Part:BBa_K2980000

Cry2

Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact with CIB1 (Part:BBa K2980002). Technically, we use 488nm laser of confocal microscope, which also activate GFP, to stimulate the binding of two light-control element. Functionally, it is used to fuse with other protein and bring them together into phase under light.

Usage and Biology

Photoreceptor that mediates primarily blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation, and regulates other light responses, including circadian rhythms, tropic growth, stomata opening, guard cell development, root development, bacterial and viral pathogen responses, abiotic stress responses, cell cycles, programmed cell death, apical dominance, fruit and ovule development, seed dormancy, and magnetoreception. Photoexcited cryptochromes interact with signaling partner proteins to alter gene expression at both transcriptional and post-translational levels and, consequently, regulate the corresponding metabolic and developmental programs[1], or via an alternative electron transport that involves small metabolites, including NADPH, NADH, and ATP. The half-life of the activated signaling state is about 16 minutes[2].

Reference

[1] Yu, X., Liu, H., Klejnot, J., & Lin, C. (2010). The Cryptochrome Blue Light Receptors. The Arabidopsis Book, 8(8). doi:10.1199/tab.0135

[2] Engelhard, C., Wang, X., Robles, D., Moldt, J., Essen, L., Batschauer, A., ... & Ahmad, M. (2014). Cellular Metabolites Enhance the Light Sensitivity of Arabidopsis Cryptochrome through Alternate Electron Transfer Pathways. The Plant Cell, 26(11), 4519-4531.

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 393
  Illegal BglII site found at 852
  Illegal BamHI site found at 1331
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 277
  Illegal AgeI site found at 1006
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 629
  Illegal BsaI.rc site found at 38
  Illegal SapI.rc site found at 146
  

Contribution: NUDT_CHINA 2021

In our project for iGEM2021, we use this basic part with CIB1(Part:BBa_K2980002) to fuse with other proteins to introduce a blue light-induced switch to our protein degradation system. Herein, we constructed plasmids based on the mechanism of tet-on system, co-transfected the plasmids into HEK293T cells, applied the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48h and then conducted SEAP assay to validate the interaction between this part and CIB1.

Method

We separately expressed tetR fused with CIB1 and CRY2 fused with VP64 so that only by applying blue light stimulus would the two proteins bind to each other through CRY2-CIB1 interaction.Meanwhile, we utilized a translational control element (TCE) which consists of seven tetO to enable the transcription of reporter gene (SEAP) downstream once bound with tetR and VP64. Under this assumption, the expression level of SEAP can illustrate whether CRY2 interacts with CIB1 under blue light and we can further demonstrate the interaction level through SEAP assay.

Figure1. Experimental validation approach of CRY2 and CIB1

Result

Figure2. SEAP activity in the culture medium collect from cells transfected with tetR-Cry2, CIB1-VP64 and tetO7-SEAP expressing plasmids under either blue light illumination or dark condition. Cells were illuminated with 3 mW/cm2 of 405nm blue light, the illumination was programed as the repeat of [2 s ON /28 s OFF] cycle for 48 hours. BDL represents below detection limits, Data represent mean ± s.e.m. (n=3 biological replicates)

As expected, HEK-293 cells co-transfected with tetR-Cry2, CIB1-VP64 and tetO7-SEAP expressing plasmids showed significantly increased SEAP production upon 24h or 48 h of blue light illumination comparing to control cells transfected with the same plasmids placed in a dark incubator.