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<p><u>From gel intesity (rough esimation)</u>:</p>
 
<p><u>From gel intesity (rough esimation)</u>:</p>
  
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         <img src="https://static.igem.org/mediawiki/parts/1/11/T--IISc-Bangalore--imcherry_trun_gel.png" width=100% style="border: 1px solid black;">
 
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         <figcaption>The scatter plot showing the replicates, average and standard deviation for mCherry with gel data.
 
         <figcaption>The scatter plot showing the replicates, average and standard deviation for mCherry with gel data.
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<p style="clear:right;"><u>From the calculations combining gel intensity and fluorescense</u>:</p>
 
<p style="clear:right;"><u>From the calculations combining gel intensity and fluorescense</u>:</p>
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         <figcaption>The scatter plot showing the replicates, average and standard deviation for mCherry with Gel+Fluorescnse data.
 
         <figcaption>The scatter plot showing the replicates, average and standard deviation for mCherry with Gel+Fluorescnse data.

Revision as of 22:23, 16 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 idea of imroving the part.

Characterization

Expression with BBa_K2609016

The protein was expressed under T7 promoter in E.coli BL21(DE3) with 6x-His tag at the N-terminal. The culture was induced at 37°C for three hours with a final IPTG concentration of 500μM. The cells were then lysed to obtain 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 its truncated counterpart.

SDS PAGE with the cell lysate. The top band is the non-truncated protein and the the bottom band is the truncated protein.

Purification using Ni-NTA with BBa_K2609016

The cell lysate thus obtained was purified using Ni-NTA beads which only bind to proteins with a 6x-His tag, which is absent in the truncated protein. Ideally, the supernatant after binding should have the truncated protein and the elution after purification should have the non-truncated protein. This however is not true because the binding of 6xHis to Ni-NTA is not perfect.

SDS PAGE of fractions from Ni-NTA purification. The top band is the non-truncated protein and the bottom band is the protein truncated at the internal start codon (see arrowheads).

Fluoroscence

Excitation Spectrum

The excitation 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

Tht excitation and emission spectra of imCherry after normalizing it with WT BL21 (DE3) lysate.
Note: The kinks in the graph are an artifact of the normalization procedure to eliminate source fluoroscence.
mCherry and imCherry colonies on media lacking IPTG. imCherry seems to have a higher basal fluorescence in the absence of an inducer. This might be due to a increase in over translation rate from the proper initiation site after the removal of the internal RBS.

Quantification of Truncation

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

  • By analysing the intensity of the truncated and non-truncated protein bands after SDS PAGE.
  • By combining the fluorescence 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 fluorescence. The fluorescence of each of the above samples was divided into fluorescence due to truncated and non-truncated protein based on their corresponding 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

From gel intesity (rough esimation):

% of protein (imCherry)
Truncated Non-Truncated
Replicate 1 19.9 80.1
Replicate 2 34.2 65.8
Replicate 3 24 76
Replicate 4 21.7 78.3
Replicate 5 20 80
Average 23.96 76.04
Std.dev 5.33 5.33
The scatter plot showing the replicates, average and standard deviation for mCherry with gel data.




From this, mcherry is estimated to have a truncation of 39.73 % +/- 7.95 %

From the calculations combining gel intensity and fluorescense:

% of protein (imCherry)
Truncated Non-Truncated
Replicate 1 16.35 83.65
Replicate 2 22.82 77.18
Replicate 3 20.53 79.47
Replicate 4 21.81 78.19
Replicate 5 20.74 79.26
Average 20.45 79.55
Std.dev 2.21 2.21
The scatter plot showing the replicates, average and standard deviation for mCherry with Gel+Fluorescnse data.




From this, mcherry is estimated to have a truncation of 38.82 % +/- 2.52 %

For more information see Team:IISc-Bangalore/Improve
Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]