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PLoS ONE. 2008 Sep 12;3(9):e3200.
An Optimized, Chemically Regulated Gene Expression System for Chlamydomonas.
Ferrante P, Catalanotti C, Bonente G, Giuliano G.
Italian National Agency for New Technologies, Energy and the Environment (ENEA),
Casaccia Research Center, Rome, Italy.
BACKGROUND: Chlamydomonas reinhardtii is a model system for algal and cell
biology and is used for biotechnological applications, such as molecular farming
or biological hydrogen production. The Chlamydomonas metal-responsive CYC6
promoter is repressed by copper and induced by nickel ions. However, induction
by nickel is weak in some strains, poorly reversible by chelating agents like
EDTA, and causes, at high concentrations, toxicity side effects on Chlamydomonas
growth. Removal of these bottlenecks will encourage the wide use of this
promoter as a chemically regulated gene expression system. METHODOLOGY: Using a
codon-optimized Renilla luciferase as a reporter gene, we explored several
strategies to improve the strength and reversibility of CYC6 promoter induction.
Use of the first intron of the RBCS2 gene or of a modified TAP medium increases
the strength of CYC6 induction up to 20-fold. In the modified medium, induction
is also obtained after addition of specific copper chelators, like TETA. At low
concentrations (up to 10 microM) TETA is a more efficient inducer than Ni, which
becomes a very efficient inducer at higher concentrations (50 microM). Neither
TETA nor Ni show toxicity effects at the concentrations used. Unlike induction
by Ni, induction by TETA is completely reversible by micromolar copper
concentrations, thus resulting in a transient "wave" in luciferase activity,
which can be repeated in subsequent growth cycles. CONCLUSIONS: We have worked
out a chemically regulated gene expression system that can be finely tuned to
produce temporally controlled "waves" in gene expression. The use of cassettes
containing the CYC6 promoter, and of modified growth media, is a reliable and
economically sustainable system for the temporally controlled expression of
foreign genes in Chlamydomonas.
PMID: 18787710 [PubMed - as supplied by publisher]
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