Kloniranje mutiranih različic antitoksina 1067 iz cianobakterije Microcystis aeruginosa PCC 7806.

Nemanič, Vivian (avtor), Klemenčič, Marina (mentor)

Sistemi toksin-antitoksin (TA) so genetski elementi, ki so sestavljeni iz zapisa za toksin in za kompatibilen antitoksin ter so pogosto prisotni pri bakterijah in arhejah. Sestavljeni so iz toksina, ki inhibira rast celic, ter iz antitoksina, ki onemogoča delovanje toksina. Najbolj pogosti in najbolje okarakterizirani sistemi TA so sistemi tipa II, pri katerih sta toksin in antitoksin proteina in skupaj tvorita tesen kompleks. Microcystis aeruginosa je cianobakterija, ki naseljuje raznolike ekološke niše in v okolju zelo dobro uspeva. To ji omogočajo različni genetski elementi, med katere spadajo tudi sistemi toksin-antitoksin. En par sistema TA tipa II v M. aeruginosa je zanimiv zato, ker je v genomu M. aeruginosa seva PCC 7806 v neposredni bližini gena, ki zapisuje za eno izmed šestih ortokaspaz, ki so prisotne pri tem sevu, MaOCA-Ia (prej MaOC1). Domnevni par TA je sestavljen iz toksina, ki je podoben toksinom družine RelE/ParE, ter iz sorodnega antitoksina. MaOCA-Ia je proteolizno aktivna proteaza, ki preferenčno cepi za skupki bazičnih aminokislinskih ostankov. Takšna zaporedja najdemo tudi pri antitoksinu 1067, in sicer sta v aminokislinskem zaporedju dve regiji, ki vsebujeta skupke dveh oziroma treh argininov. V sklopu te diplomske naloge nas je zanimalo, ali MaOCA-Ia cepi antitoksin 1067 za omenjenima dvema regijama v aminokislinskem zaporedju. Ker tega predhodno ni bilo mogoče ugotoviti s protitelesi proti oznaki His6, smo cepitev poskušali dokazati tako, da smo pripravili mutirane antitoksine na ravni DNA. Predhodno je bilo torej potrebno divjemu tipu antitoksina 1067 spremeniti zaporedje, kar smo naredili tako, da smo obe omenjeni regiji mutirali. Po indukciji izražanja smo pridobili mutirane antitoksine, ki so nam omogočili analizo cepitvenih mest. V sklopu tega diplomskega dela smo pripravili zapise za tri mutirane različice antitoksina 1067 z inverzno verižno reakcijo s polimerazo (PCR). Pravilnost zaporedij z uvedenimi mutacijami smo preverili s sekvenciranjem in kasneje z bioinformacijsko analizo zapisov.

• Jezik: Slovenski jezik
• Ključne besede: Microcystis aeruginosa PCC 7806, antitoksin, toksin, MaOCA-Ia
• Vrsta gradiva: Diplomsko delo/naloga
• Tipologija: 2.11 - Diplomsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-138966
• COBISS.SI-ID: 120808451
• Datum objave v RUL: 26.08.2022

NEMANIČ, Vivian, 2022, Kloniranje mutiranih različic antitoksina 1067 iz cianobakterije Microcystis aeruginosa PCC 7806. [na spletu]. Diplomsko delo. [Dostopano 4 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=138966

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Cloning of mutated variants of the 1067 antitoxin from the cyanobacterium Microcystis aeruginosa PCC 7806.

Izvleček

Toxin-antitoxin (TA) systems are genetic elements encoding a toxin and a compatible antitoxin, and are commonly present in bacteria and archaea. They consist of a toxin, that inhibits cell growth, and an antitoxin, that disables the action of the toxin. The most common and best characterised TA systems are type II systems, in which both the toxin and antitoxin are proteins which form a tight complex. Microcystis aeruginosa is a cyanobacterium that inhabits diverse ecological niches and thrives very well in the environment. This is made possible by various genetic elements, including toxin-antitoxin systems. One pair of type II TA systems in M. aeruginosa is especially interesting, because in the genome of strain PCC 7806 it is located in close proximity to the gene that codes for one of the six orthocaspases present in this strain, MaOCA-Ia (formerly MaOC1). The putative TA pair consists of a toxin similar to those of the RelE/ParE family of toxins, as well as the cognate antitoxin. MaOCA-Ia codes for a proteolytically active protease, that preferentially cleaves after clusters of basic amino acid residues. Such sequences are also found in antitoxin 1067, with two regions containing two and three arginine clusters, respectively. As a part of this thesis, we were interested in whether MaOCA-Ia cleaves antitoxin 1067 after these two regions. As this could not be previously confirmed with antibodies, we attempted to demonstrate cleavage by preparing mutant antitoxins. Therefore, mutations had to be intoduced into the DNA sequence of the wild-type antitoxin 1067. After expression of the mutant forms and their isolation, we were able to analyse the cleavage sites. In the context of this thesis, we prepared at the DNA level three different mutant versions of antitoxin 1067 by inverse polymerase chain reaction (PCR). The correctness of the sequences with the introduced mutations was verified by sequencing and by bioinformatic analysis of the sequences.

• Ključne besede: Microcystis aeruginosa PCC 7806, antitoxin, toxin, MaOCA-Ia