Difference between revisions of "Part:BBa K5322003"

Revision as of 14:55, 30 September 2024

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 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