Top Count Measurement

The Packard TopCount Microplate Scintillation and Luminescence Counter utilizes single photomultiplier tubes to measure the light produced from recombinant luxAB or luc reporter cyanobacteria in counts per second (cps). The advantage of using the TopCount to measure circadian bioluminescence is multifold: one can screen hundreds of strains at a time, and the automated counting protocol allows for rhythms to be monitored 24 h a day, for weeks at a time, with little attention from an otherwise sleep-deprived worker.

3.3.1. Plate Preparation

1. Sterilize a black 96-well microtiter plate (ThermoLabsystems; see Subheading 3.3.3.) and clear plastic lid with 70% ethanol (EtOH; see Note 5). Evaporate the EtOH in a laminar flow hood under UV light for at least 30 min before use.

2. Prepare 50 mL of BG-11M-2X agar. Place in 65°C water bath to bring to temperature and add 50 mL of BG-11M-2X salts that have been warmed to 65°C. Add antibiotics directly to melted mixture and mix well.

3. In a laminar flow hood, use a multichannel micropipet to add 300 ^L of agar medium to each well of a 96-well black plate. Cover with lid and let solidify at room temperature for at least 30 min.

3.3.2. Sample Preparation

1. Inoculate each well with test strains. If using liquid culture, add 20 to 40 ^L of cell suspension (recommended OD750 = 0.7) to desired wells. Alternatively, cultures growing on solid medium may be streaked onto the agar pad of each well using a sterile toothpick. If using luc strains, we have found that inoculation of TopCount sample plates with liquid cultures provides superior traces.

2. Lay a flat toothpick on either side of the black plate immediately outside the outer wells and place the clear lid on top of the toothpicks to prevent the lid from directly contacting the samples. This will prevent mixing of cell cultures from adjacent wells.

3. Incubate in constant light at 30°C overnight. To synchronize the cells' clocks, a 12-h dark treatment is typically used (see Note 6).

4. After dark treatment, in a laminar flow hood, replace the clear lid with a plastic Packard TopSeal (PerkinElmer Life Sciences), being careful not to have any tape hang over the edges of the plate (see Note 7). If using luc reporter strains, add 10 ^L of a 5 mM D-luciferin solution to each well before applying the TopSeal.

5. Using a 16-gage sterile needle, poke a hole in the plastic seal above each well that contains a sample to allow gas exchange throughout the TopCount run, being careful not to touch the samples with the needle.

3.3.3. TopCount Protocol and Interpretation of Results

S. elongatus is an obligate photoautotroph, so constant light conditions are used for circadian monitoring. A frame that surrounds the TopCount stackers was designed by D. Denke (see Note 8) to provide light to the samples. We use a light source consisting of eight 40 W compact fluorescent bulbs (four bulbs on each side) that create a light intensity of about 1300 ^E/m2s at the outer edge of the stacker and about 1000 ^E/m2s in the middle of the stacker, which maintains a light gradient within the wells of the sample plate that ranges from about 230 ^E/m2s in the outer wells that are closest to the light source to about lights

Fig. 2. Schematic drawing of the Packard TopCount Microplate Scintillation and Luminescence Counter. A set of four compact fluorescent light bulbs lines each side of the TopCount stackers. Stackers hold black sample plates separated by three clear plates to allow sufficient light penetration into sample wells. To reduce the heat supplied by the lights, a fan is placed in front of the stackers to maintain a temperature of 30°C. Each black sample plate is taken into the machine approximately every 2 h and bioluminescence is measured for each well. Data are stored on the TopCount computer until retrieved; rhythmic data can be interpreted using the Import and Analysis program.

Fig. 2. Schematic drawing of the Packard TopCount Microplate Scintillation and Luminescence Counter. A set of four compact fluorescent light bulbs lines each side of the TopCount stackers. Stackers hold black sample plates separated by three clear plates to allow sufficient light penetration into sample wells. To reduce the heat supplied by the lights, a fan is placed in front of the stackers to maintain a temperature of 30°C. Each black sample plate is taken into the machine approximately every 2 h and bioluminescence is measured for each well. Data are stored on the TopCount computer until retrieved; rhythmic data can be interpreted using the Import and Analysis program.

50 ^E/m2s in the inner wells (9). Because the high-intensity lamps cause an increase in temperature, we place a fan in front of the stackers, set at its lowest speed, to maintain a temperature of 30°C across the stackers. The temperature within the measuring chamber is controlled automatically.

