Lactic acid bacteria are gram-positive bacteria that have been an integral part of human life for centuries and are recognized as safe by the World Health Organization. They appear as components of the human diet, as probiotics, and also as an integral part of the individual's intestinal microbiota. With genetic engineering, lactic acid bacteria can be used as vectors for the delivery of antigens, recombinant proteins and DNA for the purpose of vaccination or neutralization of inflammatory mediators and toxins. The model bacterium Lactococcus lactis also serves as a host organism for the expression of recombinant proteins. For this purpose, a nisin-controlled system (NICE) for protein expression which can be upgraded. Namely, modern, improved plasmid vectors are needed to express recombinant proteins. Among the possible improvements is the ability to easily insert multiple genes with the goal of simultaneously expressing multiple proteins. In the research work, we adapted the NICE system for simultaneous dual protein expression. Plasmids pNZ8148, pJET1.2 and pGEM were used. The selected genes were excised with restriction enzymes, separated by agarose electrophoresis and combined by ligation with gene constructs for the expression of the desired proteins. Lactococcal plasmids prepared in this way were introduced into the bacterium L. lactis NZ9000 using the electroporation technique. Plasmid pNZDual, which contains two nisin promoters and two cloning sites, plasmid pNZDualTT, in which another transcription terminator is added to plasmid pNZDual, and plasmid pNZPolycyst, which contains a single nisin promoter and two cloning sites separated by a ribosome-binding site, were obtained. Genes for two model proteins, namely infrared fluorescent protein irfp713 (IRFP) and human IgG-binding designed ankyrin repeat protein (DARPIN) I07, were inserted into the newly generated plasmids. Genes were cloned into plasmids in all possible combinations to evaluate the effect on the amount of expression. The results were evaluated by absorption and fluorescence measurements, as well as by whole-cell ELISA and flow cytometry. We found that the plasmid pNZDual with two promoters enables the equal expression of both selected model proteins. With this, we have developed a new series of plasmids for advanced genetic modification of the lactic acid bacterium L. lactis.
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