Difference between revisions of "Part:BBa K2609006"

Revision as of 17:50, 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

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.

Quantification of Truncation

The truncation of imCherry was determined by through two orthogonal methods:

• By analaysing the intensity of the truncated and non-truncated protein bands in the SDS PAGE.
• By combining the fluorescense and gel intensity data of the Ni-NTA purification products(supernatant after binding, wash and elution).This is done assuming that truncated and non-truncated protein has the same fluoresence. The calculation is as follows

  	% Intensity of bands		Fluorescence
  	Truncated	Non-Truncated	Truncated	Non-Truncated
  Supernatant	Ist	Isn	Fst	Fsn
  Wash	Iwt	Iwn	Fwt	Fwn
  Elution	Iet	Ien	Fet	Fen