Difference between revisions of "Part:BBa K1859020"
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| − | We designed this generator[ | + | We designed this generator[BBa_K1859020] to express long-chain protein with keeping functionality of the protein, giving the red fluorescence. |
</p> | </p> | ||
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| − | The generator | + | The generator is comprised of Circular parts combined the linker( |
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859007" > [BBa_K1859007] </a> | <a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859007" > [BBa_K1859007] </a> | ||
and | and | ||
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859011" > [BBa_K1859011] </a> | <a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859011" > [BBa_K1859011] </a> | ||
), His-RFP without stop codon | ), His-RFP without stop codon | ||
| − | <a href= "https://parts.igem.org/wiki/index.php?title=Part: | + | <a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1332002" >[BBa_K1332002] </a> |
, Lacl | , Lacl | ||
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_R0010" >[BBa_R0010] </a> | <a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_R0010" >[BBa_R0010] </a> | ||
, RBS | , RBS | ||
| − | <a href= "https://parts.igem.org/wiki/index.php?title=Part: | + | <a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034" >[BBa_B0034] </a> |
and DT | and DT | ||
| − | <a href= "https://parts.igem.org/wiki/index.php?title=Part: | + | <a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_B0015" >[BBa_B0015] </a> |
. | . | ||
</p> | </p> | ||
| + | <br> | ||
<p> | <p> | ||
| − | + | We inserted this generator into <i>E. coli</i> K-12 JM109.<br> | |
| + | We cultivated this bacteria and performed SDS-PAGE without boiling.<br> | ||
| + | Lane E shows this sample.<br> | ||
| + | </p> | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2015/0/04/Gifu-sds-before.jpeg" width="" height="240" vspace="10" hspace="50" align=""></img> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/a/ab/Gifu-sds-dye.jpg" width="" height="240" vspace="10" hspace="50" align=""></img> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/6/60/Gifu-sds-page-combine.png" width="" height="240" vspace="10" hspace="50" align=""></img> | ||
| + | <br>left: SDS-PAGE before dye / center: SDS-PAGE after dye / right: overlay | ||
| + | <br><br> | ||
| + | |||
| + | <p> | ||
| + | There was no polymer RFP at the top of these picture. We found that this generator doesn’t make long-chain protein. | ||
</p> | </p> | ||
<p> | <p> | ||
Linker sequence which we introduced changed structure of ribozyme related to splicing and lost their ability in function is considered as a cause. | Linker sequence which we introduced changed structure of ribozyme related to splicing and lost their ability in function is considered as a cause. | ||
</p> | </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.