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 so as to introduce a blue light-induced switch to our protein degradation system. However, we find this part document lack experimental validation to prove its interaction with CIB1 under blue light stimulation. Therefore, 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

• Here is the protocol for SEAP assay:

1.Cell transfection and stimulation

(1)Seed HEK293T cells into 24-well cell culture plates. (Approximately 5 cells per well)

(2)Culture for 16 h before transfection

(3)Total plasmid mixes of 800ng per well are mixed thoroughly in DMEM before a polyethylenimine (PEI) solution (1 mg/ml) is added into the plasmid mixture in a ratio of 1:5 (plasmid weight/PEI weight)

(4)The plasmid–PEI mixture is vortexed and incubated at room temperature for 15 min. The mixture is then added into the cells and incubated for at least 6 h.

(5)Cells are then changed into fresh medium and apply the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48 h before sampling and analysis assay. Culture the control group in the dark for the same period.

2.SEAP assay in vitro

(1)Sample 200 ul culture medium from each well, heat inactivate at 65 ℃ for 30 min

(2)During the heat inactivation procedure, warm up 2 SEAP buffer (100 ul/well) at 37 ℃

(3)Add 1/5 buffer volume of pNPP (20 μL/well) substrate into the 2x buffer to prepare the “Detection Mixture.”

(4)Measure absorption at 405 nm, 37 s per read for 10 reads.

(5)Calculate enzymatic activity.

Result

Figure2. SEAP activity of CRY2-CIB1 under dark and 0/24/48/72h cell culturing with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval).

The interaction between the two proteins can be detected by SEAP analysis. The result showed a significantly high SEAP activity of the experimental group compared to the control group, proving the interaction with CRY2 and CIB1 under blue light can be observed.