Difference between revisions of "Part:BBa K4248013"

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pET28a- T7 pro-6His -Sparamosin26-54-T7 ter

pET28a- T7 pro-6His -Sparamosin26-54-T7 ter

Contribution

Penicillin is the first anti-infective drug used in clinical practice in the world. After years of development, more and more antibiotics have sprung up, but the problem of drug resistance caused by the large-scale and large-scale use of antibiotics has gradually become prominent. . Antibacterial peptides are considered to have broad application prospects due to their high antibacterial activity, wide antibacterial spectrum, wide variety, wide selection range, and difficulty in producing resistance mutations in target strains. The most prevalent mechanism of action of antimicrobial peptides is through their direct activity on bacterial cell membranes. Briefly, antimicrobial peptide binding leads to disruption of membrane potential, alteration of membrane permeability, and leakage of metabolites, ultimately leading to bacterial cell death. Through literature research, we found a variety of other novel antimicrobial peptides, one of which is Sparamosinsub26-54. We uploaded its DNA sequence information and basic introduction information into the registry of standard biological parts, and did the corresponding molecular construction to provide more antimicrobial peptide choices for future iGEM teams.

Figure 1. The map of the recombinant plasmids pET28a(+)-Sparamosin26-54 .

Engineering Success

Sparamosin26-54 is the new antimicrobial peptide that was found at Scylla’s gonad. By continued research about this new peptide, it was found that the sparamosin1-25 and Sparamosin26-54 were good antibacterial to the fungi cryptococcosis that is a disease that fatality rate can reached 40%. This kind of antimicrobial peptide might be an important medicine in the future.

1. Construction of the antimicrobial peptide expression plasmids

We design the plasmid: The DNA sequence of the four antimicrobial peptide Sparamosin26-54, was inserted into the pET-28a(+) vector. We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure2.), and the plasmid was successfully constructed. So far, we have successfully obtained five recombinant plasmids, which were respectively on the pET28a(+) vector, which can be used to express antimicrobial peptide proteins.

Figure 2. The sequencing blast results of the recombinant plasmids pET28a(+)-Sparamosin26-54 .

2. Protein expression and purification

In order to obtain the five antimicrobial peptide proteins, we transferred the recombinant plasmids into E.coli BL21(DE3), expanded the culture in the LB medium, and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column purification to purify the antibacterial peptide protein we wanted. The concentration of Sparamosin26-54 protein was measured as:0.42mg/mL。 At this point, we got the five antimicrobial peptide protein solutions we wanted.

3. Antibacterial ability test

To confirm the ability of the our purified antimicrobial peptide to inhibit bacterial growth, we used E.coli DH5-alpha as bacteria, and antibiotics as a positive control for bacteriostatic test experiments. To better show the relationship between the concentration of antimicrobial peptides and the inhibition of bacterial growth, we added 100 μL of DH5α and 100 μL of different concentrations of the antimicrobial peptide to each of the five test tubes. Our five test tubes were filled with the antimicrobial peptide stock solution and diluted 1, 5, 25, 125, and 625 times solution, and repeated three times for each concentration to form the average data graph with error bars. The results showed that the antimicrobial peptide proteins had significant antibacterial effects at double dilution concentrations. A single antimicrobial peptide protein can be seen to inhibit the growth of about 60% of bacteria in the range of 5 to 25 times dilution. A detailed analysis of the antibacterial effect of each antimicrobial peptide is given below.

Figure 3. Test results of protein Sparamosin26-54 inhibiting bacterial growth.

We performed dilution of the antimicrobial peptide Sparamosin26-54 using the same procedure and operation. By comparing the height of the histograms and the margin of error, we learned that the antimicrobial peptide sterilization after 625-fold LB solution dilution was not competent because its height was higher than the image height of the negative control. The samples diluted by 125x and 5x were similar in sterilization ability, but the 25x was a bit worse than both. Among all models, the Sparamosin26-54 stock solution had the best antimicrobial effect, which could reach 76.20%.

Sequence and Features