Part:BBa_K3165049

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

This part is used to generate i²mCherry (Leu) which overcomes the problems in the existing mCherry sequence which undergoes significant truncation in the protein. The 2 single-base mutations at the 46th and 48th base causes conversion of the internal start codon into CTT (coding for Leu).

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 (Leu) is an improved version of mCherry (BBa_J19832), 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 2 single-base mutations at the 46th and 48th nucleotides to convert ATG to CTT (coding for Leu). In the absence of a start codon, no transcription is expected, thus eliminating the chances of any truncated products.
i²mCherry(Leu) was developed to overcome the shortcomings of the existing mCherry (BBa_J19832) 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.