Part:BBa_K4711050

Gal4(AD)+linker+Fhy1

Usage and Biology

Due to the small amount of benzo (a) pyrene in kitchen waste oil, due to its strong carcinogenic ability, it has adverse effects on the environment and human health. Therefore, we designed a related circuit to degrade benzo (a) pyrene. At the same time, in order to achieve controllability of degradation, we designed a red light switch to control the expression of degradation enzymes [1,2,6,7]. The gene circuit is shown below:

Fig 1 Benzo[a]pyrene degradation pathway

Among them, phyA is a functional phytochrome receptor composed of an apolipoprotein and a phytochrome mobile protein (chromophore). Since chromophore cannot be synthesized in yeast, a similar compound, phycocyanobilin (PCB), purified from cyanobacteria, was added to the medium. PCB is readily taken up by yeast cells and is bound by the phytochrome apoprotein to form phytochrome photoreceptors.

Under red light (λmax = 660 nm) or far-red light (λmax = 730 nm), PhyA reversibly changes its conformation to be able to bind to FHY1, which binds PhyA to BD (DNA binding domain) and PHY1 to AD (transcription activation domain) via linker. The principle of yeast double hybrid [12-13] was used to realize the expression of the target gene under the induction of red light.

Fig 2 Red light system schematic

References

[1]Sorokina, O., Kapus, A., Terecskei, K. et al. A switchable light-input, light-output system modelled and constructed in yeast. J Biol Eng 3, 15 (2009). https://doi.org/10.1186/1754-1611-3-15 [2]Hochrein L, Machens F, Messerschmidt K, Mueller-Roeber B. PhiReX: a programmable and red light-regulated protein expression switch for yeast. Nucleic Acids Res. 2017 Sep 6;45(15):9193-9205. doi: 10.1093/nar/gkx610. PMID: 28911120; PMCID: PMC5587811. [3]Shimizu-Sato S , Huq E , Tepperman J M ,et al.A light-switchable gene promoter system[J].Nature Biotechnology, 2002, 20(10):1041-1044.DOI:10.1038/nbt734. [4]Levskaya, A., Weiner, O., Lim, W. et al. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 997–1001 (2009). https://doi.org/10.1038/nature08446 [5]Li, H., Qin, X., Song, P., et al. A LexA-based yeast two-hybrid system for studying light-switchable interactions of phytochromes with their interacting partners. Biotechnology Bulletin: English Edition, 2021, 2(2), 12. DOI: 10.1007/s42994-021-00034-5.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]