Part:BBa_I757004

Fhy1_120-202_FusionPart

• Fhy1 is an interaction partner of PhyA
• this part codes for Amino acids 120-202, encompassing the potential interaction domain with PhyA and additional N-terminal amino acids
• SwissProt: Q8W565 , backtranslated
• Purpose: design of a light switchable interaction
• NgoMIV / AgeI protein fusion part
• 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

Characterization

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 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 needs to be characterized, that is why Sorokina et al. wanted to provide a real-time, detectable in vivo reporter system. The GAL4-responsive GAL1 promoter was fused to the firefly luciferase gene (GAL1:LUC) and stably integrated into the yeast genome as described in [6]. Separate sets of yeast were illuminated with red light (R) or far-red light (FR), or R immediately followed by FR (R/FR), or were kept in darkness. As you can observe in figure 3, luminescence reached a maximum between 14-16h under the R light, followed by a graduate decrease. On the contrary, FR light alone or R/FR treatments induced very low levels of luciferase activity.

Fig. 3(adapted from [6]): Yeast cells harboring the GAL1:LUC reporter and expressing PHYA-GBD/GAD-FHY1 fusion protein-pairs were grown in darkness to form patches (merged colonies) for two days at 30°C, treated with 2.5 mM luciferin and transferred to 22°C for 17.5 h. Separate yeast patches were irradiated with single red (R), or far-red (FR) light pulses, or with red pulses immediately followed by far-red pulses (R/FR), or were kept in darkness (Dark). Luminescence values normalized to the pre-pulse levels are shown; time 0h is the start of the light treatment.

Plant phytochromes are chromoproteins composed of the apoprotein and a covalently linked, linear tetrapyrrole chromophore, phytochromobilin (PΦB) [7]. As mentioned above, the PhyA follows the same rule and is brought into the activated state through the light absorption mediated by the covalently bound PΦB. In the absence of PΦB, PhyA can not absorb light and therefore do not show any light-induced conformation changes [8]. Since the yeast does not produce naturally PΦB, in the experience, shown in figure 3, Sorokina et al. added an exogenous chromophore from Cyanobacteria, called PCB. PCB is generally used as an added chromophore for plant phytochromes since it has strong similarities with PΦB structure [9,10].

At the same time, to the surprise, significant R induction could also be observed in the absence of exogenously added PCB (fig.4). The fold-induction was reduced to 30% compared to the results with added PCB in figure 3. This outcome demonstrates the existence of an unidentified compound naturally present in yeast that can serve as a chromophore for PhyA expressed in a heterologous system. Therefore PhyA/FHY1 optogenetic regulation system is particularly interesting for the biotechnological or iGEM applications in yeast.

Fig. 4(adapted from [6]): Yeast cells harboring the GAL1:LUC reporter and expressing PHYA-GBD and GAD-FHY1 fusion proteins were grown in darkness at 30°C for two days on media without PCB. Luciferin and light treatments and imaging were performed as in Fig. 3.

References for Toulouse_INSA-UPS 2020's contribution

• [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]Emborg, T. J., Walker, J. M., Noh, B., & Vierstra, R. D. (2006). Multiple Heme Oxygenase Family Members Contribute to the Biosynthesis of the Phytochrome Chromophore in Arabidopsis. Plant Physiology, 140(3), 856–868. https://doi.org/10.1104/pp.105.074211
• [8]Legris, M., Ince, Y. Ç., & Fankhauser, C. (2019). Molecular mechanisms underlying phytochrome-controlled morphogenesis in plants. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-13045-0
• [9]Li, L., & Lagarias, J. C. (1994). Phytochrome assembly in living cells of the yeast Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, 91(26), 12535–12539. https://doi.org/10.1073/pnas.91.26.12535
• [10]Matute, R. A., Contreras, R., Pérez-Hernández, G., & González, L. (2008). The Chromophore Structure of the Cyanobacterial Phytochrome Cph1 As Predicted by Time-Dependent Density Functional Theory. The Journal of Physical Chemistry B, 112(51), 16253–16256. https://doi.org/10.1021/jp807471e

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4
  Illegal AgeI site found at 259
  
• 1000
  COMPATIBLE WITH RFC[1000]