Difference between revisions of "Part:BBa K2609006"

Revision as of 05:53, 13 October 2018

imCherry (improved mCherry)

Coding sequence of imCherry, an improved alternative to mCherry (<a href="https://parts.igem.org/Part:BBa_J18932">BBa_J18932</a>) that reduces the truncation at the N-term by around 50% thus improving its usage in fusion constructs.

Usage and Biology

Biology

imCherry is an improved version of the fluorescent protein mcherry (BBa_J18932) which is a widely used marker for protein studies. A fusion at the N-term of mCherry however is not a viable method for quantification because of the prominent truncation suffered by the protein near this termina. This is because of the presence of a strong RBS sequence upstream of the the ninth amino acid, Met, the sequence codon being a start codon. The new part, imCherry is made by modifying this RBS sequence such its translational efficiency is reduced, which thereby 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 idea of imroving the part.

Characterization

Expression with BBa_K2609016

The protein was expressed under T7 promoter in E.coli BL21(DE3) with 6x-Histag at the N-terminal. The culture was induced at 37°C for three hours with IPTG concentration of 500μM. The cells 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 25 kDa and 32 kDa. The heavier band is the non-truncated protein and the lighter one is the truncated protein.

Purification using Ni-NTA with BBa_K2609016

The cell lysate thus 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 purification would have the non-truncated protein.

Fluoroscence

Excitation Spectrum

The excitaion spectrum of the purified sample(elution) was obtained at a fixed emission wavelength of 610 nm. The excitation maxima was obtained at 576 nm.

Emission Spectrum

The emission spectrum of the purified sample(elution) was obtained at a fixed excitation wavelength of 587 nm. The emission maxima was obtained at 607 nm

Quantification of Truncation

The truncation of imCherry was determined by through two different 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 fluorescence of each of the above samples were divided into fluorescnce due to truncated and non-truncated protein based on their coressponding band intensities. The sum of fluorescence values of truncated and non-truncated protein were then used as a measure of their concentration to determine truncation.

Truncation Data