa 145N),BBa_K1740003(coding Dronpa 145K)). See our result below
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<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_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_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_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_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"