Difference between revisions of "Part:BBa K3570004"

Latest revision as of 16:23, 26 October 2020

PhyA-FHY1 red optogenetic regulation system in S. cerevisiae

Introduction

The purpose of this biobrick is to provide S. cerevisiae with the optogenetic gene expression regulation system. This is achieved by expressing two fusion proteins PhyA-GBD and GAD-Fhy1. This construction should be put into replicative or integrative plasmid.

Design

Fig. 1: PhyA-FHY1 construct. The genes coding for PhyA and FHY1 come from Arabidopsis thaliana.

The majority of phytochromes (PhyA to PhyE) in Arabidopsis thaliana are subject to conformational changes induced by light. On the contrary, only PhyA and PhyB were found to have the interacting proteins upon the light illumination [1,2,3]. PhyA was found to have as its partner the FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) [4].

The biotechnology is always seeking gene expression regulation systems with both spatial and temporal regulation. And light could be the answer as it is not toxic, homogenous and the unicellular organisms are normally transparent to visible light. PhyA/FHY1 couple can be used as an optogenetic regulation system if PhyA is fused to Gal4 DNA binding domain (GBD), and FHY1 is fused to Gal4 activation domain (GAD). Such a system would allow activation of the transcription of the promoter Gal1/10 by exposing the biological system to red light (660nm). Since PhyA is linked to a chromophore which, under the action of wavelength 660 nm, changes the intrinsic conformation of PhyA[5]. This change of configuration allows PhyA and FHY1 to interact, and because FHY1 can be fused to GAD there will be a recruitment of transcription factor (TF) to the promoter. This is believed to activate the expression of the gene of interest (GOI) This interaction is reversible under far-red light (740 nm) or after a while in dark conditions[6] (fig. 1-2).

Fig. 1: Optogenetic regulation system based on PhyA/FHY1 effectors. Non-activated state, which corresponds to illumination by far-red light (740nm) or under dark conditions. PhyA: phytochrome A; FHY1: Far-red elongated HYpocotyl 1; GBD: Gal4 DNA binding domain; GAD: Gal4 activation domain; GOI: gene of interest.

Fig. 2: Optogenetic regulation system based on PhyA/FHY1 effectors. Activated state, which corresponds to illumination by red light (660nm). PhyA: phytochrome A; FHY1: Far-red elongated HYpocotyl 1; GBD: Gal4 DNA binding domain; GAD: Gal4 activation domain; GOI: gene of interest.

ADH1 promoter and terminator were chosen for their constitutive activity and versatile use when growing yeast on different carbon sources[7], the sequences were identified from personal communication with Dr. Anthony Henras.

Experiments

Team iGEM Toulouse 2020 did not have sufficient time to complete the cloning and hence, to test this part functionality.

References

• [1]Ni, M., Tepperman, J. M., & Quail, P. H. (1999). Binding of phytochrome B to its nuclear signaling partner PIF3 is reversibly induced by light. Nature, 400(6746), 781–784. https://doi.org/10.1038/23500
• [2]Quail, P., Boylan, M., Parks, B., Short, T., Xu, Y., & Wagner, D. (1995). Phytochromes: photosensory perception and signal transduction. Science, 268(5211), 675–680. https://doi.org/10.1126/science.7732376
• [3]Kim, J. (2003). Functional Characterization of Phytochrome Interacting Factor 3 in Phytochrome-Mediated Light Signal Transduction. THE PLANT CELL ONLINE, 15(10), 2399–2407. https://doi.org/10.1105/tpc.014498
• [4]Hiltbrunner, A., Viczián, A., Bury, E., Tscheuschler, A., Kircher, S., Tóth, R., Honsberger, A., Nagy, F., Fankhauser, C., & Schäfer, E. (2005). Nuclear Accumulation of the Phytochrome A Photoreceptor Requires FHY1. Current Biology, 15(23), 2125–2130. https://doi.org/10.1016/j.cub.2005.10.042
• [5]von Horsten, S., Straß, S., Hellwig, N., Gruth, V., Klasen, R., Mielcarek, A., Linne, U., Morgner, N., & Essen, L.-O. (2016). Mapping light-driven conformational changes within the photosensory module of plant phytochrome B. Scientific Reports, 6(1). https://doi.org/10.1038/srep34366
• [6]Sorokina, O., Kapus, A., Terecskei, K., Dixon, L. E., Kozma-Bognar, L., Nagy, F., & Millar, A. J. (2009). A switchable light-input, light-output system modelled and constructed in yeast. Journal of Biological Engineering, 3(1), 15. https://doi.org/10.1186/1754-1611-3-15
• [7]- Peng, B., Williams, T. C., Henry, M., Nielsen, L. K., & Vickers, C. E. (2015). Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities. Microbial Cell Factories, 14(1). https://doi.org/10.1186/s12934-015-0278-5