Difference between revisions of "Part:BBa K3165046"
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<partinfo>BBa_K3165046 short</partinfo>
 
<partinfo>BBa_K3165046 short</partinfo>
  
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This part is used to generate i^2mCherry which overcomes the problems in the existing mCherry sequence which undergoes significant truncation in the protein. The single-base mutation at the 48th base causes conversion of the internal start codon into ATC (coding for Ile).
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This part is used to generate i<sup>2</sup>mCherry (Ile) which overcomes the problems in the existing mCherry sequence which undergoes significant truncation in the protein. The single-base mutation at the 48th base causes conversion of the internal start codon into ATC (coding for Ile).
  
 
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===Usage and Biology===
 
===Usage and Biology===
  
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i2mCherry (Ile) is an improved version of mCherry(BBa_J19832), 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 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.
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i<sup>2</sup>mCherry (Ile) is an improved version of mCherry(BBa_J19832), 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 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.
 
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.<br>
 
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.<br>

Revision as of 10:38, 20 October 2019


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

This part is used to generate i2mCherry (Ile) which overcomes the problems in the existing mCherry sequence which undergoes significant truncation in the protein. The single-base mutation at the 48th base causes 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_J19832), 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 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.