Team:IISc-Bangalore/Improve

Improve

Making a better mCherry

Introduction

mCherry is one of the most widely used fluoroscent reporters in the field of cell biology. This popularity stems from the comparatively bright emission on proper excitation and remarkably fast maturation times. Thus it is possible to record fluorescence outputs extremely quickly once the production process is triggered. It is also highly photo-stable and resistant to photo-bleaching under normal lab conditions and samples containing the protein can be stored relatively easily for long duration.

To characterize the extracellular transport signals that we used with mcp-1, we decided to use mCherry because of these merits. However, on further analysis, we realised that mCherry BioBrick (BBa_J18932) suffers from a fundamental flaw when it came to protein-protein fusion. The N-term of the coding sequence in BBa_J18932 has an internal start codon with a Shine-Dalgarno like sequence upstream of it. This start codon translates with a considerable efficiency leading to a truncation of around 50% (unpublished) of the protein produced from the transcript. The truncated protein is equally bright and similar in characteristics to the nontruncated mCherry. Any fusion at the N-term however, will lead to a production of mCherry with and without the tag in almost equal quantities. This is a major problem when this fusion protein is being used to report important parameters (like transport in our case). As such, we decided to use an in-silico strategy to improve the mCherry BioBrick for reduced truncation.

N-term truncation of mCherry CDS with a N-term tag

Modification

We used the Salis lab RBS strength calculator[1] to calculate the RBS translation efficiency from the internal start codon. We were able to decrease the predicted truncation by about 75% by making synonymous mutations right before the internal start codon.

Sequence modifications for improved mCherry

Parts

The following parts were used to validate the improvement experimentally. The assembly process can be found on the assembly page.

BBa_K2319009 - Generator for BBa_J18932
6xHis - mCherry under T7 expression system
BBa_K2609016 - Generator for BBa_K2609006
6xHis - imCherry under T7 expression system

The data presented here has also been added to the registry pages of the following parts: BBa_J18932, BBa_K2609006, BBa_K2319009 and BBa_K2609016

Validation Strategy

The first part of the validation procedure was to come up with a method to quantify the truncation produced by the internal start codon. For this purpose, we created a composite part containing a 6xHis tag fused to the N-terminal of mCherry coding sequence (BBa_K2319009). This was placed under a T7 promoter-terminator adapter (with HindIII/NheI sites for easy cloning). A similar part was designed with the imCherry coding sequence replacing mCherry (BBa_K2609016).

The 6x-Histag allows separation of the truncated protein from its non-truncated counterpart. The relative abundances of the two was first roughly estimated by measuring the SDS PAGE band intensity of the truncated and non-truncated protein bands from the crude lysate (The bands labelled truncated and non-truncated in the figure below). To make sure the numbers are correct we also used SDS-PAGE band intensities of each Ni-NTA purification fractions to divide the fluorescence for each of them into constituent truncated and non-truncated fractions. These fluorescence values were then summed across the flow-through, wash and elution fractions to find the exact ratio in the cell lysate.(As shown in the calculation in the figure below)

Schematic for quantifying mCherry/imCherry truncation

Gel images

SDS PAGE gel for mCherry with supernatant, wash, crude lysate and elution. The top band is the non-truncated protein and the bottom band is the truncated protein.
SDS PAGE gel for imCherry with crude lysate, supernatant, wash and elution. The top band is the non-truncated protein and the bottom band is the truncated protein.
SDS PAGE gel showing difference in truncation between mCherry and imCherry. The top band is the non-truncated protein and the bottom band is the truncated protein.

Spectral Characterization

The mCherry and imCherry generators were transformed into BL21 (DE3) cells and the 6x-Histagged protein was purified for spectral characterization.

Snaps of the plates: left mCherry and right imCherry

We noticed an increased expression of the imCherry part in BL21 (DE3) in the absence of IPTG compared to mCherry. This could be attributed to an increase in overall translation rates after removal of the internal RBS on top of the leaky T7 promoter. This difference in expression was NOT quantified in our experiments.

Excitation and Emission spectrum of mCherry

The emission spectrum was obtained at a fixed excitation wavelength of 587nm and excitation spectrum was obtained at a fixed wavelength of 610nm.

