Composite

Part:BBa_K3895009

Designed by: Rui Zhu   Group: iGEM21_SZ_SHD   (2021-10-01)
Revision as of 13:47, 15 October 2021 by Tigerjiang (Talk | contribs)


kerBlMKU3_dna_pET-28a(+)

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Construction

Keratinase KerBlMKU3 (BBa_K3895005) is a serine keratinase originally secreted by Bacillus licheniformis MKU3. It can work at 37°C for 48 h in nutrient broth or on feather medium [1]. The keratinase was compared with the other 3 keratinases (BBa_K3895004; BBa_K3895005;BBa_K3895006) with the same construction method. 6x His-tags were added to both sides of the sequence for purification and constructed into PET-28a(+). The constitutive T7 promoter (BBa_I719005) derived from the T7 bacteriophage allows high expression of proteins.

T--SZ SHD--KU3plasmid.jpg
Name of keratinase IPTG concentration/mM Temperature/°C Time/h
KerBIMKU3 0.4 37 4
Figure 1. PCR and gel electrophoresis of KerBlMKU3.
Figure 2. SDS-PAGE of purified protein.Protein molecular marker; Lane1. Empty PET-28a vector; Lane2. The crude enzyme (kerBlER15); Lane3. The crude enzyme (kerAvDZ50); Lane4. The purified enzyme (kerBlER15); Lane5. The purified enzyme (kerAvDZ50); Lane6. Empty PET-28a vector; Lane7. The crude enzyme (kerBlMKU3); Lane3. Crude enzyme (kerBteQ7); Lane8. The purified enzyme (kerBlMKU3); Lane5. The purified enzyme (kerBteQ7).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 606
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 411
    Illegal AgeI site found at 1149
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 351


[edit]
Categories
//awards/composite_part/nominee
Parameters
None