Part:BBa_K4711051

SV40 NLS+PhyA+linker+LexA(BD)

Usage and Biology

Transcription activation proteins can bind to specific sequences on DNA to initiate the transcription reaction of corresponding genes. The Binding Domain (BD) and Activation Domain (AD) are two independent domains on the transcription activation protein, and both of them are required for gene transcription activation. Based on this principle, yeast two-hybrid experiments have been designed to verify the interaction between the two proteins. Currently, yeast two-hybrid experiments have used two systems, LexA system and Gal4 system.

Following the design of the 2012TU_Munich team, we chose LexA as our DNA-binding domain. This is because, in contrast to GAL4-based systems, no knockdown of the yeast endogenous GAL4/GAL80 gene is required. It therefore also does not interfere with endogenous yeast metabolism and signaling systems because it recognizes only one specific prokaryotic DNA sequence, the so-called LexA binding site. Unlike the GAL4-based system, we do not need a special strain carrying a GAL4/80 deletion, so theoretically every yeast strain could work.

Meanwhile, based on the study of Oxana Sorokina et al. (2009), we found that the combination of PhyA and FHY1 had a faster and better response to red light. Therefore, PhyA and FHY1 were selected as the two main proteins in the red light system among the many phytochrome and its interaction factors.

Fig 1 Different photosensitive pigments and their interacting factors

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]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.

[4]Zhu, K., Zhang, J., Liu, S. Research progress on the yeast transcription factor Gal4. Chinese Journal of Biotechnology, 2011, 31(01), 81-85. DOI: 10.13523/j.cb.20110116. Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 2469
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]