Difference between revisions of "Part:BBa K1859020"
 
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Histidine tag and RFP linker [GGSGGS*2] semi-permanent generator
 
Histidine tag and RFP linker [GGSGGS*2] semi-permanent generator
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</p>
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<p>
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We designed this generator[BBa_K1859020] to express long-chain protein with keeping functionality of the protein, giving the red fluorescence.
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</p>
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<p>
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In the study of iGEM Gifu 2014, we made long-chain protein from Circular mRNA, but it lost functionality.
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It is thought that folding of the protein failed for a cause．
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</p>
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<p>
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Therefore we designed some linkers to fix the folding of the protein.
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Then, we combined circular parts, the 3'side of the intron
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1332005" > [BBa_K1332005] </a>
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and the 5'side of the intron
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1332003" > [BBa_K1332003] </a>
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, with those linkers and made them parts.
  
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このgenerator[K1859020]はタンパク質の機能性を保ったまま長鎖タンパク質を発現できるように設計した。
 
  
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In the study of iGEM Gifu 2014, we made long-chain protein from Circular mRNA, but it lost functionality. It is thought that folding of the protein failed for a cause． Therefore we designed some linkers to fix the folding of the protein.
 
  
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その後、それらのリンカーとサーキュラーパーツ,the 3'side of the intron[BBa_    ] and the 5'side of the intron[BBa_] 　　　,を結合させ、パーツ化した。
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The generator is comprised of Circular parts combined the linker(
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859007" > [BBa_K1859007] </a>
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and  
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859011" > [BBa_K1859011] </a>
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), His-RFP without stop codon
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1332002" >[BBa_K1332002] </a>
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, Lacl
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_R0010" >[BBa_R0010] </a>
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, RBS
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034" >[BBa_B0034] </a>
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and DT
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_B0015" >[BBa_B0015] </a>
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.
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</p>
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<br>
  
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The generator consists of リンカーを結合させたサーキュラーパーツ([BBa_]と[BBa_])、His-RFP without stop codon[BBa_K1332002]、Lacl[]、RBS[]、DT[].
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<p>
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We inserted this generator into <i>E. coli</i> K-12 JM109.<br>
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We cultivated this bacteria and performed SDS-PAGE without boiling.<br>
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Lane E shows this sample.<br>
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</p>
  
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<img src="https://static.igem.org/mediawiki/2015/0/04/Gifu-sds-before.jpeg" width="" height="240" vspace="10" hspace="50" align=""></img>
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<img src="https://static.igem.org/mediawiki/2015/a/ab/Gifu-sds-dye.jpg" width="" height="240" vspace="10" hspace="50" align=""></img>
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<img src="https://static.igem.org/mediawiki/2015/6/60/Gifu-sds-page-combine.png" width="" height="240" vspace="10" hspace="50" align=""></img>
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<br>left: SDS-PAGE before dye / center: SDS-PAGE after dye / right: overlay
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<br><br>
  
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このgeneratorを導入した大腸菌で発現させたRFPは長鎖化されない。
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<p>
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There was no polymer RFP at the top of these picture. We found that this generator doesn’t make long-chain protein.
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</p>
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<p>
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Linker sequence which we introduced changed structure of ribozyme related to splicing and lost their ability in function is considered as a cause.
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</p>

Latest revision as of 09:51, 22 September 2015

Histidine tag and RFP linker [GGSGGS*2] semi-permanent generator

We designed this generator[BBa_K1859020] to express long-chain protein with keeping functionality of the protein, giving the red fluorescence.

In the study of iGEM Gifu 2014, we made long-chain protein from Circular mRNA, but it lost functionality. It is thought that folding of the protein failed for a cause．

Therefore we designed some linkers to fix the folding of the protein. Then, we combined circular parts, the 3'side of the intron [BBa_K1332005] and the 5'side of the intron [BBa_K1332003] , with those linkers and made them parts. The generator is comprised of Circular parts combined the linker( [BBa_K1859007] and [BBa_K1859011] ), His-RFP without stop codon [BBa_K1332002] , Lacl [BBa_R0010] , RBS [BBa_B0034] and DT [BBa_B0015] .


We inserted this generator into E. coli K-12 JM109.
We cultivated this bacteria and performed SDS-PAGE without boiling.
Lane E shows this sample.


left: SDS-PAGE before dye / center: SDS-PAGE after dye / right: overlay

There was no polymer RFP at the top of these picture. We found that this generator doesn’t make long-chain protein.

Linker sequence which we introduced changed structure of ribozyme related to splicing and lost their ability in function is considered as a cause.