Coding

Part:BBa_K2450101:Design

Designed by: Zoe Ford   Group: iGEM17_Oxford   (2017-09-28)
Revision as of 14:58, 21 October 2017 by Tinyref (Talk | contribs) (→‎Design Notes)

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TEV protease tagged with mCherry


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 1444
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We wanted to be able to purify TEV with and without the mCherry, so we had the His tag on the other terminus of the protein.

'Compared with the time course curve of TEV-His6, we found that sfGFP-TEV-His6 Nd1–6 had different degrees of loss of catalytic activity. Among them, Nd2 had the closest curve to TEV-His6. Ranking the cleavage rate at 60 minutes, the second highest ranked Nd2 could digest around 66% substrate, which retained about 95% catalytic activity of TEV-His6.' (from reference below)

The TEV protease is an anti-self-cleavage TEV.

We used mCherry so it would be compatible with our parts K2450201 and K2450301, and easily observable with our fluorescent microscope as it does not have much spectral overlap with CFP and eYFP.

Source

mCherry from BBa_J06504

TEV protease from BBa_K1319004

Linker is Nd2 from https://www.hindawi.com/journals/bmri/2009/591923/tab2/

References

Xudong Wu, Di Wu, Zhisheng Lu, Wentao Chen, Xiaojian Hu, and Yu Ding, “A Novel Method for High-Level Production of TEV Protease by Superfolder GFP Tag,” Journal of Biomedicine and Biotechnology, vol. 2009, Article ID 591923, 8 pages, 2009. doi:10.1155/2009/591923