Excitation-Emission spectrum of mcherry
mCherry (BBa_J18932)
Excitation maxima: 567nm
Emission maxima: 605nm
Excitation and Emission spectrum of imCherry

The emission spectrum was obtained at a fixed excitation wavelength of 587nm and excitation spectrum was obtained at a fixed emission wavelength of 610nm.

Excitation-Emission spectrum of imcherry
imCherry (BBa_K2609006)
Excitation maxima: 576nm
Emission maxima: 607nm

Truncation characterization

Results from gel intensity data (Rough calculation)
mCherry imCherry
Relative gel intensity (or) % of protein Relative gel intensity (or) % of protein
Truncated Non-Truncated Truncated Non-Truncated
Replicate 1 30.25 69.75 19.9 80.1
Replicate 2 48.6 51.4 34.2 65.8
Replicate 3 35.8 64.2 24 76
Replicate 4 34.2 65.8 21.7 78.3
Replicate 5 49.8 50.2 20 80
Average 39.73 60.27 23.96 76.04
Std. dev. 7.95 7.95 5.33 5.33
The scatter plot showing the replicates, average and standard deviation for imCherry and mCherry with gel data. From the statistical significance analysis of the two means, we got a p-value of P=0.0062.

From this rough gel intensity estimation, we have a percentage truncation of 39.73 % ± 7.95 % for mCherry and 23.96 % ± 5.33 % for imCherry

Results from combining gel intensity and fluorescence data
mCherry imCherry
% of protein % of protein
Truncated Non-Truncated Truncated Non-Truncated
Replicate 1 34.66 65.34 16.35 83.65
Replicate 2 38,91 61.09 22.82 77.18
Replicate 3 42.53 57.47 20.53 79.47
Replicate 4 39.51 60.49 21.81 78.19
Replicate 5 38.47 61.53 20.73 79.26
Average 38.82 61.18 20.45 79.55
Std. dev. 2.52 2.52 2.21 2.21
The scatter plot showing the replicates, average and standard deviation for imCherry and mCherry with gel+fluorescence data. From the statistical significance analysis of the two means, we got a p-value of P < 0.0001 .
mCherry
Ifnt Ift Iwnt Iwt Ient Iet Ff Fw Fe % of truncated protein % of non-truncated protein
Replicate 1 22.06 77.94 100 0 100 0 891.74 473.91 639.57 34.66 65.34
Replicate 2 3.4 96.6 85.6 14.4 100 0 1099.02 1081.70 948.02 38.91 61.09
Replicate 3 2.5 97.5 85.2 14.8 100 0 1077.32 843.28 842.41 42.53 57.47
Replicate 4 0 100 81.4 18.6 100 0 690.62 640.25 718.62 39.51 60.49
Replicate 5 0 100 82.6 17.4 100 0 952.35 947.2 1004.64 38.47 61.53
imCherry
Ifnt Ift Iwnt Iwt Ient Iet Ff Fw Fe % of truncated protein % of non-truncated protein
Replicate 1 32.4 67.6 79.73 20.27 100 0 163.01 234.77 567.31 16.35 83.65
Replicate 2 15 85 90.7 9.3 100 0 860.28 1523.21 1441.96 22.82 77.18
Replicate 3 0 100 92 8 100 0 218.83 399.74 522.93 21.97 78.03
Replicate 4 8.5 91.5 89.6 10.4 100 0 590.06 1106.52 1306.53 21.81 78.19
Replicate 5 9.4 90.6 93.4 6.6 100 0 679.35 1383.86 1345.60 20.74 79.26

From this rough gel intensity estimation, we have a percentage trunction of 38.82 % ± 2.52 % for mCherry and 20.45 % ± 2.21 % for imCherry

Improvement in the part - Reduction in truncation

From the fine measurements combining gel intensity data and fluorescence data:

  • mCherry was observed to have a truncation of  38.82 % ± 2.52 %
  • The improved version of mcherry - imCherry - was observed to have a truncation of  20.45 % ± 2.21 %
  • Therefore, our modified mCherry sequence - called imCherry - reduces the truncation of the protein by   47.4%   of the original (p < 0.0001).

References

[1]https://salislab.net/software/:Salis Lab - The Ribosome Binding Site Calculator