Detection of promoter induction via beta-galactosidase activity is a well characterised method in S. cerevisiae however it requires time-consuming sampling and in vitro analysis. In order to provide a real-time, in vivo detectable reporter in our system, the GAL4-responsive GAL1 promoter was fused to the firefly luciferase gene (GAL1:LUC) (Fig 1). Figure 1B shows the resulting gene circuit in the community standard Systems Biology Graphic Notation (SBGN) 21. As a constitutive control, the ADH1:LUC (ALCOCHOL DEHYDROGENASE I) construct was prepared and stably integrated into the genome. Yeast colonies prepared as described in Materials and Methods reached a steady state of luminescence 16-18 hr after luciferin was applied (Additional file 1). As expected, ADH1:LUC produced much higher light emission than GAL1:LUC independent of the GBD/GAD fusion proteins expressed (Additional files 1 and 2). Separate set of patches were irradiated with red light (R) or far-red light (FR), or R immediately followed by FR (R/FR), or were kept in darkness. R light induced a rapid increase of luminescence in the case of yeast patches expressing GAL1:LUC, but not in the ADH1:LUC-expressing patches or in patches expressing GAL1:LUC without GBD/GAD fusion proteins (Fig 2 and Additional files 1 and 2). Luminescence reached a maximum 14-16 hr after the R light, followed by a slow decrease. In contrast, FR light alone induced very low levels of luciferase activity, which was essentially the same, when R light treatments were followed by FR light immediately (Fig 2). Since the relative (fold) induction was the highest in yeast cells having the GAL1:LUC reporter and expressing PHYA-GBD and FHY1-GAD (Fig 2A), this set of interacting proteins were used in further experiments. These results demonstrate that (i) appropriate LUC markers can be used to report phytochrome photoconversion and light-induced protein-protein interactions in our system; (ii) LUC enzyme activity is unaffected by light in yeast and (iii) yeast patches grown on solid media and treated with luciferin represent stable and reliable experimental material for luminescence imaging.

In order to calculate changes in the rate of transcription from real-time luminescence data, it was necessary to determine the relationship between transcription and enzyme activity.

The copper inducible CUP1 promoter was fused to luciferase gene, expression was induced and CUP1: LUC RNA and luciferase activity were tested over 7 hours time course. Figure 3 shows a 3-4 h delay in the induction of LUC activity relative to LUC mRNA expression. These data contributed to determine the kinetic parameters (mRNA half-life, translation rate) for the Luc reporter model.

Phytochromes are chromoproteins consisting of the apoprotein and a covalently linked, linear tetrapyrrole chromophore, phytochromobilin (PΦB) 22. In the absence of chromophore, phytochromes cannot absorb light, do not show light dependent conformation changes and, therefore, do not function as photoreceptors. Phytochrome apoproteins are synthesised in the Pr form in plants and after autoligation of PΦB are capable of light absorption and photoconversion into the Pfr conformer. Cyanobacteria (Synechococcus and Synechocystis sp.) harbour phytochrome-like photoreceptors, which use a chromophore (PCB) with similar structure to that of PΦB 23. Plant phytochromes binding PCB are fully functional photoreceptors and, because of the relative ease of PCB purification it is generally used as an exogenously added chromophore 24. However, it was unclear whether yeast cultures contained chromophore-like compounds that could serve as the chromophore for plant phytochromes. To test this, yeast cells with the GAL1: LUC reporter and expressing PHYA-GBD and FHY1-GAD were grown on media lacking PCB and the same light treatments were administered as in Fig 2A. To our surprise, significant R induction was detected in the absence of PCB (Fig 4). The fold-induction was reduced to 30% compared to the results with added PCB (Fig 4 vs. Fig 2A). Moreover, Figure 4 shows that FR light alone, or R followed by FR light also gave qualitatively very similar results to the photoreceptor with PCB. The basal expression level of GAL1:LUC was not affected significantly by the presence or absence of PCB (Additional file 2). These results demonstrate that an unidentified compound naturally present in yeast, can serve as a chromophore for phytochromes expressed in this heterologous system.

To use our regulatory system for synthetic biology we developed an ordinary differential equation (ODE) model of its function based on kinetic data from the literature and experimentally determined parameter values (Fig 1A, B and Additional file 3). The model describes all the known phytochrome properties (e.g. photoconversion, dark reversion, sequestration, etc), using yeast phytochrome data to provide a realistic description of the light-switch function (for the detailed model description and structure, see Methods).

