Difference between revisions of "Part:BBa J18932"

Revision as of 22:08, 16 October 2018

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 up to 50% of protein produced in E.coli will be truncated (R. Grünberg, unpublished)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 <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2319009>BBa_K2319009</a>

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.

       <img src="" width=100% style="border: 1px solid black;">
   <figcaption>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.</figcaption>

Purification using Ni-NTA with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2319009>BBa_K2319009</a>

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.

       <img src="" width=100% style="border: 1px solid black;">
   <figcaption>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).</figcaption>

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

       <img src="" width=100% style="border: 1px solid black;">

       Note: The kinks in the graph are an artifact of the normalization procedure to eliminate source fluoroscence.
       </figcaption>
   </figure>

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 crdue 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 intesity (rough esimation):

<figure style="width: 20%; float:right; max-height: 300px text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; padding: 0.5em;">

       <img src="" width=100% style="border: 1px solid black;">
       <figcaption>The scatter plot showing the replicates, average and standard deviation for mCherry with gel data.
       </figcaption>
   </figure>

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

From the calculations combining gel intensity and fluorescense:

<figure style="width: 20%; float:right; max-height: 300px text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; padding: 0.5em;">

       <img src="" width=100% style="border: 1px solid black;">
       <figcaption>The scatter plot showing the replicates, average and standard deviation for mCherry with Gel+Fluorescnse data.
       </figcaption>
   </figure>

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

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Sequence and Features