Part:BBa_K3924032

Pro-LAC, specifically labelling RNA by sgRNA guiding under blue light illumination

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 43
  Illegal PstI site found at 682
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 43
  Illegal NheI site found at 4
  Illegal PstI site found at 682
  Illegal NotI site found at 1102
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 43
  Illegal BamHI site found at 37
  Illegal XhoI site found at 1086
  Illegal XhoI site found at 1111
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 43
  Illegal PstI site found at 682
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 43
  Illegal PstI site found at 682
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 950
  

Profile

Name: Pro-LAC
Base Pairs: 1137bp
Origin: No natural comparator
Properties: A fusion protein which can bind to sgRNA and specifically label miRNA to play the role of bio marker detection

Usage and Biology

Pro-LAC is a fusion protein inspired by CIRT system (CRIPSR-Cas Inspired RNA Targeting system)^[1] and we used miniSOG, an engineered flavoprotein which can non-specific label RNA after blue light exposed,^[2]-[4] to be the effector domain. For our Pro-LAC, the miniSOG domain can be located to miRNA through the binding ability among ssRNA binding protein, RNA hairpin binding protein and sgRNA. With the high labeling ability exposed in blue light, trace amount of miRNA can be highly effectively labeled in specific way. By this part, we can achieve a universal, convenient and sensitive RNA detection system in vitro.

Design and Construction

This part consists of three domains effector domain, ssRNA binding protein and RNA hairpin binding protein. We noticed that there are several choices for RNA hairpin binding protein and ssRNA binding protein, but we only test ORF5(ssRNA binding) and TBP (RNA hairpin binding) as an example. For the effector domain, miniSOG, we got this sequence thanks to a grant from the Peng Zou lab, Peking University. Due to the time cost of obtaining the above sequence, the all plasmid sequences was submitted to the company for synthesis after codon optimization and we sequenced it to verify the correctness of the synthetic sequence.

File:T--Tsinghua--PRO-LAC.png

Figure 1: The design of Pro-LAC

Functional Verification

Validation based on protein expression level

After sequencing of synthetic plasmid (by company), We then transform the palsmid into BL21 chemocompetent cells and plates on ampicillin agar plates, followed by picking single colony, expanding, and inducing. After bacteria collected, lysed it, and the proteins were affinity-purified using Ni-NTA or Co-NTA beads. Then we ran the SDS-PAGE gel to Coomassie brilliant blue staining and Western Blotting. The results are shown in Fig 3. Both Coomassie dyeing and Western Blotting show positive result. For the Coomassie dyeing, the results not only show the expression of Pro-LAC but also prove the purified efficiency. For the Western Blotting result, it shows the reliability of induction and there was a little leaking without the inducer.

Figure 2: The Coomassie dyeing result

Validation based on protein activity

For validation based on protein activity, we mainly did two experiments which were the non-specific and the specific labelling test. More details were showed in result:

Reference

[1] Rauch S, He E, Srienc M, Zhou H, Zhang Z, Dickinson BC. Programmable RNA-Guided RNA Effector Proteins Built from Human Parts. Cell. 2019 Jun 27;178(1):122-134.e12. doi: 10.1016/j.cell.2019.05.049.
[2] 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. https://doi.org/10.1038/s41589-019-0368-5
[3] 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. https://doi.org/10.1002/anie.202005486
[4] 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. https://doi.org/10.1021/ja4020524