In summary, the model assumptions are:

1) Overall concentrations of Phys and PIF3/FHY1/FHL are constant,

2) Before the light impulse all the phytochromes are in the inactive (Pr) form, and sequestered in a slow acting pool. This might be related to their inclusion in SAPs(Sequestrated Areas of Phytochromes)-like structures similar to those observed by microscopy in cytosol 15 (see Methods for more details).

3) The dark reversion rate is the same for the free Phy and the Phy-FHY1/FHL complex.

4) Photoconversion or dark reversion of the Pfr_FHY1/FHL complex creates an intermediate step (Pr_FHY1/FHL) with distinct kinetics, prior to dissociation of the complex.

5) The luciferase - luciferin subsystem is approximated as a steady state before light treatments.

6) The initial sharp decrease in luminescence following the application of luciferin is due to the diffusion of luciferin from the point of application on the yeast patch into the agar medium.

This enables the model to provide a good fit to conceptually similar systems with different interaction partners. For example, the adapted model fits Shimizu-Sato's data with good accuracy using parameter values derived from the literature 671625. This model is simpler, for the main part because the slow LacZ degradation obscures the long-term kinetics (see the model equations in Methods and simulation results in Additional file 4).

To account for the initial difference in cell density for each yeast patch that affects luminescence intensity (Fig 5) and for the non-uniformity of the solid media, the initial conditions were set for each patch (each experiment) individually, so that each experimental curve is considered for two regimes, "diffusion" and "phytochrome". The former starts from the application of luciferin with initially decreasing luminescence, which approaches an approximate steady state after 17-18 hours. The latter begins from the light treatment and continues to the end (Fig 6A). The first regime fits separately to the diffusion part of the model and provides the initial luciferin level at the time of light application. Such decomposition of the initial conditions is introduced to describe the temporally changing substrate availability that emerges from the solid culture conditions. Modelling the solid media allows a much wider variety of experimental applications in conditions that are most common in yeast and synthetic biology. The diffusion coefficient in our experiments corresponds to diffusion rate of 3 to 10 mm/h, which is in good agreement with literature data for agar gel 26.

Parameter values for model equations were obtained from fitting the model to all the timeseries data from luciferase imaging (Fig 6A, B), within the parameres ranges derived from the literature (Table 1). The parameter data for luciferase protein degradation rate was supported by additional experiments using cycloheximide treatment of yeast cultures constitutively expressing the luciferase gene (data not shown). The degradation rate constant is estimated to be 0.2-0.8, which corresponds to a 0.8-3 hour half-life. This is similar to the value measured in yeast and mammalian cell cultures and plants 272829.

Plasmids expressing PHYA-GBD, PHYBNT-GBD, GAD-PIF3, GAD-FHY1 and GAD-FHL fusion proteins have been described 61112. PHYBNT corresponds to an N-terminal fragment of PHYB containing residues 1-621. GAL1, CUP1 and ADH1 promoters containing full 5' un-translated regions and the 3' un-translated region (terminator) of the GAL2 and ADH1 gene were amplified from S. cerevisiae PJ69-4A genomic DNA using the following primers:

GAL1 Fwd: 5'-AAA

GTCGAC

GAL1 Rev: 5'-TT

GAATTC

CUP1 Fwd: 5'-CGG

GTCGAC

CUP1 Rev: 5'-CGCC

GAATTC

ADH1 Fwd: 5'-CTG

GGATCC

ADH1 Rev: 5'-CGC

GAATTC

GAL2t Fwd: 5'-ATA

CTGCAG

GAL2t Rev: 5'-ATA

CCCGGG

ADH1t Fwd: 5'-CGC

CTGCAG

ADH1t Rev: 5'-TAT

CCCGGG

The firefly luciferase gene was amplified from plasmid pGL3 (Promega) using the following primers:

LUC Fwd: 5'-ATGAATTCATGGAAGACGCCAAAAACATA-3'

LUC Rev: 5'-ATACTGCAGTTACACGGCGATCTTTCC-3'

