Coding

Part:BBa_I757007

Designed by: Freiburg iGEM 2007 team   Group: iGEM07_Freiburg   (2007-10-24)
Revision as of 07:52, 8 October 2020 by Antonmykhailiuk (Talk | contribs)

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 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,

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 normalised to the pre-pulse levels are shown; time 0 h is the start of the light treatment.

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


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 AgeI site found at 1219
  • 1000
    COMPATIBLE WITH RFC[1000]


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