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.
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