Vzpostavitev sistema za spremljanje regulacije izražanja toksin-antitoksin modulov in vivo.

Živič, Zala (avtor), Hadži, San (mentor), Klemenčič, Marina (komentor)

Sistemi toksin/antitoksin so bakterijski in arhejski operoni, ki so namenjeni ohranjanju celične populacije. Zapisujejo za proteinski toksin, ki s svojim delovanjem vodi v celično smrt ali pa zastoj metabolizma in proteinski/RNA antitoksin, ki nasprotuje delovanju toksina. V okviru magistrskega dela smo se osredotočali na sistem tipa II higBA2. Vsebuje zapis za proteina toksin HigB2 in antitoksin HigA2. Predlagan model samoregulacije tega sistema toksin/antitoksin predvideva mehanizem uravnavanja preko razpoložljivosti neurejenega N-konca HigA2. V prisotnosti toksina HigB2 je ta vezan nanj kar ošibi vezavo HigA2 na DNA. Promotor phigB-2 se sprosti in izražanje operona je omogočeno. Model regulacije sistema je bil določen in vitro. Želeli smo sestaviti sistem za preverjanje modela in vivo s pomočjo reporterskega proteina mRFP1 pod nadzorom phigB-2 ter HigB2 in HigA2 pod ločenimi konstitutivnimi promotorji. V ta namen smo želeli z molekularno biološkimi metodami povečati nabor že narejenih vektorjev. Prav tako smo pripravili vektor s toksinom pod inducibilnim promotorjem. Sama restrikcija in mutageneza sta bili težavni. Pogosto se je z vektorja izgubil zapis za toksin, česar nismo želeli. Uspešno smo pridobili enega izmed vektorjev z zapisom za toksin in brez zapisa za antitoksin. Mutageneza ni bila uspešna. Sklepali smo, da je lahko težavna toksičnost toksina kljub temu, da se izraža njegova encimsko neaktivna mutanta. Razlog bi lahko bil v vezavi HigB2 na ribosom. Zato smo pripravili vektor s toksinom pod inducibilnim promotorjem, kjer pride do izražanja gena za toksin šele ob dodatku L-arabinoze. Izvedli smo preliminarne poskuse izražanja ter potrdili prisotnost proteina po indukciji s točkovnim nanosom. V sklopu tega dela smo torej uspeli pripraviti ustrezen nabor vektorjev, ki omogočajo izvebo indukcije in merjenje fluorescence mRFP1 z namenom karakterizacije pripravljenih sistemov in vivo.

• Jezik: Slovenski jezik
• Ključne besede: toksin/antitoksin, regulacija izražanja, higBA2, HigB2, HigA2
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2020
• PID: 20.500.12556/RUL-118580
• COBISS.SI-ID: 27029763
• Datum objave v RUL: 27.08.2020

ŽIVIČ, Zala, 2020, Vzpostavitev sistema za spremljanje regulacije izražanja toksin-antitoksin modulov in vivo. [na spletu]. Magistrsko delo. [Dostopano 6 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=118580

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Establishing a tracking system for expression regulation of toxin-antitoxin modules in vivo.

Izvleček

Toxin/antitoxin systems represent bacterial and archaeal operons meant to preserve cell populations. They encode for protein toxins which can lead to cell death or stop cell metabolism and protein/RNA antitoxins which counteract the effect of toxins. In this work we focused on the type II toxin/antitoxin system higBA2. It encodes for a protein toxin HigB2 and a protein antitoxin HigA2. A suggested model for the regulation of the higBA2 system predicts a mechanism involving the availability of HigA2’s intrinsically disordered N-tail. In the presence of HigB2 the N-tail binds to it and therefore weakens the HigA2-DNA interaction. The promotor phigB-2 is released and expression is enabled. The regulation model has been established in vitro. With our work we aimed to construct an in vivo system to confirm this model with the use of mRFP1 under regulation of phigB-2 and HigA2 as well as HigB2 regulated by their own separate constitutive promoters. To achieve this goal, we aimed to increase the pool of already made vectors through biomolecular methods. We also produced a vector with the toxin under an inducible promoter. Restriction and mutagenesis were difficult. Often, the vectors would lose the toxin sequence, which is something we did not want. We have successfully produced a vector with the toxin sequence and without the antitoxin sequence. Mutagenesis was not successful. We speculated that the toxicity of the catalytically inactive toxin mutant might still be significant. The reason for that could be HigB2 binding to the ribosome. Therefore, we established a vector containing the toxin under an inducible promoter, where the protein is expressed only after the addition of L-arabinose. Preliminary induction experiments and the use of a DotBlot experiment show successful protein expression. Within our work we successfully established vectors which allow us to induce protein expression and measure fluorescence of mRFP1 in order to characterise the studied systems in vivo.

• Ključne besede: toxin/antitoxin, expression regulation, higBA2, HigB2, HigA2