Difference between revisions of "Part:BBa K2003012:Design"

 
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===Design Notes===
 
===Design Notes===
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<b>NOTE THAT THE PART HAS <a href=https://parts.igem.org/Help:Assembly_standard_25>RFC25</a> PREFIX AND SUFFIX!</b></br>
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In the original paper (Kumagai et al., 2013) the authors obtained the UnaG sequence from a cDNA library of unagi eel muscle. Since we lack the budget to go fishing in Japan, we ordered the nucleotide sequence from IDT. This not only gave the possibility to address codon bias, but through synonymous base substitutions to eliminate all restriction sites as per the iGEM BioBrick guidelines. An exception is an NsiI site in the N-terminal 6xHis tag, but a BioBrick without it was also made. In addition to the affinity tag, the original sequence was also modified to include C-terminal flexible linker (GSG)<sub>2</sub> in case the protein would be part of a fusion construct. By default a double TAA stop-codon is present right before this flexible linker, which prevents it from expressing and thus potentially affecting protein function. This does not interfere with 3A assembly, but a variant of the BioBrick without the flexible linker also exists.
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The original UnaG sequence was ordered from IDT with a 6xhistidine tag on the 5' end and a flexible linker on the 3' end. However, the first two codons before the flexible linker were stop codons, meaning that the default UnaG does not translate the flexible linker. The histidine tag and the stop codons were removed from the sequence by PCR, resulting in a UnaG sequence without the histidine tag and with the flexible linker.  
 
The original UnaG sequence was ordered from IDT with a 6xhistidine tag on the 5' end and a flexible linker on the 3' end. However, the first two codons before the flexible linker were stop codons, meaning that the default UnaG does not translate the flexible linker. The histidine tag and the stop codons were removed from the sequence by PCR, resulting in a UnaG sequence without the histidine tag and with the flexible linker.  
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===Source===
 
===Source===
  
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Synthesized by PCR mutagenesis from UnaG sequence designed and ordered from IDT.  
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Synthesized by PCR mutagenesis with UnaG BioBrick BBa_K2003011 as template.  
  
 
===References===
 
===References===
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Kumagai, A., Ando, R., Miyatake, H., Greimel, P., Kobayashi, T., Hirabayashi, Y., Shimogori, T., and Miyawaki, A. (2013).</br>
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A Bilirubin-Inducible Fluorescent Protein from Eel Muscle. Cell 153, 1602–1611.
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Revision as of 12:13, 16 October 2016


Inducible Green Fluorescent Protein UnaG+Flexible Linker


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 437
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

NOTE THAT THE PART HAS RFC25 PREFIX AND SUFFIX!
In the original paper (Kumagai et al., 2013) the authors obtained the UnaG sequence from a cDNA library of unagi eel muscle. Since we lack the budget to go fishing in Japan, we ordered the nucleotide sequence from IDT. This not only gave the possibility to address codon bias, but through synonymous base substitutions to eliminate all restriction sites as per the iGEM BioBrick guidelines. An exception is an NsiI site in the N-terminal 6xHis tag, but a BioBrick without it was also made. In addition to the affinity tag, the original sequence was also modified to include C-terminal flexible linker (GSG)2 in case the protein would be part of a fusion construct. By default a double TAA stop-codon is present right before this flexible linker, which prevents it from expressing and thus potentially affecting protein function. This does not interfere with 3A assembly, but a variant of the BioBrick without the flexible linker also exists.

The original UnaG sequence was ordered from IDT with a 6xhistidine tag on the 5' end and a flexible linker on the 3' end. However, the first two codons before the flexible linker were stop codons, meaning that the default UnaG does not translate the flexible linker. The histidine tag and the stop codons were removed from the sequence by PCR, resulting in a UnaG sequence without the histidine tag and with the flexible linker.


Source

Synthesized by PCR mutagenesis with UnaG BioBrick BBa_K2003011 as template.

References

Kumagai, A., Ando, R., Miyatake, H., Greimel, P., Kobayashi, T., Hirabayashi, Y., Shimogori, T., and Miyawaki, A. (2013).
A Bilirubin-Inducible Fluorescent Protein from Eel Muscle. Cell 153, 1602–1611.