BLT Stages

Abstract Bachelor Project 1 FBT 2020-2021: Role of the peroxiredoxin Tsa1 in amino acid metabolism and protein biosynthesis

In yeast, the major peroxiredoxin (Prx) is Tsa1, which has both a specific antioxidant function to protect cells against oxidative stress and a role in protecting cells from misfolding and aggregation of new proteins. Tsa1 is crucial for resistance against exogenously added hydrogen peroxide (H2O2) and is required during normal aerobic growth conditions. When Tsa1 is absent cells accumulate a higher level of spontaneous mutations. Molin and Picazo found a connections between Tsa1 and protein synthesis. They studied three genes encoding tRNA modification enzymes (DEG1, UBA4 and ELP6) that share negative genetic interaction with Tsa1. They noticed that in the H2O2 resistant strain with an extra copy of TSA1, a deg1Δ mutant made cells H2O2 sensitive despite overexpressing TSA1. Therefore, in this work, different plasmid containing the gene DEG1 were constructed to attempt to overexpress the DEG1 gene. Moreover, recent unpublished studies (Molin & Picazo, 2021) have shown that the levels of intracellular methionine, lysine, glutamate an glutamine are higher in tsa1∆ mutant compared to the wildtype strain. To further explore this result, in this thesis the question whether there is a connection between Tsa1 and enzymes associated with tRNA synthetase or tRNA modification was asked. To answer this question, the gene expression levels of genes encoding enzymes in these biosynthesis pathway, was checked. The studied genes expression levels through RT-qPCR do not show big differences between the different cells strains under H2O2 conditions. This because in the tsa1∆ mutant, Tsa1 may regulate amino acid biosynthesis at the level of tRNA and not at the level of gene expression. The performed spot tests with different constructs overexpressing DEG1 suggested that overexpression of DEG1 from its endogenous promotor boosted cellular H2O2 resistance. However, this occur at the absence of TSA1 suggesting a close functional interaction between Deg1 and Tsa1 in cellular H2O2 resistance. 

Abstract Bachelor Project 2 FBT 2020-2021: Optical mapping of plasmid carrying antibiotic resistance gene on glass

The inefficiency of antibiotics due to antibiotic resistance remains recurrent, and this poses a major global health problem. Infections from a bacterium carrying an antibiotic-resistance gene can induce antibiotics inefficiency to the host. When such an infection is related to a multi-drug resistant bacterium, which can survive last-resort-antibiotics, there exists no proper treatment. The World Health Organisation (WHO) esteems that antibiotic resistance has negatively impacted global health and there is need for rapid actions. A method for detecting a multi-drug resistant gene present in bacteria, for example the MCR-1 gene, that circulates through horizontal gene transfer amongst Enterobacteriaceae is optical DNA mapping (ODM). ODM is based on fluorescently labelling and stretching single DNA molecules to check for the presence of a gene. In this assay, we (1) used CRISPR/Cas9 having a guide-RNA (gRNA) sequence complementary to the MCR-1 gene to cut the plasmids thereafter, applied a competitive binding using YOYO-1 and netropsin, (2) use silane solutions to functionalize glass coverslips and (3) uniformly stretched the plasmids on the silane coated glass surfaces for fluorescence microscopy. Surface stretching on glass is new compared to nanochannels stretching which is standard. The YOYO-1 and netropsin binding patterns on the plasmids could reveal sequence information on the plasmids carrying the resistance gene that permitted the building of the plasmid barcode. Using ODM on glass, we could determine sizes (93 - 115 kilo base pair) of plasmids carrying the MCR-1 antibiotic resistance gene and device a barcode for the plasmids. When the same samples were stretched using nanofluidic channels, their barcodes showed similarity to when samples were stretched on glass coverslips. The results attained indicate that ODM on glass stands as a prospective tool for detecting antimicrobial resistance genes present in bacteria.