A switchable light-input, light-output system modelled and constructed in yeast

Journal of Biological Engineering BioMed Central Research Open Access A switchable light-input, light-output system modelled and constructed in yeast Oxana Sorokina†1, Anita Kapus†2, Kata Terecskei2, Laura E Dixon1,3, Laszlo Kozma-Bognar2, Ferenc Nagy1,2 and Andrew J Millar*1,3 Address: 1Institute of Molecular Plant Sciences, The University of Edinburgh, Kings Buildings, Mayfield Road, Edinburgh EH9 3JH, UK, 2Institute of Plant Biology, Biological Research Center, Temesvari krt. 62, H-6726, Szeged, Hungary and 3Centre for Systems Biology at Edinburgh, C.H. Waddington Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JD, UK Email: Oxana Sorokina - ; Anita Kapus - ; Kata Terecskei - ; Laura E Dixon - ; Laszlo Kozma-Bognar - ; Ferenc Nagy - ; Andrew J Millar* - * Corresponding author †Equal contributors Published: 17 September 2009 Journal of Biological Engineering 2009, 3:15 doi:10.1186/1754-1611-3-15 Received: 28 April 2009 Accepted: 17 September 2009 This article is available from: http://www.jbioleng.org/content/3/1/15 © 2009 Sorokina et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Advances in synthetic biology will require spatio-temporal regulation of biological processes in heterologous host cells. We develop a light-switchable, two-hybrid interaction in yeast, based upon the Arabidopsis proteins PHYTOCHROME A and FAR-RED ELONGATED HYPOCOTYL 1-LIKE. Light input to this regulatory module allows dynamic control of a lightemitting LUCIFERASE reporter gene, which we detect by real-time imaging of yeast colonies on solid media. Results: The reversible activation of the phytochrome by red light, and its inactivation by far-red light, is retained. We use this quantitative readout to construct a mathematical model that matches the system's behaviour and predicts the molecular targets for future manipulation. Conclusion: Our model, methods and materials together constitute a novel system for a eukaryotic host with the potential to convert a dynamic pattern of light input into a predictable gene expression response. This system could be applied for the regulation of genetic networks both known and synthetic. Background Gene expression systems with both spatial and temporal regulation are key components of engineered and synthetic biological networks. Engineered systems generally use a controlled external stimulus to signal to a specific promoter element, producing a rapid and dose-dependent response [1]. The external stimulus, used at the level of both the whole organism and cell culture, has often been a small, cell permeable molecule, which functions as an activator for the corresponding promoters [2-4]. Heat shock gene promoter systems can also be utilised for conditional gene expression using heat or irradiation as the stimulus [5]. The yeast artificial light switchable promoter system proposed by Shimizu-Sato et al. demonstrates many of the advantages of inducible systems, including low background expression, high inducibility, reversibility and dose-dependence [6]. It combines these desirable features with non-toxicity and a lack of pleiotropic and unanticiPage 1 of 16 (page number not for citation purposes) Journal of Biological Engineering 2009, 3:15 pated effects which are inherent properties of chemically inducible systems. This system is based on the properties of the plant phytochrome B photoreceptor (PhyB), which reversibly changes its conformation in response to red (λmax = 660 nm) or far-red light (λmax = 730 nm). The farred light absorbing conformer (PhyB Pfr) binds to the phytochrome interacting factor 3 (PIF3) protein, whereas interaction between the red light absorbing conformer (PhyB Pr) and PIF3 is much less efficient [7]. In the proposed system, PhyB and PIF3 are expressed as chimeric proteins, fused to the DNA-binding (GBD) or the transcriptional activator (GAD) domain of the GAL4 transcription factor, respectively, giving a typical two - hybrid interaction assay. The cis component of the system is the lacZ reporter gene controlled by a GAL4-responsive artificial promoter. In darkness, PhyB-GBD binds the promoter, but does not induce transcription. Red light illumination converts PhyB into the Pfr form, therefore facilitating PhyB-PIF3 interaction, which recruits PIF3GAD to the GAL4-dependent promoter resulting in the activation of transcription. Subsequent far-red light illumination coverts PhyB Pfr to Pr and this is followed by the dissociation of the PhyB-GBD - PIF3-GAD complex and abrogation of transcription. The authors demonstrated the dose-dependent response of the system and the dynamics of photoreversible activation of the lacZ reporter gene, derived from quantitative liquid culture assays. Recently, another genetically encoded signalling system based on PhyB - PIF3 interaction, with different chimeric proteins, has been successfully used for photoswitching of actin assembly through the Cdc42-WASP-Arp2/3 pathway in E.coli [8]. All phytochromes (PhyA-E) in the model plant Arabidopsis thaliana are capable of light-dependent conformational changes, but interacting proteins have only been investigated for the two most abundant phytochromes (PhyA and PhyB) [7,9,10]. FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) and FHY1 LIKE (FHL) proteins control the nuclear import of PhyA via specific interactions with the Pfr conformer [11,12]. It follows that, besides the PhyB-PIF3 pair, other phytochrome-interacting protein combinations could be employed as the "light sensing" module of the expression system. Functional phytochrome receptors consist of the apoprotein and the covalently linked chromophore called phytochromobilin. Since the chromophore is not synthesised in yeast, an analogous compound, phycocyanobilin (PCB), purified from cyanobacteria, is added to the media. PCB is taken up readily by yeast cells and is autoligated by phytochrome apoproteins resulting in photochemically functional phytochrome photoreceptors [13-15]. When http://www.jbioleng.org/content/3/1/15 expressed in yeast with PCB, PhyA behaves like other phytochrome receptors: the Pr ↔ Pfr conversion is controlled by red and far-red light [15-17]. The light switch described by Shimizu-Sato at al., translates light-dependent protein interactions into transcriptional regulation of a selected gene [6]. Beta-galactosidase is the most widely used reporter gene in yeast; however, the protein has a half-life of more than 20 hours in this system, and it can be detected in vitro only [18]. By comparison, the firefly luciferase has a 1.5 hour half-life in yeast, and luciferase activity (luminescence) can be monitored in real-time and in vivo, which makes this reporter a better tool for monitoring dynamic (...truncated)