Difference between revisions of "Part:BBa K5292361"

Latest revision as of 07:08, 2 October 2024

MnfaA-25

In order to improve the expression of the PET-degrading enzyme ICCG, we utilized a Markov transfer frequency matrix to modify the H region of the nfaA signal peptide, resulting in the design of the MnfaA signal peptide. The MnfaA signal peptide was designed to optimize the secretion efficiency of ICCG, and was characterized by its fusion with the ICCG-GFP protein. However, during the experiments, the performance of the MnfaA signal peptide did not fully meet the expected improvement. Nevertheless, this design provides valuable reference information for further optimization and adjustment, especially in exploring the influence of signal peptides on protein secretion. Future work will focus on deeper improvements based on these preliminary results, continuing to explore methods to enhance ICCG secretion and expression.

Sequence and Features

Abstract

MnfaA is a signal peptide obtained by modifying and optimizing the H region of the nfaA signal peptide. The nfaA signal peptide is derived from Thermobifida fusca, a bacterium that secretes proteins extracellularly. During the design of MnfaA, we utilized the Markov transfer frequency matrix to introduce mutations in its key region, aiming to enhance the secretion efficiency of the ICCG protein in Escherichia coli. By combining MnfaA with the ICCG-GFP fusion protein, experimental results demonstrated the effectiveness of this signal peptide in secretion expression. MnfaA provides a valuable tool for projects requiring enhanced protein secretion, especially for recombinant protein expression in synthetic biology and industrial applications.

Profile

Name：MnfaA25
Base Pairs: 84
Origin: Thermobifida fusca
Properties: Mainly used to guide target proteins through the inner membrane for secretion to the extracellular space or the periplasmic space, the H region of the MnfaA signal peptide has been optimized to have greater hydrophobicity and an appropriate length under specific conditions.

Usage and Biology

MnfaA is a signal peptide that has been modified and optimized based on the nfaA signal peptide, aimed at improving the secretion efficiency of target proteins. In our experiment, MnfaA was added to the N-terminus of the ICCG-GFP fusion protein, and fluorescence intensity was used to characterize ICCG secretion levels. Although the original goal was to enhance ICCG secretion, the experimental results showed that MnfaA did not fully achieve the expected improvement. Nevertheless, this design provided valuable insights for further optimization of the signal peptide.
MnfaA is derived from the nfaA signal peptide of Thermobifida fusca, with its H region optimized to increase hydrophobicity and appropriate length for better guidance of target protein translocation. In the experiment, MnfaA was added to the N-terminus of the ICCG-GFP fusion protein to guide ICCG secretion. While there was some improvement in secretion, it did not fully meet the expected enhancement. This indicates that MnfaA has potential, but further adjustment and optimization may be needed in specific expression systems to achieve the desired secretion efficiency.

Design Notes

We used PCR amplification to obtain a linearized vector and seamless cloning to obtain recombinant plasmids carrying the mutated nfaA signal peptide. The recombinant plasmids was then transformed into the Escherichia coli strain DH5α. We then sequenced the amplified recombinant plasmid and obtained the correctly constructed recombinant plasmid.

Characterization

To characterize the activity of the MnfaA signal peptide, we constructed an ICCG-GFP fusion protein, using fluorescence intensity as a quantitative measure of expression. The following steps were taken to characterize the mutants:
- The constructed MnfaA signal peptide mutant plasmids were transformed into Escherichia coli BL21 (DE3) expression strains.
- The transformed strains were cultured in a high-throughput screening system, and the expression levels of different mutants were compared by measuring fluorescence/OD600.

The high-throughput screening system is as follows:
Induce Temperature (25℃), Induce Time (12 h), Concentration of IPTG (1.0 mM)
1.  All the signal peptides and promoters mutants which we had designed were constructed on pET26-ICCG-GFP and were transformed into BL21 (DE3), and were selected for screening.
2. The above BL21 (DE3) strains containing recombinant protein-encoding plasmids were selected and grown in 96-deep-well plates with 600 μL of LB liquid medium with corresponding resistance per well for 6 h at 37 °C.
3.  Then, 8 μL of the bacterial culture were transferred into 96-deep-well plates with 800 μL of LB liquid medium per well and grown for 6 h at 37 °C.
4.  100 μL samples were transferred into transparent 96-well microplates for OD600 measurements before the inducer was added.
5.  Then, 7 μL inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) for each wells was added to a final concentration of 1.0 mM, and the cultivation was continued at 25℃ for 12 h.
6.  Take 100 μL from each well add to test the OD600 before add the inducer.
7.  100 μL samples were transferred into transparent 96-well microplates for OD600 measurements and 100 μL samples were transferred into 96-well black microplates for GFP fluorescence measurement.
8.  The fluorescence intensity was measured using an excitation wavelength of 485 nm and an emission wavelength of 520 nm by using EnVision Multilabel Reader.
The secretion levels of ICCG were characterized by the fluorescence intensity of the ICCG-GFP fusion protein. The experimental data showed that while the MnfaA signal peptide did slightly enhance ICCG secretion, the secretion level did not achieve the expected significant improvement. Compared to the control group, the fluorescence signal exhibited only minor changes, indicating that MnfaA's effect on improving ICCG secretion efficiency was limited.

Figure 1. Normalized fluorescence intensity characterization of the mutants.

Although MnfaA did promote ICCG secretion to some extent, it did not show significant improvement compared to the original nfaA signal peptide. This suggests that MnfaA may require further optimization, particularly in the context of the Escherichia coli expression system, where unforeseen factors may be limiting its efficiency.

Although the experimental results indicated that MnfaA did function as a signal peptide, its performance in this experiment did not meet the goal of significantly enhancing ICCG secretion. Further optimization may be necessary, particularly in adjusting the signal peptide's amino acid sequence, protein folding, or host system selection.

In summary, while MnfaA showed some potential, the experimental results indicated room for improvement in secretion efficiency. Future experiments will focus on optimizing the structure of the signal peptide and expression conditions.

Reference

1.Zhang, S., Liu, D., Mao, Z., Mao, Y., Ma, H., Chen, T., Zhao, X., & Wang, Z. (2018). Model-based reconstruction of synthetic promoter libraryin Corynebacterium glutamicum. Biotechnology Letters, 40(5), 819–827.
2.Van Brempt, M., Clauwaert, J., Mey, F., Stock, M., Maertens, J., Waegeman, W., & De Mey, M. (2020). Predictive design of sigma factor-specific promoters. Nature Communications, 11, 5822.
3.Li, Z.-J., Zhang, Z.-X., Xu, Y., Shi, T.-Q., Ye, C., Sun, X.-M., & Huang, H. (2022). CRISPR-based construction of a BL21 (DE3)-derived variant strain library to rapidly improve recombinant protein production. ACS Synt, 11(1), 352-352.