Difference between revisions of "Part:BBa K1859020"

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K1859020で発現させたRFPは長鎖化されなかった
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<html>
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<p>
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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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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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<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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<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.