Composite

Part:BBa_K3895007

Designed by: Rui Zhu   Group: iGEM21_SZ_SHD   (2021-10-01)
Revision as of 08:03, 15 October 2021 by Auskuld (Talk | contribs) (→‎Protein purification)


kerAvDZ50_dna_pET-28a(+)

Construction

Keratinase KerAVDZ50 (BBa_K3895003) is an extracellular serine thiol alkaline protease from the Actinomadura viridilutea strain, which can be isolated from the Algerian fishing port [1]. As a thermo-alkaline keratinases, it can function at a pH range of 7–12 and a temperature range of 35–80 °C. 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--Z50plasmid.jpg

Protein purification

Name of keratinase IPTG concentration/mM Temperature/°C Time/h
KerAVDZ50 0.67139 16 12


Figure 1. Plasmid construction of KerAVDZ50.
Figure 2. PCR and gel electrophoresis of KerAVDZ50.



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


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