High-throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence

Abstract

The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise and generate meaningful quantitative measurements of clock output levels for advanced analysis.

Keywords: Kai proteins; Luciferase; Single colony bioluminescence; Synechococcus elongatus; Temporal automated measurement.

Figure 1. Fully constructed turntable

(A) Photograph of assembled computer-controlled turntable. (B) Cross-sectional schematic of assembled computer-controlled turntable.

Figure 2. Turntable components

(A) Schematic of turntable surface (referred to in text as Table Top). (B) Schematic of Aluminum Light Shield. (C) Photograph of Rotary Table attached to Aluminum Disc Spacers, Aluminum Base Plate and Copy Stand base. (D) Photograph of Aluminum Light Shield on turntable surface. The PVC pipe is moved from its final location to show the hole in the center of the Aluminum Light Shield.

Figure 3. Example data from time course experiment

(A) The image on the left is a raw image of a plate with luxAB-luxCDE expressing cyanobacteria. The image in the center is the mask generated by the RCFinder.R script. Each white spot represents an identified colony. Those spots that are numbered and circled in red were identified as rhythmic. The number is displaced down and to the right of the spot. The right image is an overlay of the first two images to show which colonies on the plate are rhythmic. (B) Bioluminescence data for five rhythmic colonies found in (A). Colony intensity is a measure of the average pixel intensity for a colony object and varies between 0 and 1 (arbitrary units).

Figure 4

Flow chart for quantitative bioluminescence sample preparation.

Figure 5. Example data from time course experiment

Default high bioluminescence level for class 1 promoter P_kaiBC measured from both the luc (A) and lux constructs (B). Time-dependence of bioluminescence from the WT (black squares) and mutants that carry disruptions of KaiC (blue diamonds) or KaiABC (purple circles) following a 1-d and 2-d entrainment period for (A) and (B) respectively. The bioluminescence using luc was measured with the TopCount (Mackey et al., 2007), whereas the bioluminescence using lux was measured on the turntable as described above. Averages of the replicates and the standard error of the means for the bioluminescence values are indicated. Bioluminescence was converted to counts per pixel in (B) for easier comparison with the TopCount (Paddock et al., 2013). Each genetic background showed the same general behavior with both reporter systems, even though the luciferase systems and the detection systems are distinct. Thus, the constitutively high levels of bioluminescence observed in the knockouts strains are attributed to the genetic lesions and are not a consequence of detection methods or luciferase reporter constructions. This figure was adapted from Proc. Natl. Acad. Sci. U S A. 110(40):E3849–E3857; M.L. Paddock, J.S. Boyd, D.M. Adin, and S.S. Golden; The active output state of the Synechococcus Kai circadian oscillator; Copyright (2013), with permission from the National Academy of Sciences.