Part:BBa_K4817004
OmpA-Bac
Biology and Usage
This part is composed of the signal peptide OmpA from Escherichia coli and the antibacterial peptide Bactenecin from bovine neutrophils. The antibacterial peptide Bactenecin has an inhibitory effect on Gram negative bacteria such as sulfate reducing bacteria1,2. The signal peptide OmpA can transport the antibacterial peptide to the extracellular space3, and the two parts are connected by amino acids A-S-A, which is the cleavage site of the signal peptidase, allowing the antibacterial peptide to be cut off from the fusion protein without affecting its original function. We have our engineered Escherichia coli express the OmpA-Bac fusion protein to inhibit the growth of sulfate reducing bacteria in the sewer.| OmpA-Bac | |
| Function | Inhibit sulfate reducing bacteria |
| Use in | Gram negative bacillus |
| Backbone | pET28a |
| Derived from | Bovine neutrophils |
Design and Properties:
Efficient expression of extracellular antimicrobial peptide bactenecin expression elements induced by IPTG under the regulation of T7 promoter.The signal peptide OmpA fused at the N-terminus of bactenecin cleaves and separates from each other at the C-terminus ASA sequence of OmpA during the process of assisting bactenecin exocrine secretion.
Figure 1 The inhibitory effect of bactenecin on Escherichia coli at different concentrations
As shown in Figure 1, we extracted the supernatant of engineering bacteria that secrete extracellular Bactenecin and treated E. coli with three different concentration gradients of 1, 2/3, 1/3 (corresponding to Bac-1, Bac-2, and Bac-3, respectively). The culture supernatant of E. coli BL21 was used as a negative control, ampicillin antibiotic treatment was used as a positive control, and E. coli cultured in LB medium was used as a blank control group.The experimental results showed that the antibacterial peptide Bactenecin was successfully secreted into the culture medium system, and the antibacterial effect increased with increasing concentration.
Figure 2 The inhibitory effect of antibacterial peptide Bactenecin on the growth of SRB (Sulfate Reducing Bacteria: Desulfovibrio vulgaris)
As shown in Figure 2, we investigated the inhibitory effect of antimicrobial peptide Bactenecin on SRB. The concentration of the concentrated Bactenecin treatment group was twice that of the normal Bactenecin group. We completed the concentration process using a vacuum centrifuge concentration instrument and a freeze-drying machine. The experimental results showed that the treatment effect of the concentrated antimicrobial peptide Bactenecin was better than that of the normal concentration group.
Figure 3 In order to investigate whether the secretion of antimicrobial peptides will affect the growth of Escherichia coli BL21, we added IPTG to the engineering bacterial culture medium to induce the expression of antimicrobial peptides. The OD600 of the bacterial solution was measured at regular intervals, compared with the engineering bacteria that did not secrete antimicrobial peptides or the Escherichia coli BL21 strain that did not introduce plasmid vectors. The results showed that adding IPTG induction would slightly slow down the growth rate of bacteria, and the impact on bacterial growth was not significant.
Experimental approach:
1. Transform the plasmids into E. coli BL21 competent cells.2. The engineered bacteria are cultured in 60mL LB medium overnight at 37℃, 250rpm;
3. When the OD600 of the culture medium reaches 0.6, add 1M IPTG (1:1000) and induce at 16 ℃ for 12 hours at 150 rpm.
4. Escherichia coli DH5 that has been cultured to OD600 within the range of 0.6-0.8 α The strain culture solution was used as the test bacterial solution, and 3ml of each tube was separately packed into centrifuge tubes at 12000 rpm, 4 ℃, and centrifuged for 3 minutes. Discard the supernatant and retain the bacterial sediment.
5. Configure the experimental group system according to different concentration gradients, add the supernatant of engineering bacteria according to the ratio requirements of different concentration groups (extraction conditions are the same as the treatment conditions of the test bacterial solution), and fill it with LB culture medium to 6ml.
6. Use a spectrophotometer to measure at OD600 every 1 hour, a total of five times (0h, 1h, 2h, 3h, 4h), plot curves, and observe changes in the growth of Escherichia coli.
References
1. Stillger, L., Viau, L., Kamm, L., Holtmann, D. & Müller, D. Optimization of antimicrobial peptides for the application against biocorrosive bacteria. Appl Microbiol Biotechnol 107, 4041–4049 (2023).2. Jayaraman, A., Mansfeld, F. B. & Wood, T. K. Inhibiting sulfate-reducing bacteria in biofilms by expressing the antimicrobial peptides indolicidin and bactenecin. Journal of Industrial Microbiology and Biotechnology 22, 167–175 (1999).
3. Pechsrichuang, P. et al. OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. SpringerPlus 5, 1200 (2016)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 64
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |