Difference between revisions of "Part:BBa K2609016"
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<p style="clear:left; display: inline-block; margin-top: 150px;"><b> From this, mcherry is estimated to have a truncation of 39.73 % +/- 7.95 %</b></p>
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<p style="clear:left; display: inline-block; margin-top: 150px;"><b> From this, imcherry is estimated to have a truncation of 23.96 % +/- 5.33 %</b></p>
  
  
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<p style="clear:left; display: inline-block; margin-top: 150px; margin-bottom: 150px;"><b> From this, mcherry is estimated to have a truncation of 38.82 % +/- 2.52 %</b></p>
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<p style="clear:left; display: inline-block; margin-top: 150px; margin-bottom: 150px;"><b> From this, imcherry is estimated to have a truncation of 20.45 % +/- 2.21 %</b></p>
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<h4>Truncation reduction</h4>
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    <p> From the fine measurements combinig gel intensity data and fluorescense data, mCherry is observed to have truncation of 38.82 % +/- 2.52 % (From <a href=https://parts.igem.org/Part:BBa_K2319009>BBa_K2319009</a>). The improved version of mcherry, imCherry has truncation of 20.45 % +/- 2.21 %. Therefore, the percentage reduction of truncation in imCherry from mCherry is about 47% within the error limits. </p>
 
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<div style="display: block; width:35%;";>For more information see <a href="http://2018.igem.org/Team:IISc-Bangalore/Improve">Team:IISc-Bangalore/Improve</a></div>
 
<div style="display: block; width:35%;";>For more information see <a href="http://2018.igem.org/Team:IISc-Bangalore/Improve">Team:IISc-Bangalore/Improve</a></div>
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Revision as of 22:44, 16 October 2018


6xHis-imCherry under T7 expression system

This part generates imCherry (an improved version of BBa_J18932 with reduced truncation) when transformed into a T7 expression strain like Bl21 (DE3).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 768
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

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 terminal. This is caused by the presence of a strong RBS sequence upstream of the the ninth amino acid, a methionine encoded by a start codon that acts as a site of initiation. The new part, imCherry is made by modifying this internal RBS sequence such that its translational efficiency is reduced, which thereby reduces truncation. The new part is shown to have truncation reduced by about 50%.(http://2018.igem.org/Team:IISc-Bangalore/Improve)

Usage

The mCherry part,BBa_J18932, was to be used by the 2018 IISc-Bangalore iGEM team for characterization of a N-term signal peptide when they realised that an estimate obtained from truncation prone mCherry would not be accurate. imCherry was created to answer this problem. The sequence at the internal RBS was modified in-silico, and the truncation characterized experimentally using a combination of PAGE and Fluorescent quantification. (See http://2018.igem.org/Team:IISc-Bangalore/Improve).

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 imCherry with gel data. From the statistical significance analysis of the two means, we got a p-value of P=0.0062 with (**).




From this, imcherry is estimated to have a truncation of 23.96 % +/- 5.33 %

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 imCherry with gel data. From the statistical significance analysis of the two means, we got a p-value of P<0.0001 with (***).




From this, imcherry is estimated to have a truncation of 20.45 % +/- 2.21 %

Truncation reduction

From the fine measurements combinig gel intensity data and fluorescense data, mCherry is observed to have truncation of 38.82 % +/- 2.52 % (From BBa_K2319009). The improved version of mcherry, imCherry has truncation of 20.45 % +/- 2.21 %. Therefore, the percentage reduction of truncation in imCherry from mCherry is about 47% within the error limits.

For more information see Team:IISc-Bangalore/Improve