Part:BBa_K5322005

NO-inducible Mfp3 Expression System

Usage and Biology

The plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp3-T7 utilizes the pET29a vector for high-level expression in Escherichia coli. To achieve better intestinal mucosal repair effects in the high NO environment associated with enteritis, we employed the oxidative stress-responsive SoxR/SoxS promoter, targeted specifically at the inflamed intestinal region for therapeutic purposes. The expression of SoxR is controlled by the strong constitutive promoter J23119, which responds to NO and activates the SoxR promoter, thereby regulating the expression of the mussel foot protein Mfp3. The ribosome binding site (RBS) ensures efficient translation of mRNA, while the T7 terminator provides a clean and efficient end for transcription. This system is designed for the efficient expression of Mfp3 in the high NO environment of enteritis, enhancing its adhesive properties.

Construction of the plasmid

Due to the absence of endotoxins in Escherichia coli Nissle 1917 (EcN), we chose to use EcN as the chassis cell to enhance safety. To efficiently express the adhesin, we selected the strong constitutive promoter J23119 as the regulatory element. To achieve better intestinal mucosal repair effects in the high NO environment associated with enteritis, we utilized the oxidative stress-responsive SoxR/SoxS promoter, targeting the inflamed intestinal region for treatment. As shown in Figure 2-1, we designed the plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp3-T7. Through homologous recombination, we integrated this plasmid into EcN and selected individual E. coli colonies from several transformation plates for plasmid extraction. We performed PCR verification using specific primers targeting a 1075 bp fragment, as illustrated in Figure 2-2. The plasmids with correctly positioned bands were subjected to sequencing. The sequencing results in Figure 2-3 confirmed the successful construction of the plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp3-T7.

Figure 2-1 Plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp3-T7

Figure 2-2 Colony PCR gel electrophoresis of plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp3-T7(1075bp)

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

Protein Expression Validation

Given that the mussel adhesive proteins used in this study are intended for application in the treatment of mammalian enteritis, we selected 37°C, the temperature closest to body temperature, as the optimal induction temperature. The induction time was identified as a crucial factor influencing protein characterization. The recombinant strain EcN-pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp3-T7 was cultured until an OD₆₀₀ of 0.8 was reached, followed by the addition of 100μM sodium nitroprusside (SNP) for induction. The recombinant protein was expressed for 16 hours in the EcN strain, and detection was carried out using Tricine-SDS-PAGE, as shown in Figure 3-1.Finally, we performed a Western Blot, as shown in Figure 3-2, which confirmed that the NO-inducible Mfp3 can be expressed in a high NO environment.

Figure 3-1 Tricine-SDS-PAGE analysis of NO-Mfp3

Figure 3-2 Western Blot analysis of NO-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 552
  Illegal NheI site found at 836
  Illegal NotI site found at 800
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]