Difference between revisions of "Part:BBa K2609006"
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===Usage and Biology=== | ===Usage and Biology=== | ||
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Revision as of 14:58, 11 October 2018
imCherry (improved mCherry)
An improved alternative to mCherry (BBa_J18932) that reduces the truncation by 50% improving usage in fusion proteins.
Usage and Biology
Usage and Biology
Biology
imCherry is an improved version of the fluroscent protein mcherry (BBa_J18932. mcherry has truncation at its N-terminal due to a strong RBS sequence in front of the the ninth amino acid, Met. The new part, imCherry is made by modifying this RBS sequence such its translational efficiency is reduced, which reduces truncation. The new part is shown to have a reduction in truncation by about 50%.http://2018.igem.org/Team:IISc-Bangalore/Improve
Usage
The idea of Imcherry came into the picture when the 2018 IISc-Bangalore iGem team decided to check the efficieny of their N-terminal signal peptide PelB using mCherry. They found out that mcherry gets truncated by about 50%, which led to the improvement of the part.
Characterization
Expression with BBa_K2609016
The protein was expressed under T7 promoter in E.coliBL21(DE3) with 6x-Histag at the N-terminal. The culture was induced at 37°C for three hours with IPTG concentration of 500μM. The cell were then lysed to obatin the protein The size of the complete protein with 6x-Histag is about 26kDa. We observed two bands in the induced sample between
Purification using Ni-NTA with BBa_K2609016
The cell lysate obatined was purified using Ni-NTA beads which only binds to the proteins with 6x-Histag, which is absent in the truncated protein. So the supernatant after binding would have the truncated protein and the elution after the purification would have the non-truncated protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]