Part:BBa_K1859020

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.