Difference between revisions of "Part:BBa J18932"
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<h3>Improvement by IISc-Bangalore 2018</h3>
 
<h3>Improvement by IISc-Bangalore 2018</h3>
 
The sequence was modified to reduce the strength of the internal RBS and made into <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609006">BBa_K2609006</a>.  
 
The sequence was modified to reduce the strength of the internal RBS and made into <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609006">BBa_K2609006</a>.  
 +
 +
<h3>Improvement by IISc-Bangalore 2019</h3>
 +
The sequence was modified to reduce the truncation by replacement of the internal start codon with Leucine or Isoleucine and made into <a href="https://parts.igem.org/Part:BBa_K3165012">BBa_K3165012</a> and <a href="https://parts.igem.org/Part:BBa_K3165013">BBa_K3165013</a> respectively.
  
 
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Revision as of 18:40, 20 October 2019

mCherry RFP

Red fluorescent protein derived from DsRed.

Advantages:

  • fast folding and maturation
  • bright and photo-stable

Purity issues (update):

  • Ajo-Franklin...Silver (2007) report multiple bands for mCherry purifications in E. coli
  • Contains a hidden translation start site in the N-terminal -> up to 50% of protein produced in E.coli will be truncated (R. Grünberg, unpublished)
  • SDS treatment and boiling before PAGE hydrolyzes the chromophore at F//MYG splitting the protein in half (discussed in Gross et al. (2000) The structure of the chromophore within DsRed protein from coral.)
  • Best maturation for expression at 20 or 25 C (rather than 37)

Usage and Biology

Characterization

Expression with BBa_K2319009

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 for WT uninduced, WT induced, K2319009 uninduced, K2319009 induced. The top band is the non-truncated protein and the the bottom band is the truncated protein.

Purification using Ni-NTA with BBa_K2319009

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 567 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 605 nm

Tht excitation and emission spectra of mCherry after normalizing it with WT BL21 (DE3) lysate.
Note: The kinks in the graph are an artifact of the normalization procedure to eliminate source fluorescence.

Quantification of Truncation

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

  • By analysing the intensity of the truncated and non-truncated protein bands from the SDS PAGE of the crude lysate.(rough estimation)
  • By combining the fluorescence and gel intensity data of the Ni-NTA purification fractions (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 fractions 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 intensity (rough estimation):

% of protein
Truncated Non-Truncated
Replicate 1 30.25 69.75
Replicate 2 48.6 51.4
Replicate 3 35.8 64.2
Replicate 4 34.2 65.8
Replicate 5 49.8 50.2
Average 39.73 60.27
Std.dev 7.95 7.95
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 fluorescence:

% of protein
Truncated Non-Truncated
Replicate 1 34.66 65.34
Replicate 2 38.91 61.09
Replicate 3 42.53 57.47
Replicate 4 39.51 60.49
Replicate 5 38.47 61.53
Average 38.82 61.18
Std.dev 2.52 2.53
The scatter plot showing the replicates, average and standard deviation for mCherry with Gel+Fluorescense data.




From this, mcherry is estimated to have a truncation of 38.82 % ± 2.52 %

For more information see Team:IISc-Bangalore/Improve

Improvement by IISc-Bangalore 2018

The sequence was modified to reduce the strength of the internal RBS and made into BBa_K2609006.

Improvement by IISc-Bangalore 2019

The sequence was modified to reduce the truncation by replacement of the internal start codon with Leucine or Isoleucine and made into BBa_K3165012 and BBa_K3165013 respectively.

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]