Part:BBa_K5322002
Constitutive Mfp5 Expression System
Contents
Usage and Biology
The plasmid pET29a-J23119-RBS-Mfp5-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 Mfp5. 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 Mfp5 under conditions not influenced by environmental factors, allowing it to exhibit its adhesive properties.
Construction of the plasmid
To express eukaryotic proteins in prokaryotic systems, we chose Escherichia coli BL21(DE3) as the chassis cell, using the strong promoter J23119 as the regulatory element for efficient expression of the adhesive protein. As shown in Figure 2-1, we designed the plasmid pET29a-J23119-RBS-Mfp5-T7. The plasmid was transferred to BL21(DE3) via homologous recombination, and single colonies of E. coli were selected on several transformation plates for plasmid extraction. We performed PCR verification using specific primers targeting a 512 bp fragment, as illustrated in Figure 2-2. Plasmids with correctly positioned bands were sequenced, and the sequencing results in Figure 2-3 confirmed the successful construction of the plasmid pET29a-J23119-RBS-Mfp5-T7.
Figure 2-1 Plasmid pET29a-J23119-RBS-Mfp5-T7
Figure 2-2 Colony PCR gel electrophoresis of plasmid pET29a-J23119-RBS-Mfp5-T7(512bp)
Figure 2-3 Sequencing results of plasmid pET29a-J23119-RBS-Mfp5-T7
Protein Expression Validation
We verified the performance of the lysis module through dry experiments and plan to complete wet experimental validation of its lytic function in the future. Through literature review and numerical modeling validation, we found that after inducing the expression of antimicrobial peptides and lysis proteins with NO for twenty minutes, the engineered strain will be lysed by the lysis protein and release the antimicrobial peptides. Mathematical modeling confirmed that at this time, the concentration of antimicrobial peptides is sufficient to reach an effective inhibitory concentration.
Figure 3-1 Tricine-SDS-PAGE analysis of Mfp5
Figure 3-2 Western Blot analysis of Mfp5
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 300 - 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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