Revision as of 06:45, 8 October 2020

phyA1-406_N_part

Toulouse_INSA-UPS 2020contributed to the characterisation of this part by adding a new documentation learned form literature on how to use Fhy1 and PhyA as a transcriptoinal optogenetic effectors.
(--antonmykhailiuk 15:26, 07 October 2020 (UTC+2))

• PhyA is an interaction partner of Fhy1 (parts BBa_I757002, I757004)
• Purpose: design of a light switchable interaction
• this part codes for Amino acids 1-406, encompassing the potential Fhy1-interaction domain
• N-terminus of this protein should not be fused for fusions to avoid disturbance of folding and potential Fhy1 interaction ("N-part")
• SwissProt: P14712
• NgoMIV / AgeI protein fusion part
• iGEM Team Freiburg 2007
• synthetic DNA by GeneArt optimized for E.coli
• Part in pGA4 vector (AmpR, ColEI ori)
  

Contribution from other teams

Toulouse_INSA-UPS 2020's contribution

Characterisation

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 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 the 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.

Such an interaction -two photos from desing -article sorokina -article structure-conformation+photo -FHY1

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

Sequence and Features