Part:BBa_K5322003
Constitutive Mfp53 Expression System
Usage and Biology
The plasmid pET29a-J23119-RBS-Mfp53-T7 utilizes the pET29a vector for high-level expression in Escherichia coli. This system is controlled by the strong constitutive promoter J23119, which regulates the expression of the mussel foot protein 5+mussel foot protein 3( Mfp53) . The ribosome binding site (RBS) ensures efficient translation of the mRNA, while the T7 terminator provides a clean and efficient termination for transcription. This system is designed for the effective expression of Mfp53 under conditions not influenced by environmental factors, allowing it to exhibit its adhesive properties.
Construction of the plasmid
To express eukaryotic proteins using prokaryotic systems, we selected Escherichia coli BL21(DE3) as the host cell. To ensure efficient expression of the adhesive proteins, we employed the strong constitutive promoter J23119 as a regulatory element and used a protein linker (GGGGS) to connect Mfp3 and Mfp5. As shown in Figure 2-1, we designed the plasmid pET29a-J23119-RBS-Mfp53-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 680 bp fragment, as illustrated in Figure 2-2. The plasmids with correctly localized bands were sequenced, and the sequencing results in Figure 2-3 confirmed the successful construction of the plasmid pET29a-J23119-RBS-Mfp53-T7.
Figure 2-1 Plasmid pET29a-J23119-RBS-Mfp53-T7
Figure 2-2 Colony PCR gel electrophoresis of plasmid pET29a-J23119-RBS-Mfp53-T7(680bp)
Figure 2-3 plasmid pET29a-J23119-RBS-Mfp53-T7 sequencing result
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 487
Illegal NotI site found at 451 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 137
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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