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Mlečnokislinske bakterije so grampozitivne bakterije, ki spremljajo ljudi že več stoletij in jih Svetovna zdravstvena organizacija priznava kot varne. Pojavljajo se kot sestavine prehrane, kot probiotiki, poleg tega so sestavni del posameznikove črevesne mikrobiote. S pomočjo genskega inženiringa lahko mlečnokislinske bakterije izkoristimo kot vektorje za dostavo antigenov, rekombinantnih proteinov in dostavo DNA z namenom cepljenja ali nevtralizacije vnetnih mediatorjev ter toksinov. Modelna bakterija Lactococcus lactis služi tudi kot gostiteljski organizem za izražanje rekombinantnih proteinov. V ta namen se pri bakteriji L. lactis najpogosteje uporablja z nizinom nadzorovan sistem za izražanje proteinov (NICE), ki ga je možno nadgraditi. Za izražanje rekombinantnih proteinov so namreč potrebni sodobni, izboljšani plazmidni vektorji. Med možnimi izboljšavami je zmožnost enostavne vstavitve več genov s ciljem hkratnega izražanja več proteinov. V raziskovalnem delu smo sistem NICE prilagodili za sočasno izražanje dveh proteinov. Uporabili smo plazmide pNZ8148, pJET1.2 in pGEM. Izbrane gene smo izrezali z restrikcijskimi encimi, jih ločili z agarozno elektroforezo in jih z ligacijo združili z genskimi konstrukti za izražanje željenih proteinov. Tako pripravljene laktokokne plazmide smo s tehniko elektroporacije vnesli v bakterijo L. lactis NZ9000. Pridobili smo plazmid pNZDual, ki vsebuje dva nizinska promotorja in dve klonirni mesti, plazmid pNZDualTT, pri katerem je plazmidu pNZDual dodan še en transkripcijski terminator ter plazmid pNZPolycist, ki vsebuje en sam nizinski promotor in dve klonirni mesti, ločeni z ribosom-vezavnim mestom. V novo nastale plazmide smo vstavili gene za dva modelna proteina, in sicer infrardeči fluorescenčni protein irfp713 (angl. infrared fluorescent protein IRFP) in humani IgG-vezavni načrtovani protein z ankirinskimi ponovitvami (angl. designed ankyrin repeat protein DARPIN) I07. Gene smo vstavili v plazmide v vseh možnih zaporedjih z namenom ocene vpliva na količino izražanja. Vrednotenje rezultatov je bilo opravljeno z meritvami absorpcije in fluorescence ter z metodama celične ELISE in pretočne citometrije. Ugotovili smo, da plazmid pNZDual z dvema promotorjema omogoča enakomerno izražanje obeh izbranih modelnih proteinov. S tem smo razvili novo serijo plazmidov za napredno gensko spreminjanje mlečnokislinske bakterije L. lactis. 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. |
