Part:BBa_K2924032

T7 + RBS + α-s1-casein + terminator

T7-lacO promoter (BBa_K2406020) and RBS expressing α-s1-casein (BBa_K2924026) with 6xHis-tag and T7-Terminator (BBa_B0012) in E. coli

Usage and Biology

Fig. 1: Scheme of α-s1-casein overexpression construct. The insert, containing the promoter P_T7 (BBa_K2406020), α-s1-casein gene (BBa_K2924026) fused with a 6x His-tag and the T7 terminator (BBa_B0012), was cloned into the pET22b backbone

Using the strong T7 expression system, the P_T7 promoter (BBa_K2406020) expresses α-s1-casein (BBa_K2924026) with the T7 terminator (BBa_B0012). The transcription of the T7 promoter is carried out by the T7-RNA polymerase, whose expression is inhibited by lacI and can be induced by either lactose or IPTG¹. The lac-operator is also present between the promoter and the gene to reduce the leakiness of the expression by requiring derepression of the T7-RNA polymerase as well as the target gene’s expression. In addition the used pET22b contains the Ribosome-binding site (BBa_K2924053)². Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 756
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

α-s1-casein was cloned into the T7 promoter-based vector pET22b which is a high copy plasmid and contains the pelB signal sequence, a specific amino acid sequence that is fused to the protein and facilitates the export into the periplasm. The plasmid contains a fused histidine tag (6xHis) for easier purification of overexpressed proteins.

The strain BL21(DE3) was used for expression. This strain is a common strain for expression because it contains T7 polymerase in its genome and expresses fewer proteases, which might degrade the product³. For the IPTG-induced overexpression 100 ml LB containing Ampicillin [100mg/ml] were inoculated with 1 ml of overnight culture.

The cells were incubated at 37° C at 180 rpm until an OD600 of 0.6 was reached. IPTG [0.5 mM] was added and the cultures were incubated for another 2 h at 37° C at 180 rpm. The cultures were centrifuged at 8000 x g for 20 min at 4°C to separate the cell pellet from the medium, both were kept on ice. The cell pellet was lysed with a lysis reagent. The samples were used for Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to prove the heterologous expressed α-s1-casein which has a size of approximately 25.4 kDa size (Fig. 2).