Difference between revisions of "Part:BBa K2003010:Design"

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===Design Notes===
 
===Design Notes===
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As part of this iGEM project, research has been done on the newly discovered green fluorescent protein called UnaG. This protein is interesting foremost because it is about half the size of other fluorescent proteins that are being used today, making it suitable as a fusion protein. In addition it fluoresces only when a bilirubin molecule is bound non-covalently to the protein. This feature could make UnaG suitable as an inducible marker by the addition and removal of bilirubin to cells. Moreover, unlike all other GFP derivatives used today, UnaG does not need molecular oxygen in order to fluoresce, meaning that it can be used as a marker protein in research under hypoxic conditions. These cool features are among the reasons that motivated us to add this protein to the iGEM toolbox. During the summer we created three different UnaG BioBricks: one with a hexahistidine tag suitable for affinity chromatography (BBa_K2003010), one biobrick with only the flexible linker attached to UnaG suitable for fusing to other proteins (BBa_K2003012), and a third with the hexahistidine tag and a flexible linker of six amino acids (BBa_K2003011).
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NOTE THAT THE PART HAS RFC25 PREFIX AND SUFFIX!
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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)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.
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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===
 
===Source===

Revision as of 11:59, 16 October 2016


Inducible Green Fluorescent Protein UnaG+6xHis-tag


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

Eel

References