Part:BBa_K3165046

i^2mCherry (Ile) Coding Device (under T7 expression)

This part is used to generate i²mCherry (Ile) which overcomes the problems in the existing mCherry (BBa_J18932) sequence which undergoes significant truncation in the protein. The single-base mutation at the 48th base causes the conversion of the internal start codon into ATC (coding for Ile).

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 738
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

i²mCherry (Ile) is an improved version of mCherry (BBa_J18932), which is widely used as a fluorescent marker. However, the N-terminal fusion of proteins with mCherry is not suited for studying various signal peptides due to the significant truncation that arises due to the presence of an RBS like sequence upstream of the ninth amino acid, Met, which happens to be coded by the start codon (ATG). The RBS like sequence along with the start codon causes the transcription to begin at an internal site, resulting in significant truncation during protein expression. The 2018 IISc-Bangalore team attempted to reduce the truncation of the mCherry sequence by modifying the internal RBS like sequence and decreasing its ability to pair with the ribosome. Their improved part imCherry (BBa_K2609006) reported a considerable decrease in the truncation but they couldn't get rid of the truncated product entirely. In order to completely shut down the truncation caused by mCherry, we modified the internal start codon (ATG) via a single base mutation at then 48th nucleotide to convert ATG to ATC (coding for Ile). In the absence of a start codon, no transcription is expected, thus eliminating the chances of any truncated products.
I²mCherry(Ile) was developed to overcome the shortcomings of the existing mCherry (BBa_J18932) BioBrick which is not suitable for protein fusion studies due to the truncation faced at the N-terminal. As a consequence of reduced truncation, I²mCherry can be used for studying signal peptides and other N-terminal protein fusion components.

Characterization

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 37^oC 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.

Truncation Reduction

The Truncation was reduced from (48.5 ∓ 2.0)% in mCherry(<a href="https://parts.igem.org/Part:BBa_J18932">BBa_J18932</a>) to (5.03 ∓ 0.5)% in i²mCherry (Ile).