Difference between revisions of "Part:BBa K1859020"
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| + | <p><font size="4"> At result</font></p> | ||
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We inserted this generator into <i>E. coli</i> K-12 JM109.<br> | We inserted this generator into <i>E. coli</i> K-12 JM109.<br> | ||
We cultivated this bacteria and performed SDS-PAGE without boiling.<br> | We cultivated this bacteria and performed SDS-PAGE without boiling.<br> | ||
| − | Lane E shows this sample. | + | Lane E shows this sample.<br> |
</p> | </p> | ||
Revision as of 09:50, 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] .
At result
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.