In this work, parameters of induction and deactivation of the iron-responsive Fea1 and the ammonium/nitrate-responsive Nit1 promoters were analyzed over time in the green alga Chlamydmonas reinhardtii. The constructs used contain the easily verifiable reporter gene mCherry, linked to the resistance gene ble via the self-cleaving peptide 2A from the foot-andmouth disease virus. The data presented allow drawing comparisons between the promoters as well as two strains, CC 400 and CC 4351. The Fea1 promoter was successfully deactivated upon transferring the cells to medium containing 10 and 20 μM Fe3+. Within 120 h, cells showed only 1.7 - 6 % of the initial fluorescence. In contrast, lower concentrations of iron (4 - 6 μM) allowed the promoter to return to its active state before the end of the cultivation. Activation of the Fea1 promoter occurred promptly and accentuatedly when cells were transferred to iron-deplete medium. However, growth was impaired, motivating the investigation of an alternative way of depleting iron from the medium after high biomass density occurred. This could be achieved with the use of the iron chelator deferoxamine that was proven efficient in removing iron from the culture medium and promoting the consequent activation of Fea1. Upon transferring the cells to chelator-free iron-replete medium, cells resumed growth and the promoter switched off. The non-damaging effect of the concentrations of deferoxamine used and the reversibility of promoter activity show the potential of establishing a cyclic process that alternates biomass production and product formation. In general, a pronounced difference between the active and the inactive states of the Fea1 promoter was observed. High expression rates were obtained in a strain-dependent way (up to 724-fold above wild type (WT) cells for strain CC 400 and up to about 50-fold for strain CC 4351). The Fea1 promoter was even more efficient than the robust Hsp70A/Rbcs2 tandem promoter. The Nit1 promoter was also able to drive expression of the ...


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    Titel :

    Evaluating inducible promoter systems for controlled nuclear transgene expression in the green alga Chlamydomonas reinhardtii ; Evaluierung induzierbarer Promotorsysteme zur kontrollierten nukleären Transgenexpression in der Grünalge Chlamydomonas reinhardtii



    Erscheinungsdatum :

    2018-01-01


    Medientyp :

    Hochschulschrift


    Format :

    Elektronische Ressource


    Sprache :

    Englisch


    Schlagwörter :

    Klassifikation :

    DDC:    629




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