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FIGURE 5.1
Analysis of nitrogen-starved primary transformants cultured in a 96-well plate by a
fluorescence plate reader. Excitation was set at 485 nm, emission at 535 nm. The ratio of
fluorescence in the presence of Nile red divided by fluorescence prior to Nile red addition
is shown. The culture in well # 1 is wild type. Putative mutants with strong reductions in
fluorescence ratios (well # 85 and 89) are present.
7 days of growth (e.g., Fig. 5.1 ). This second measurement may recover mutants that
cannot produce oil or generate oil droplets. In these loss-of-function mutants, genes
encoding structural and regulatory genes essential for oil biosynthesis and storage
may be disrupted. Colonies with abnormal Nile red fluorescence ratios from both
measurements are subjected to further analysis by fluorescence microscopy. The pu-
tative mutants are rescreened by thin-layer chromatography in combination with mi-
croscopic examination. We screened approximately 7000 individual colonies in 4
months and identified more than 10 putative mutants with decreased oil content
( Fig. 5.2 A) or changes in oil droplet morphology. Shown in Fig. 5.2 B is one mutant
line with fewer but larger oil droplets in comparison with the wild type strain cultured
under identical conditions. The disrupted genes can be identified by a combination of
plasmid rescue and PCR-based approaches ( Dent et al., 2005 ). A detailed protocol
for lipid mutant screening in Chlamydomonas is described below.
5.1.1 Materials
1. Mutant colonies.
2. Sterile toothpicks.
3. Sterile 96-well liquid culture plates (2 mL per well).
4. Black 96-well plates for fluorescence applications with no well-to-well
interference
5. Nile red stock solution, 1 mg/mL in acetone.
6. Sterile 10 mM NH 4 þ TAP medium.
7. Sterile 1 mM NH 4 þ TAP medium.
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