Difference between revisions of "Part:BBa K2003011:Design"

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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.
 
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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Revision as of 11:14, 16 October 2016


Inducible Green Fluorescent Protein UnaG+6xHis-tag+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
    COMPATIBLE WITH RFC[25]
  • 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.

IDT sequence the entire pUCIDT-Amp vector including the ordered insert as part of their quality control workflow, so their results were used as a reference when doing modification to the plasmid.

Source

Rational design and synthesis.

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.