| 摘要 |
The unicellular green alga Chlamydomonas reinhardtii is currently used in many biotechnological applications for the production of biopharmaceuticals and of biohydrogen. These applications require the tightly controlled expression of genes. The present invention consists in the development and use of gene cassettes and culture growth media to obtain inducible, reversible expression of genes in Chlamydomonas. These cassettes contain the Chlamydomonas cytochrome c6 (Cyc6) promoter. It is already known that the Cyc6 promoter, joined to a DNA sequence, controls its transcription on the basis of the concentrations of copper (Cu2+) and nickel (Ni2+) ions in the growth medium (Quinn et al., 2003). In the present invention, the Cyc6 promoter activity was increased up to 20 times, as measured by the activity of an enzyme placed under its control, through the use of transcriptional enhancers, as for example the first intron of Rbcs2 gene and growth media with reduced amounts of transition metals and EDTA. These expedients allow to increase the induction levels of the Cyc6 promoter upon Ni2+ supplement and to induce its expression through the addition of Cu2+ specific chelators. The induction by chelators is reversible through the addition of Cu2+, allowing the expression, followed by the subsequent repression, of an exogenous gene in precise moments of Chlamydomonas cell growth cycle. The described induction followed by repression has never been described before in algal, plant or animal cells and allows the reversible expression of gene products at precise moments of the Chlamydomonas growth cycle.
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