Part:BBa_K3924031

miniSOG, mutagenesis from Arabidopsis phototropin 2

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 43
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 43
  Illegal NheI site found at 4
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 43
  Illegal BamHI site found at 37
  Illegal XhoI site found at 364
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 43
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 43
  
• 1000
  COMPATIBLE WITH RFC[1000]

Profile

Name: miniSOG
Base Pairs: 369bp
Origin: Arabidopsis thaliana
Properties: A mutagenesis phototropin protein which can generate ROS after being exposed to blue light

Usage and Biology

miniSOG was engineered by-site specific mutagenesis from Arabidopsis phototropin 2, which naturally binds FMN but does not generate ROS ^[1]-[3]. By mutation, it can generate ROS after being exposed to blue light and ROS can oxidize guanosine into an intermediate, which could be attacked by primary amine to form covalent bond with NH2-containing probes which could be used to label RNA.

Design and Construction

For this part, we got the amino acid sequence thanks to a grant from the Peng Zou lab, Peking University. Then we optimized the protein codon and synthesized the sequence by company.

Functional Verification

After getting the synthetic plasmid, we test it by DNA sequencing and the result showed correctness of the sequence. Then we transformed the plasmid into BL21 chemocompetent cells and expressed miniSOG successfully. We first test the expression of miniSOG by Coomassie bright blue staining and the result is below (Figure 1.). Then the purified miniSOG was used to test it labelling ability and more experiment details were shown in result:

Figure 1: The Coomassie dyeing result

Reference

[1] Wang, P., Tang, W., Li, Z., Zou, Z., Zhou, Y., Li, R., Xiong, T., Wang, J., & Zou, P. (2019). Mapping spatial transcriptome with light-activated proximity-dependent RNA labeling. Nat. Chem. Biol., 15(11), 1110–1119.
[2] Ding, T., Zhu, L., Fang, Y., Liu, Y., Tang, W., & Zou, P. (2020). Chromophore-Assisted Proximity Labeling of DNA Reveals Chromosomal Organization in Living Cells. Angew. Chem. Int. Ed., 59(51), 22933–22937.
[3] Ruiz-González, R., Cortajarena, A. L., Mejias, S. H., Agut, M., Nonell, S., & Flors, C. (2013). Singlet oxygen generation by the genetically encoded tag miniSOG. J. Am. Chem. Soc., 135(26), 9564–9567.