Method

1
Method
Results
1. CultivationThe cells were grown in BG-11
medium, under continuous light and shaking. When
the culture reached an OD750 of 0.4, ethanol or
retinal were added every two days. The OD750 was
monitored daily.
2
1
10uM
5uM
Supernatant of cell culture sample (1.35uM, 14
detected)
1uM
0.1uM
2. Analysis of Retinal UptakeRetinal was
extracted from culture supernatant and analyzed
by HPLC.
HPLC analysis of retinal uptake. Different known
concentrations of retinal were separated by
reverse phased HPLC , and the peak was used to
generate a standard curve . HPLC detected both
retinal (major peak), and retinol (minor peak).
Retinal in the culture supernatant was tested.
Supernatants of cell culture samples were taken a
few minutes after addition of 10 µM retinal to
the culture. The HPLC analysis shows that 14 of
the added retinal can be detected in the
supernatant.
3. Pigment ExtractionPigments were extracted
after cultivation for 4 weeks, and quantified
relative to dry cell weight
3

• 4. Analysis of Gene Expression
• RNA extraction
• cDNA
• Real-Time PCR

Green bar Retinal Yellow barControl
Retinal
Control
Growth curves of Synechococcus elongates PCC 7942
cultures with or without added retinal. Cultures
were grown under constant illumination (70 µE).
When the cultures reached log phase, either
ethanol (control) or retinal (in ethanol) was
added. Retinal showed a positive effect on
growth during the first 10 days of cultivation
(A). A less pronounced effect was seen after
prolonged cultivation times (B C).
Retinal
Control
Conclusion
Extracted pigments of Synechococcus elongates PCC
7942 cell cultures. Pigments were extracted from
30 mL of cell cultures (normalized to an OD750
0.8). The culture with retinal contained
significantly more carotenoids than the control
cultures (significance based on Students T-test
with a confidence level of 95). Chlorophyll
levels were not significantly different between
the two cultures.

• Carotenoid pigments were produced more in
  cultures containing retinal.
• Retinal might act as signaling molecule in the
  carotenoid biosynthesis pathway.
• Retinal addition affects carotenoids levels and
  its biosynthesis. Further study is needed.

4
Retinal induced changes of cell pigmentation of
Synechococcus elongates PCC 7942 after 65 days
of growth. A control, no retinal added
(yellow), B 5 µM retinal added (yellow-green),
C 10 µM retinal added (green), D 20 µM
retinal added (dark green).
A B C D
References Acknowledgments I would like to
thank Erin Marasco for her enormous help and
support me throughout this study, Dr. Claudia
Schmidt-Dannert for advising, Cassy Rislov for
her initiated retinal stress experiment, and
everyone in Schmidt-Dannert Laboratory for their
support. 1Marasco, Erin K., Kim-leng Vay and
Claudia Schmidt-Dannert. (2006). Identification
of Carotenoid Cleavage Dioxygenases from Nostoc
PCC 7120 with Different Cleavage Activities. J
Bio Chem. 271 (42) 31583-93.