Difference between revisions of "Part:BBa K3165013"

 
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<partinfo>BBa_K3165013 short</partinfo>
 
<partinfo>BBa_K3165013 short</partinfo>
  
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i^2mCherry was developed to limit the truncation caused by the existing mCherry sequence in the iGEM registry. To reduce the truncation to the minimum possible value we replace the internal start codon (ATG) to ATC coding for Isoleucine. It results from a single base mutation at the 48th base pair.
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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.
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'><b>Sequence and Features</b></span>
 
<partinfo>BBa_K3165013 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3165013 SequenceAndFeatures</partinfo>
  
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===Usage and Biology===
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<h2> Biology </h2>
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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.<br>
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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. <br>
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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.
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<h2> Usage </h2>
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i<sup>2</sup>mCherry(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, i<sup>2</sup>mCherry can be used for studying signal peptides and other N-terminal protein fusion components.
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<h3> References : </h3>
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(1) https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609006
  
 
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Revision as of 07:18, 19 October 2019


i^2mCherry (Ile)

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]

Usage and Biology

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.

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