Team:IISc-Bangalore/Improve

Improvement


INTRODUCTION

mCherry, a monomeric red fluorescent protein derived from DsRed (obtained naturally from the coral species of Discosoma) is one of the most extensively used fluorophores in all molecular biology studies. mCherry was developed as an attempt to overcome the shortcomings of the initial DsRed fluorophore (wild-type fluorescent proteins happened to be obligately tetrameric and often toxic and disruptive). The directed mutations in the wild type DsRed gene involving Q66M, V7I, M182K, M163Q, N6aD, R17H, K194N, T195V & D196N led to the final construction of mCherry as a functional fluorophore.
Having one of the fastest maturation time of all known fluorophores (T½ for maturation at 37˚C = 15 min) and relatively high brightness on appropriate excitation (Extinction coefficient = 72,000 M-1 cm-1 and fluorescence quantum yield = 0.22), mCherry qualifies as a suitable fluorophore for various applications. It is also quite photostable (t½ for photobleaching = 68 s) and has a lifetime of around 1.4 ns making the storage and usage of the protein much more convenient.

Due to these several advantages we opted for mCherry to be used as a reporter protein for some of our composite systems. In order to characterise the functioning of Opto T7 system and the T7 promoter, we planned to use mCherry as a reporter protein to quantify the strength of the promoter at different intensities of blue light.
Last year, the iGEM team IISc-Bangalore (2018) ventured to improve a major flaw in the mCherry sequence (BBa_J18932) which caused severe truncation [unpublished] in protein expression due to an internal start codon with a Shine Dalgarno like sequence upstream of it.
In an attempt to correct this imperfection, they came up with an improved version of the fluorophore – imCherry (BBa_K2609016) with modifications in the ribosome binding site upstream of the internal ATG codon. An estimated 75% decrease in truncation was expected with better expression of His-tagged imCherry. The team achieved considerable success by reducing the truncation from 38.82 % ± 2.52 % for mCherry to 20.45 % ± 2.21 %.

Fig (1) - Truncation in mCherry

Although the amount of protein produced without the N-terminal tag was reduced to a measurable extent, the truncation was still significant for imCherry. In order to completely eliminate the truncated protein produced, we used an in-silico method to improve the existing mCherry.

MODIFICATION

To totally terminate the expression of the truncated part we modified the internal start codon ATG to CTT and ATC coding for the amino acids Leucine and Isoleucine respectively. The choice of the amino acids Leucine and Isoleucine was based on the structural similarities between them and methionine (coded by ATG). By removing the internal start codon we intended to put an end to the truncation (absence of any start codon will prohibit the expression of the truncated product).


Fig (2) : Modifications in the mCherry sequence (BBa_J18932)



Fig (3) : Modification in sequence of mCherry sequence

PARTS

The parts used to validate the improvement experimentally are listed below. The details of their assembly can be found on the Assembly Page.

BBa_K2319009 : Generator for BBa_J18932

BBa_K3165046 : Generator for BBa_K3165013

The data corresponding to the characterisation of these parts have been added to the Registry Pages of the following parts : (BBa_J18392), (BBa_K2319009), (BBa_K3165012), (BBa_K3165013), (BBa_K3165046) & (BBa_K3165049).

VALIDATION STRATEGY

To validate the improvement in mCherry, we required a method to quantify the amount of truncated protein in the crude cell lysate.
For this purpose, we created a composite part by the addition of a 6xHistag fused at the N-terminal of the improved mCherry coding sequence. We placed this construct under a T7 promoter and a suitable double terminator. The truncated protein being transcribed from an internal sequence lacks the N-terminal 6xHistag and this difference in sequence is the basis for our truncation quantification. At the end of the Ni-NTA purification procedure, the 6xHistagged protein is found mostly in the elution fraction whereas the truncated protein appears in the wash and supernatant.
Moreover, we can approximately estimate the ratios of the truncated and non-truncated protein by the intensity of their bands on the SDS PAGE.

GEL IMAGES

Expression with BBa_K3165046

The protein was expressed under the T7 promoter in Escherichia coli (BL21DE3) with 6xHistag at the N-terminal. The transformed bacteria were incubated at 37oC for over 4 hours. The cells were lysed by sonication and the lysate was collected via centrifugation. The lysate was run on an SDS PAGE which showed two bands. The size of the protein of interest along with the 6xHistag is around 27 kDa. The upper band corresponds to the non-truncated protein while the lower band represents the truncated product.
Fig(1) : SDS PAGE for mCherry and i2mCherry (Ile)
From the SDS PAGE, we clearly observe two bands when using mCherry, while i2mCherry (Ile) gave one very large spot on the polyacrylamide gel with a trailing smear kind of band. The observed dark band is seen at the desired size (roughly around 27 kDa) confirming that the bands correspond to our protein of interest.

Purification using Ni-NTA with BBa_K3165046

The cell lysate thus obtained was purified using Ni-NTA beads as only the non-truncated protein having 6xHistag can bind to the beads. Ideally, the supernatant after binding should have the truncated protein while the eluted fraction should contain the non-truncated protein. This idealisation does not hold true as the binding of Ni-NTA is not perfect.
Fig (2) : SDS PAGE for Ni-NTA Purification of i2mCherry (Ile)

FLUORESCENCE ANALYSIS

Wavelengths scans of the protein lysate were performed to obtain the excitation and emission spectra of the fluorescent protein. The fluorescence data obtained were corrected for the blank (untransformed BL21DE3 protein lysate) and the data so obtained is presented below :
Fig(1) : Excitation and Emission Spectra of mCherry
Fig(2) : Excitation and Emission Spectra of i2mCherry (Ile)

From the fluorescence data, we found out that the excitation λ for mCherry is 585 nm whereas its emisiion λ is 602 nm.
Similarly for i2mCherry (Ile) the experimental excitation λ was found to be 575 nm while the emission λ is found to be 617 nm.

QUANTIFICATION OF TRUNCATION

The truncation of i2mCherry was quantified by the two following ways :

  1. By analysis of the intensity of the truncated and non-truncated protein bands as seen on the SDS PAGE.

  2. By combining the data of fluorescence and the gel intensity data of the Ni-NTA purification products (supernatant, wash and elution). Assuming the fluorescence of the truncated and non-truncated parts to be similar, we divide the fluorescence of each sample into two parts: one due to the truncated product and the other due to the non-truncated protein. The sum of the fluorescence of the two fractions was then used as a measure to estimate their concentration to determine truncation.
Fig(3) : Scatter Plot for the percentage truncation of mCherry and i2mCherry (Ile)
It is evident from the data that some amount of truncated protein was still produced irrespective of the presence or absence of the internal start codon. The presence of the RBS like sequence in the gene might be the reason behind the continued production of the truncated product.

REDUCTION IN TRUNCATION

The Truncation was reduced from (48.5 ∓ 2.0)% in mCherry(BBa_J18932) to (5.03 ∓ 0.5)% in i2mCherry (Ile).

REFERENCES

  1. Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N. G., Palmer, A. E., & Tsien, R. Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnology, 22(12), 1567–1572. doi:10.1038/nbt1037
  2. https://www.fpbase.org/protein/mcherry/