Search

Bionanotehnologija je obsežno področje, ki dobiva v družbi zaradi številnih gospodarskih, okoljskih, zdravstvenih in socialnih razlogov vedno več pozornosti. Pomemben del bionanotehnologije predstavlja razvoj novih materialov, ki bi jih lahko uporabili za različne namene. S sintezno biologijo, s katero uvajamo v biotehnologijo inženirske pristope, lahko na osnovi naravnih proteinskih gradnikov in takšnih, ki ne obstajajo v naravi, načrtujemo in pripravljamo popolnoma nove bionanomateriale. Če želimo pripraviti bionanostrukture, ki se same sestavijo v načrtovano strukturo, je potrebno najprej natančno načrtovati gradnike, ki bodo sestavljali načrtovano nanostrukturo ali material, nato pa te osnovne gradnike pridobiti in okarakterizirati, na kar smo se osredotočili v tem magistrskem delu. Polipeptidne gradnike 34cald56, različico 34cald56 s fluorescenčnim proteinom in 3APH4 smo pripravili za namene gradnje dveh načrtovanih dvodimenzionalnih proteinskih mrež, ki imata različno geometrijo in se samosestavita iz enega polipeptidnega gradnika. Vse tri proteine smo uspešno pripravili v bakteriji E. coli, izolirali in očistili ter okarakterizirali z biokemijskimi metodami. Potrdili smo, da je sekundarna struktura polipeptidnih gradnikov 34cald56 in 3APH4 v skladu z načrtovano. Iz podatkov meritev cirkularnega dikroizma smo zaključili tudi, da je temperaturna stabilnost proteina 34cald56 višja od temperaturne stabilnosti 3APH4. Z meritvami dinamičnega sipanja svetlobe smo ugotovili, da se zvita polipeptida 34cald56 in 3APH4 v raztopini samosestavita v dimere in večje nanostrukture, ki postanejo netopne in se oborijo iz raztopine, lahko pa se sestavita tudi v topne nanostrukture, ki so v primeru 34cald56 velike približno 120 nm, v primeru 3APH4 pa 60 nm. Bionanotechnology is a broad field that is receiving increasing attention in society for many economic, environmental, health and social reasons. An important part of bionanotechnology is the development of new materials that could be used for various purposes. With synthetic biology, which introduces engineering approaches to biotechnology, we can design and prepare completely new bionanomaterials based on natural protein building blocks and those that do not exist in nature. In order to prepare bionanostructures that self-assemble into the planned structure, it is necessary to first carefully design the building blocks for the planned nanostructure or material, and then to acquire and characterize these basic building blocks, which is the focus of this master's thesis. The 34cald56, the 34cald56 version with fluorescence protein and the 3APH4 polypeptide building blocks were prepared for the purpose of constructing two designed two-dimensional protein lattices having different geometries and self-assembling from one polypeptide building block. All three proteins were successfully produced in bacteria E. coli, isolated and characterized with biochemical methods. We have confirmed that the secondary structure of the 34cald56 and the 3APH4 polypeptide building blocks is in accordance with the designed. From the data of the circular dichroism measurements, we concluded that the temperature stability of the protein 34cald56 is higher than the temperature stability of the 3APH4. Measurements of the dynamic light scattering have revealed that the folded polypeptides 34cald56 and 3APH4 in solution self-assemble into dimers and larger nanostructures that become insoluble and precipitate out of solution, but can also be assembled into soluble nanostructures, which in the case of 34cald56 are large around 120 nm, and in the case of 3APH4 about 60 nm.