Black sample plates are necessary so that luminescence from neighboring wells does not interfere with measurements. We place 6 to 8 sample plates in the TopCount stackers and separate each black sample plate with three clear 96-well plates to allow sufficient light to reach the cells (Fig. 2). Each plate is read every 1.5 to 2 h depending on the number of plates used. The plates enter the machine and are kept in darkness for 3 min at 30°C to allow fluorescence from the photosynthetic apparatus to dissipate before measuring bioluminescence. It takes approx 10 min for the TopCount to count and record bioluminescence from each 96-well plate, thus placing the cells in the dark for a total of 13 min every 2 h, if using eight sample plates. This brief introduction to darkness does not have an effect on the rhythms of gene expression (as far as synchronization, entrainment, or resetting of the cultures) as displayed by the phase

amplitude

Fig. 3. Characteristics of the Synechococcus elongatus PCC 7942 circadian rhythm. Negative time denotes time during light-dark (LD) cycles that synchronize the cells. The black bar indicates time in darkness during the LD cycle; hatched bars indicate "subjective" dark during constant light (LL) conditions. Properties of the curve that are typically measured are the period (the time between occurrence of peaks or troughs), phase (the relative positioning of the curve with respect to time entering LL), and amplitude (the expression level measured from the midline of the curve to either the peak or trough) of the rhythm. Time points from a PkaiBC::luxAB reporter (AMC462; closed squares) show a class 1 phase rhythm, peaking at the light to dark transition, or subjective dusk. PpurF::luxAB (AMC408; open squares) represent a class 2 phase reporter that peaks at subjective dawn, 12 h out of phase from class 1 rhythms in constant conditions.

phase f\ period

o amplitude

Fig. 3. Characteristics of the Synechococcus elongatus PCC 7942 circadian rhythm. Negative time denotes time during light-dark (LD) cycles that synchronize the cells. The black bar indicates time in darkness during the LD cycle; hatched bars indicate "subjective" dark during constant light (LL) conditions. Properties of the curve that are typically measured are the period (the time between occurrence of peaks or troughs), phase (the relative positioning of the curve with respect to time entering LL), and amplitude (the expression level measured from the midline of the curve to either the peak or trough) of the rhythm. Time points from a PkaiBC::luxAB reporter (AMC462; closed squares) show a class 1 phase rhythm, peaking at the light to dark transition, or subjective dusk. PpurF::luxAB (AMC408; open squares) represent a class 2 phase reporter that peaks at subjective dawn, 12 h out of phase from class 1 rhythms in constant conditions.

bioluminescent reporters. Very clear rhythms have been recorded from both lux and luc reporters by the TopCount for more than 2 wk (10) with no detectable change in rhythm characteristics.

Measurements recorded by the TopCount can be downloaded and interpreted using the Import and Analysis (I&A) program designed by the S. A. Kay laboratory (available at www.scripps.edu/cb/kay/shareware/) (11). The I&A program creates a Microsoft Excel worksheet that displays the bioluminescence cps for each timepoint collected for each sample in the 96-well plate. From this worksheet, the bioluminescence emitted from the culture in each microtiter well can be graphed. Each graph displays circadian properties that can be analyzed to determine if a particular mutation or environmental cue has caused an alteration of the rhythm (Fig. 3). The period of the circadian rhythm is defined

0 24 48 72 96 120

Hours in LL

Fig. 4. Synechococcus elongatus circadian behavior obeys Aschoff's rule. Bioluminescence traces from a PpsbAI::luxAB reporter (AMC393) display a shorter period under high light (closed triangles) than under low light (open triangles).

as the amount of time between two adjacent peaks or troughs; the wild-type period for S. elongatus is between 23.5 and 25 h, depending on light intensity. This difference in periodicity occurs because the cells obey Aschoff's rule, a phenomenon of the circadian clock wherein the period of the circadian rhythm of diurnal organisms shortens with increasing light intensity (12,13). Following Aschoff's rule, the cyanobacterial cultures in the outer wells of the 96-well plate, that are closer to the light source, show consistently shorter periods than cultures in the inner wells (Fig. 4). Another noticeable element of the rhythm is relative phase, which is the positioning of the rhythm peak with respect to a reference point, such as when the culture was placed into constant light. A third characteristic is amplitude, the distance from the midline of the curve to either the peak or the trough of the rhythm. This property, though important, is the most variable of the three even among wild-type samples. These characteristics can be analyzed using the I&A computational interface fast Fourier transform-nonlinear least squares, which provides statistical period, phase, and amplitude information.

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