Comparison of sacB gene expression depending on applied plasmid type

During September 18th - 23rd our Research & Development team participated in the International Symposium on Plasmid Biology 2022, which took place in Toulouse, France. Our colleague Anna Kawa presented the results of one of the recently conducted studies on the Comparison of sacB gene expression depending on applied plasmid type.

The aim of the work was to compare levansucrase gene expression in Escherichia coli from three various plasmids, which differ in origin of replication and copy number. Tested gene has been used as a negative selection marker for engineering E. coli bacteria.??

Methods: E. coli TOP10 and E. coli wild type strain (from Proteon Pharmaceuticals collection) electrocompetent cells were transformed with high copy or low copy number plasmids including levansucrase gene and antibiotic resistance gene (positive selection marker). Transformants carrying desired plasmids were grown in medium supplemented with proper antibiotics. Then, serial dilutions were performed and cells were plated on minimal agar medium and agar medium supplemented with 5% sucrose (negative selection) simultaneously. Two different E. coli strains were used to exclude or confirm strain-specific differences in the expression of analysed gene.?

Results: No noticeable distinction in the levansucrase gene expression level between two tested E. coli strains has been observed. Visible differences in the level of?tested gene expression between analysed plasmids were noticed. The greatest reduction of colony number on 5% sucrose agar was observed in E. coli TOP10 transformed with one of the high-copy number vector. Another high-copy number vector showed slightly lower, but still easily detectable reduction of colony number on selective medium. The low copy number plasmid was characterized as the least efficient in bacterial growth reduction.??????????????????????????????????????????????????????????????????????????

The conclusion:

Type of plasmid used for gene expression determined the marker efficiency in E. coli cells. The use of stronger promoter pose to be necessary to use the levansucrase gene selection in E. coli modification.

Authors: Anna Kawa, Ma?gorzata Paszkiewicz, Magdalena Antczak, Arkadiusz Guziński , Jaroslaw Dastych

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.