Generator

Part:BBa_K2239006:Design

Designed by: Ruohong Wang   Group: iGEM17_SDSZ-China   (2017-10-01)


CBD-7alphaHSDH


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1032
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 888


Design Notes

The CBD tag is added to purify the effective enzymes. When connected with the enzyme DNA and expressed, CBD is able to bind to the cellulose on gauze. After washing off other chemicals in the solution, the enzymes are left on the gauze. As a result, they are successfully immobilized.


Source

The 7alpha-HSDH sequence is cloned from the genome of E.coli DH5alpha through PCR amplification, using the primers designed and synthesized based on its sequence. The CBD sequence is retrieved from the GenBank and artificially synthesized.


References

[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochemistry, Elsevier, 2015.

[2] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov: Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.