Difference between revisions of "Part:BBa K5322000"

Revision as of 11:09, 30 September 2024

Constitutive Mfp3 Expression System

To address the reduced mucus layer thickness and dysbiosis causing inflammation in patients with enteritis, we aim to resolve the dysregulation of the mucosal system from the ground up. A natural high-adhesion protein from marine mussels—mussel foot protein (Mfp)—has garnered our attention, particularly Mfp3, which is one of the proteins with the highest DOPA content among the six mussel proteins. We utilized Escherichia coli BL21(DE3) to establish a production system for Mfp, enhancing adhesion in damaged intestines and regulating gut microbiota. Consequently, we designed the plasmid pET29a-J23119-RBS-Mfp3-T7.

Usage and Biology

The plasmid pET29a-J23119-RBS-Mfp3-T7 utilizes the pET29a vector for high-level expression of the mussel foot protein 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-SoxR-T-pSoxS-RBS-Mfp3-T7

Figure 2-2 Colony PCR gel electrophoresis of plasmid pET29a-pJ23119-SoxR-T-pSoxS-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