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During July 3rd - 7th our Research & Development team participated in the Viruses of Microbes 2023, which took place in Tbilisi, Georgia. Our colleague Ma?gorzata Paszkiewicz presented the results of one of the recently conducted studies on the Escherichia coli prophage sequence deletion with Lambda RedBCD recombination system.

Introduction: In the post-antibiotic era bacteriophages are one of the most promising alternatives in human and animal infections treatment. For effective and safe phage multiplication an appropriate bacterial host must be selected, preferentially without prophage sequences in the genome. As prophage can excise and form an active bacteriophage, often containing some bacterial genes, the elimination of all potentially active prophages from host genome is significant, although challenging.?

Objective: The aim of the study was to delete a prophage sequence from bacterial genome in one step recombination.

Methodology: In the study E. coli strain (own collection) has been analyzed towards the presence of prophages. One potentially active 49862 bp prophage with integrase gene, crucial in phage integration and excision, was identified. Three separate knock-outs of different length have been made via Lambda RedBCD recombination. The method utilizes recBCD genes, antibiotic cassette with flippase recognition target (FRT) sites and flippase gene to induce homologous recombination. Primers complementary to the antibiotic cassette with 50-base overhangs, flanking the prophage sequence, were designed.

Results: In the first step the chosen fragment was deleted by antibiotic cassette insertion. Next,?the cassette was excised by flippase, recognising FRT sites. The prophage fragment deletion?was confirmed by PCR reaction with short primers flanking the region. Subsequently, genomic DNA of selected mutants has been sequenced (Illumina MiniSeq). All mutants have the correct mutation confirmed: integrase gene (1388 bp), 15793 bp or 38417 bp fragment deletion.

The conclusion: Lambda RedBCD has been proven as an appropriate way to remove prophage sequences from bacterial genome.

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Authors: Malgorzata Paszkiewicz, Magdalena Antczak, Anna Kawa, Arkadiusz Guziński , Patrycja Sowińska , Karolina Pospiech , Joanna Kazimierczak , Ewelina Wójcik

Proteon Pharmaceuticals is a leader in bacteriophage (phage) technology for livestock farming and aquaculture. Our mission is to eliminate the need for unnecessary antibiotic use, reducing the risk of antimicrobial resistance (AMR), as well as to increase the sustainability of protein production through the reduction of waste and improvement of on-farm efficiency. Our products function by modulating the microbiome enabling prophylactic health. We have created a precision phage product development platform using -omics technologies, molecular biology, bioinformatics, and artificial intelligence (AI) to create effective, reliable, and safe antibacterial solutions for animal and human health.