Difference between revisions of "Part:BBa K3895007"

(→‎Protein purification)
(→‎Protein purification)
Line 20: Line 20:
 
|12
 
|12
 
|}
 
|}
−
 
  
  
 
{|border=0 width="90%" align="center"
 
{|border=0 width="90%" align="center"
 
|-align="center"
 
|-align="center"
−
|[[File:T--SZ SHD--PCR2.png|540px|thumb|center|'''Figure 1.''' PCR and gel electrophoresis of KerAVDZ50.]]
+
|[[File:T--SZ SHD--PCR2.png|530px|thumb|center|'''Figure 1.''' PCR and gel electrophoresis of KerAVDZ50.]]
 
|[[File:T--SZ_SHD--SDSPAGE1.png|350px|thumb|center|''' 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).]]
 
|[[File:T--SZ_SHD--SDSPAGE1.png|350px|thumb|center|''' 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).]]
 
|}
 
|}
  
−
 
+
==Protein Concentration==
−
 
+
[[File:T--SZ_SHD--BCA1.png|center]]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 08:10, 15 October 2021


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. PCR and gel electrophoresis of KerAVDZ50.
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).

Protein Concentration

T--SZ SHD--BCA1.png

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