Part:BBa_K5322000

Constitutive Mfp3 Expression System

Usage and Biology

The plasmid pET29a-J23119-RBS-Mfp3-T7 utilizes the pET29a vector for high-level expression of the mussel foot protein 3 (Mfp3) in Escherichia coli. This system is controlled by the strong constitutive promoter J23119, which regulates Mfp3 expression. The ribosome binding site (RBS) ensures efficient translation of the mRNA, while the T7 terminator provides a clean and efficient transcriptional endpoint. This system is designed for the efficient expression of Mfp3 under stable environmental conditions, allowing it to exert its adhesive properties.

Construction of the plasmid

To enable the expression of eukaryotic proteins in prokaryotes, we selected Escherichia coli BL21(DE3) as the host cell. For efficient expression of the adhesin, we chose the strong promoter J23119 as the regulatory element. As shown in Figure 2-1, we designed the plasmid pET29a-J23119-RBS-Mfp3-T7. Through homologous recombination, we integrated this plasmid into BL21(DE3) and selected individual bacterial colonies from several transformation plates for plasmid extraction. We performed PCR verification using specific primers targeting a 422 bp fragment, as depicted in Figure 2-2. The plasmids with correctly sized bands were sequenced, and the sequencing results in Figure 2-3 confirmed the successful construction of the plasmid pET29a-J23119-RBS-Mfp3-T7.

Figure 2-1 Plasmid pET29a-pJ23119-RBS-Mfp3-T7

Figure 2-2 Colony PCR gel electrophoresis of plasmid pET29a-pJ23119-RBS-Mfp3-T7(422bp)

Figure 2-3 Sequencing results of plasmid pET29a-J23119-RBS-Mfp3-T7

Protein Expression Validation

We characterized the protein using Tricine-SDS-PAGE and Western Blot experiments, confirming the expression of Mfp3, as shown in Figures 3-1 and 3-2. The expected size of J23119-Mfp3, including the His tag, is 6.5 kDa; however, the bands at this position appear faint or even absent, while bands around 12 kDa are more prominent. We speculate that Mfp3 may have formed dimers or oligomers during the expression process.

Figure 3-1 Tricine-SDS-PAGE analysis of Mfp3

Figure 3-2 Western Blot analysis of Mfp3

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 210
  Illegal NotI site found at 174
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]