Difference between revisions of "Part:BBa K3924031"
Yiyuan-h19 (Talk | contribs) |
Yiyuan-h19 (Talk | contribs) |
||
| Line 22: | Line 22: | ||
==Functional Verification== | ==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: <!--Add the URL of Result here--><br/> | 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: <!--Add the URL of Result here--><br/> | ||
| − | [[Image: T--Tsinghua--Coomassie_of_miniSOG.png|'''Figure 1: The Coomassie dyeing result''']] | + | [[Image: T--Tsinghua--Coomassie_of_miniSOG.png|center|600px|thumb|'''Figure 1: The Coomassie dyeing result''']] |
==Reference== | ==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.<br/> | [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.<br/> | ||
Revision as of 15:25, 19 October 2021
miniSOG, mutagenesis from Arabidopsis phototropin 2
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 43
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 43
Illegal NheI site found at 4 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 43
Illegal BamHI site found at 37
Illegal XhoI site found at 364 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 43
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 43
- 1000COMPATIBLE 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:
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.