magistrsko delo
Tadej Ulčnik (Author), Helena Gradišar (Mentor), Miha Pavšič (Thesis defence commission member), Marko Novinec (Thesis defence commission member), Marko Dolinar (Co-mentor)

Abstract

Načrtovanje in priprava novih bionanomaterialov predstavljata pomembno področje hitro razvijajoče se nanotehnologije. Molekularno samosestavljanje nam omogoča pripravo novih proteinskih nanomaterialov, ki so pametni, prilagodljivi, biokompatibilni in biološko razgradljivi. Nov tip načrtovanih proteinskih nanostruktur, ki se samosestavijo iz ene polipeptidne verige, predstavlja izjemen potencial za različne aplikacije. Polipeptidna veriga je sestavljena iz več med seboj povezanih peptidnih modulov. Takšna modularna zgradba je zelo primerna za uporabo metode sestavljanja Golden Gate, ki omogoča ligacijo več delov oziroma modulov DNA naenkrat. Metoda temelji na uporabi restrikcijskih endonukleaz tipa IIs in DNA-ligaze T4. Tovrstni restrikcijski encimi režejo DNA izven svojih prepoznavnih mest, kar omogoča rezanje več različnih zaporedij DNA z uporabo enega encima. Na ta način se skrajša čas priprave konstruktov, saj poteka celoten proces v eni stopnji. Z načrtovanim vstavljanjem prepoznavnih mest za restrikcijske encime pa lahko določimo tudi vrstni red modulov. Pripravili smo zapis za protein s trikotno strukturo z molekulami fluorescenčnega proteina na ogliščih. Protein je sestavljen iz devetih modulov v definiranem vrstnem redu. Šest modulov tvori tri pare ovitih vijačnic, ki predstavljajo stranice trikotnika, trije moduli pa vsebujejo zapis za fluorescenčni protein. Vsako stranico predstavlja homodimerna ovita vijačnica, dolga 15 heptad aminokislinskih ostankov. Uporabili smo nekoliko spremenjena zaporedja naravnih homodimerov tropomiozina, Rho-kinaze ter korteksilina. Struktura ovite vijačnice zagotavlja strukturno stabilnost proteina, hkrati pa omogoča in vivo samosestavljanje proteina do načrtovane geometrijske oblike. Z načrtovanjem nukleotidnega zaporedja je bilo predhodno pripravljenih devet modulov, vsak velikosti od 300 do 700 baznih parov, z vezavnima mestoma za restrikcijsko endonukleazo tipa IIs BsaI. Z uporabo metode Golden Gate nam je uspelo izrezati in v pravilnem zaporedju ligirati v končni vektor okoli 4000 baznih parov dolg zapis iz devetih, v določenem vrstnem redu povezanih modulov. Izvedli smo tudi optimizacijo metode Golden Gate. Metodo smo želeli izboljšati do te mere, da bi se lahko uporabljala za sestavljanje zapisov iz več kot 12 modulov. Optimirali smo reakcijske pogoje, pri čemer smo spreminjali količino encimov ligaze in BsaI ter ATP. Preverili smo več različnih pristopov in najuspešnejši je bil pristop s predhodnim sestavljanjem krajših segmentov v več ločenih reakcijah. V tem primeru v ločenih reakcijah najprej med seboj sestavimo krajše segmente, ki jih nato združimo, ligiramo in vstavimo v vektor. Na ta način nam je uspelo pripraviti zapisa za proteina, ki sta sestavljena iz 12 oziroma 18 modulov.

Keywords

proteini;proteinske strukture;modularnost;proteinske nanokletke;ovite vijačnice;Golden Gate;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [T. Ulčnik]
UDC: 577.112(043.2)
COBISS: 1538446019 Link will open in a new window
Views: 592
Downloads: 141
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Other data

Secondary language: English
Secondary title: Construction of vectors encoding protein nanostructures using the Golden Gate assembly method
Secondary abstract: Designing of new bionanomaterials represents a very important field in nanotechnology, which is rapidly developing. Molecular self-assemby enables design of new artificial protein nanomaterials that are smart, adaptable, biocompatible and biodegradable. The new way of designing protein structures that self-assemble from a single polypeptide chain has high potential for many different applications. A single polypeptide chain is composed of many interconnected peptide modules. Golden Gate method is very useful for assembling modular structures of this kind, as it enables ligation of several modules in the same reaction. It uses type IIs restriction endonucleases and T4 DNA-ligase. Restriction enzymes of this type cut DNA outside of ther recognition site, which enables cutting different DNA sequences using only one restriction enzyme. The construction of DNA sequences is therefore faster, as the whole process takes place in a single reaction. With correct designing of restriction endonuclease recognition sites we can also determine the order in which the modules are assembled. We designed a sequence for a triangle-shaped protein with a fluorescent protein at the vertices. The designed protein is composed of nine modules that are interconnected in a defined order. Six modules are forming three pairs of coiled-coils that represent sides of the triangle. The remaining three modules contain sequences encoding fluorescent proteins. Each side represents a 15 heptad long coiled-coil homodimer. We used slightly modified sequences from natural homodimers tropomyosin, Rho-kinase and cortexillin. Coiled-coil structure ensures structural stability of the protein and enables in vivo self-assembly to the designed geometrical shape. We designed nucleotide sequence of nine modules, each between 300 and 700 nucleotides in length, that contain restriction endonuclease BsaI recognition sites. With the use of Golden Gate method we successfully cut and ligated in final vector on around 4000 nucleotides long sequence, that is composed of 9 modules arranged in a defined order. Additionally, we optimized the Golden Gate assembly method so that it could be used for assembly of more than 12 modules. We optimised the reaction prameters, which included the concentration of enzymes (DNA ligase and BsaI), and the concentration of ATP. We tested different approaches and the most successfull one involved preliminary assembly of shorter sequences in separate reactions. These partial constructs are in a second step joined together and ligated into the target vector. Using this approach we succeded to assemble the sequence encoding proteins composed of 12 and 18 modules, respectively.
Secondary keywords: Golden gate;modularity;coiled-coils;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 68 str.
ID: 11231234