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Browsing by Subject "BONCAT"

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  • Haapanen, Mariia Micaela (2021)
    Formation of methane as a biogas from acetate through methanogenesis could be very energy efficient and economically feasible. The acetate utilisation through aceticlastic pathway in methanogenic archaea Methanosarcina is well understood, yet the regulation of acetate utilisation is mainly unknown. The study focused on determination of the time frame of the initiation periods of protein syntheses via bioorthogonal non-canonical amino acid tagging (BONCAT) and copper catalysed click chemistry in Methanosarcina acetivorans and Methanosarcina barkeri, during a long lag phase (around 25 days) after growth substrate was shifted from methanol to acetate. In two experiments the proteins translated after the substrate shift (newly synthetised proteins) were labelled with methionine surrogates via BONCAT and tagged with fluorescent dye and a biotin tag via click chemistry for further detections. To have more complete understanding of the substrate shift acetate concentrations were observed via nuclear magnetic resonance and cell density was monitored via optical density measurements. Only rough time frames of the initiation periods of protein syntheses in both organisms could be estimated from the gel detection of fluorescent tagged proteins. The results indicate that acetate consumption and de-novo protein translation occurs early after substrate switch. In conclusion, the overall utilisation of BONCAT in the labelling of newly synthetised proteins to provide information about the beginning of protein synthesis after substrate shift in Methanosarcina was successful. I was able to detect newly synthetised proteins in both experiments and estimate time windows for beginning of protein syntheses. The information of time windows helps further research to identify the proteins in substrate shift and understand the regulation of substrate shift in Methanosarcina.