Inducible Green Fluorescent Protein UnaG+Flexible Linker
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 437
- 1000COMPATIBLE WITH RFC
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.Designing the UnaG BioBricks
The IDT plasmid contains the lac-promoter so it is possible to do test expression directly with IPTG. However due to the way the final product is created, the UnaG sequence had a 50% chance to be incorporated in reverse, which was the case. Therefore different BioBrick promoters were extracted from the 2016 distribution to test for expression. Those include a medium constitutive promoter (BBa_K608006), a strong constitutive promoter (BBa_K880005), IPTG inducible promoter (BBa_J04500), and a T7 promoter (BBa_K525998). All of those contain an RBS already assembled in order to speed up work. The UnaG sequence itself was excised from the IDT plasmid using EcoRI and PstI and ligated into pSB1C3 backbone cut with the same set of enzymes.
After successfully obtaining UnaG assembled with a variety of promoters, mutagenesis was performed in order to create several variants of the BioBrick for future use. The ‚Äústock‚ÄĚ option contains the UnaG coding sequence. Upstream of it lies a 6xHis affinity tag, separated by an additional Serine amino acid to increase flexibility between the tag and the protein. In the registry it is annotated as BBa_K2003010. Note that this part is designed with RFC25 (Freiburg Standard) prefix and suffix in mind, hence it contains additional restriction sites, that do not affect normal 3A assembly but enable in-frame protein fusion without creating stop codons. Downstream of the part lies a short Glycine-rich flexible linker to minimize the effects of possible protein fusion. However the base part still contains the double TAA codons before this flexible linker.
The stop signal has been removed with PCR in BBa_K2003011. This is part retains all the features, including the RFC25 prefix and suffix, but now the flexible linker is properly expressed and in case of fusing to another CDS would not cause premature termination.
Finally, to avoid potential interference from the 6xHistidine affinity tag, BBa_K2003012 was created based on BBa_K2003011. In this part, the six amino acids were removed (again through PCR), as well as the Serine linker in between. This part has been designed for studying the properties of the protein in vivo, since the high positive charge of the affinity tag could interfere with its function or localization. At each step of the experiments, sequencing was performed using the iGEM standard VF2 and VR verification primers to ensure CDS integrity and especially to avoid introduced frameshift mutations.
Synthesized by PCR mutagenesis with UnaG BioBrick BBa_K2003011 as template.
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‚Äď1611.