Part:BBa_K2609016
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 768
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.
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
Note: The kinks in the graph are an artifact of the normalization procedure to eliminate source fluorescence.
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 of the crude lysate.
- 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 intensity (rough estimation):
| % 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 |
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 |
From this, imcherry is estimated to have a truncation of 20.45 % ± 2.21 %
Truncation reduction
From the fine measurements combining 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% (statistical significance analysis gave p<0.0001) within the error limits.
| None |