Difference between revisions of "Part:BBa K1688020:Design"
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===References=== | ===References=== | ||
| + | Kataoka, K, Kogi H, Tsujimura S, Sakurai T. 2012. "Modifications of laccase activities of copper efflux oxidase, CueO by synergistic mutations in the first and second coordination spheres of the type I copper center" Biochemical and Biophysical Research Communications Volume 431, Issue 3, 15 February 2013, Pages 393–397 | ||
Latest revision as of 19:24, 15 September 2015
ModLac laccase with His-tag and HlyA export tag
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 249
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The modified CueO was synthesized with iGEM prefix and suffix, RBS(BBa_B0034) and a M-scar after the RBS in order to guarantee the function of the RBS. Two Nsil restriction sites were put before and after a 6xHis-tag so that the enzyme could be purified with an IMAC and so that the tag could be removed. The gene is followed by a KpnI restriction site with glycines and serines on each side, which are small amino acids which increase the flexibility of the protein. This is needed for the export tag HlyA , to make it easier for the resulting signal protein to be folded properly. A second KpnI restriction site is put after the HlyA so that the export tag can be removed easily. It has then two stop codons, and a terminator at the end.
Source
D439A/M510L CueO mutant sequence received from Professor Kunishige Kataoka and synthesized with His-tag and HlyA export tag.
References
Kataoka, K, Kogi H, Tsujimura S, Sakurai T. 2012. "Modifications of laccase activities of copper efflux oxidase, CueO by synergistic mutations in the first and second coordination spheres of the type I copper center" Biochemical and Biophysical Research Communications Volume 431, Issue 3, 15 February 2013, Pages 393–397