Part:BBa_K3165012

i^2mCherry (Leu)

An improved alternative of mCherry (BBa_J18932) designed to minimise the amount of truncated protein by modifying the internal start codon of the existing sequence.

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 679
  
• 1000
  COMPATIBLE WITH RFC[1000]

Biology

i²mCherry (Leu) is an improved version of mCherry (BBa_J18932), which is widely used as a fluorescent marker. However, 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 9^th 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.

Usage

i²mCherry(Leu) 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.

References :

(1) https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609006