Part:BBa_K5101000
Antimicrobial peptide discoverd from the Lactobacilli in the human vaginal flora
Antimicrobial peptide ORF
Usage and Biology
Antimicrobial peptides (AMP) are a large class of active oligopeptides with antimicrobial properties against harmful life forms (or pathogens) such as bacteria, fungi, parasites and viruses. In addition to their antimicrobial ability, some of these peptides can directly or indirectly participate in the immunomodulation of the host and protect the host from infection. Because they generally have a sufficient amount of positive charge and are often accompanied by hydrophobicity, they can bind to negatively charged biological membranes under electrostatic force, penetrate and destroy the membrane structure to cause cell death. Different from the traditional antibiotic single-target sterilization principle, antimicrobial peptide can carry out multi-target destruction in pathogens, which can greatly reduce the production of drug-resistant bacteria, and has a broad-spectrum antimicrobial properties, which is one of the best choices for replacing antibiotics in the future. In the current global situation, antimicrobial peptides have been used in many clinical treatment cases in bacterial infection, wound healing and cancer treatment. With the development of synthetic biology and biomedical engineering and other interdisciplinary disciplines, the molecular design and biosynthesis of antimicrobial peptides will have more ideas and technical support. Imbalance of intestinal flora and loss of tolerance to local microbiota are possible causes of inducing chronic enteritis. Therefore, regulating the balance of intestinal flora can help alleviate intestinal inflammation and improve the therapeutic effect of chronic enteritis.
We constructed suitable engineered bacterial strains to characterize a novel antimicrobial peptide newly isolated from Lactobacillus from the human vaginal flora in our Host lab (unpublished data). The antimicrobial peptide has been shown to have some bacteriostatic properties after validation with our constructed engineered bacteria. It has a wide range of bacteriostatic activity against Gram-positive and Gram-negative bacteria.
Contents
Plasmid construction of a novel antimicrobial peptide AMP
Constitutive plasmid construction
We obtained a novel antimicrobial peptide (AMP) from our host lab, and sent it to the company for gene synthesis to obtain a usable AMP gene sequence. We selected Escherichia coli BL21 (DE3) and strong constitutive promoter J23119 as chassis cell and promoter to enable long-term and efficient antimicrobial peptide expression. On this basis, we will isolate and purify the AMP expressed by our constructed engineered strains and use them as biological agents.
Based on these design principles, we designed plasmid pET29a-pJ23119-RBS-AMP-T7, as shown in Figure 1-1. Using homologous recombination integration, expression plasmid pET29a-pJ23119-RBS-AMP-T7 was constructed and transformed into DH5α. We picked several single colonies of E. coli on the transfected plates and then extracted the recombinant plasmid and performed PCR to verify that the primers were specific and the target fragment was 3275 bp, and the results are shown in Figures 1-2. We sent the plasmids with the correct positions of the bands to GENEWIZ Co. for sequencing, and the results of the sequencing were correct. The recombinant plasmid pET29a-pJ23119-RBS-AMP-T7 was successfully constructed. Sequence and Features
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