doctoral candidate from 01.01.2023 to 01.01.2026
Russian Federation
student from 01.01.2022 to 01.01.2026
VAK Russia 4.1.2
VAK Russia 4.1.3
VAK Russia 4.1.4
VAK Russia 4.1.5
VAK Russia 4.2.1
VAK Russia 4.2.2
VAK Russia 4.2.3
VAK Russia 4.2.4
VAK Russia 4.2.5
VAK Russia 4.3.3
VAK Russia 4.3.5
UDC 579.25
UDC 579.258
The aim of the study is to develop a highly efficient method for the synthesis and assembly of long codon-optimized genetic constructs for subsequent co-expression in probiotic bacterial strains. From 2024 to 2025, comprehensive studies were conducted at the Genome Center of the North Caucasus Federal University to develop a highly accurate method for gene synthesis and codon optimization for efficient co-expression of cytokines in probiotic strains of Lactobacillus spp. The objects of the study were the nuc-leotide sequences of the bovine cytokine genes IL-10 (537 bp) and IL-22 (522 bp). Bioinformatics codon optimization was performed using the GeneOptimizer program with adaptation to the codon usage profile of Lactobacillus spp., correction of the GC composition (52–55 %), and elimination of unstable elements. Three assembly methods were compared: classical phosphoramidite synthesis, hybrid assembly (similar to Gibson Assembly), and an optimized method based on the GeneOptimizer platform with integrated HPLC purification. The results of a comprehensive analysis showed that all three methodological approaches were capable of producing full-length target genetic constructs, but demonstrated significant and statistically significant differences in key parameters determining the practical applicability of the methods. These parameters included synthesis accuracy (error rate), process speed (assembly time), and overall full-length product yield, which together form an integrated performance indicator for each technology. The optimized method demonstrated maximum accuracy – 1 error per 10,000 nucleotides – with an assembly time of 16 hours and a full-length product yield of 95 %. The classical and hybrid methods had yields of 45 % and 55 %, with error rates of 1/500 and 1/2000 nt, respectively. The integration of bioinformatic design with automated synthesis and purification creates a high-precision platform for producing genetic constructs that ensure stable coexpression of immunomodulators in probiotic Lactobacillus spp. strains for veterinary applications.
codon-optimized gene synthesis, cytokine co-expression, probiotic strains of Lactobacillus spp., interleukins IL-10 and IL-22, high-precision synthesis, GeneOptimizer platform, automated gene assembly
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