The promoter:luciferase-terminator constructs were assembled in pBluescript SK plasmid using the restriction sites designed for the PCR primers (sites are underlined in the sequences of the primers above). All plasmids were transformed into E.coli by the SEM method and cultured under standard conditions 31. The GAL2 or the ADH1 terminator was used for the GAL1, CUP1:LUC or the ADH1:LUC construct, respectively. The constructs were verified by sequencing and re-cloned in the integrating plasmid pδ-UB 32. The final clones were linearized with XhoI and transformed in yeast strain PJ69-4A by standard LiAC/carrier DNA/PEG protocol. Transformants were plated on synthetic dropout media (SD) without uracil SD(-U). Selected strains carrying the GAL1:LUC construct were co-transformed with plasmids pD153 or pGADT7 (Clontech) expressing GBD- or GAD-fusion proteins, respectively 6. Transformants were selected and maintained on SD(-LW) plates. Preparation of media and transformation of yeast cells was done according to the Clontech Yeast Protocols Handbook (Clontech).

2 ml of selective SD media was inoculated with yeast cells and incubated for 16 hr at 30°C with agitation. 20 μl drops of the cultures were transferred to SD agar plates containing 10 μM PCB, irradiated with far-red light at 70 μmolm^-2s^-1 fluence rate for 10 min and incubated for 48 hr at 30°C in darkness. All further manipulations were conducted under green safety light. Yeast cells formed merged colonies (or patches) with 5-8 mm diameter. 20 μl of 2.5 mM luciferin solution was pipetted at the center of each patch and the plates were transferred in the imaging chamber at 22°C. Images were taken every 15 minutes using a liquid nitrogen-cooled CCD camera (Visitron Systems GmbH, Munich, Germany). Luminescence was quantified using the Metamorph software (Molecular Devices, Downingtown, PA). Unless stated otherwise, light treatments were administered 17-18 hr after the application of luciferin. The duration of each light treatment was 10 min and fluence rate of light (independent of wavelength) was 70 μmolm^-2s^-1. Red and far-red light was provided by Snap-Lite LED modules (Quantum Devices, Barneveld, WI).

Yeast cells carrying the CUP1:LUC construct were inoculated in 10 ml of SD(-U) media and were grown for 16 hr at 30°C with agitation. The starter cultures were diluted to a final volume of 100 ml with fresh SD(-U) media. CUP1:LUC expression was induced by adding CuSO₄ solution to a final concentration of 1.5 mM. Samples were harvested hourly from induced and non-induced cultures. 2 ml or 100 μl of the cultures were pelleted and frozen for RNA quantification or for luciferase assays, respectively. Total RNA was isolated by using the RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer's instructions. cDNA synthesis and qRT-PCR was performed as described 33. Primers for qRT-PCR were:

LUC Fwd: 5'-GGAGCACGGAAAGACGATGACGG-3'

LUC Rev: 5'-ACAAACACAACTCCTCCGCGCA-3'

ACT1 Fwd: 5'-CACCAACTGGGACGATATGGA-3'

ACT1 Rev: 5'-GGCAACTCTCAATTCGTTGTAGAA-3'

Luciferase-specific signals were normalised to ACTIN 1 (ACT1) levels for each sample. For in vitro luciferase activity measurements, frozen cell pellets were re-suspended in 100 μl of Cell Culture Lysis Buffer (Promega), and vigorously vortexed. After incubation on ice for 5 min, cell debris was pelleted by centrifugation and the supernatant was used as crude protein extract. 20 μl of protein extracts was mixed with 30 μl of the Steady-Glo Luciferase Assay Reagent (Promega) in the wells of a microtiter plate and luminescence was measured in the TopCount NXT luminometer (Perkin-Elmer) for an hour after the addition of the reagent. Counts during monitoring were averaged and normalized to total protein content of the extracts. Protein concentrations were determined by the Bradford assay 34.

Our experimental setup involves the application of a relatively small amount of luciferin substrate (20 μl) to a yeast patch, growing in a 100-mm diameter plate on an agar gel of 5-7 mm thickness. We assumed that the initial decrease in luminescence level just after luciferin application predominantly resulted from the diffusion of substrate through the gel. This was confirmed by an additional experiment (Additional file 5). Taking into account that the thickness of the gel is much smaller than the diameter of the plate, we assumed that the diffusion of luciferin could be described with the diffusion equation in polar cylindrical coordinates:

The particular solution of form

was found to fit experimental data with the best accuracy. Here, S is the cytosolic luciferin concentration, D is the diffusion coefficient, r0 is the effective colony radius, A and B are constants of integration.