Part:BBa_K3924032
Pro-LAC, specifically labelling RNA by sgRNA guiding under blue light illumination
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 43
Illegal PstI site found at 682 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 43
Illegal PstI site found at 682 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 43
Illegal PstI site found at 682 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 950
Profile
Name: Pro-LAC
Base Pairs: 1119bp
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 <a ref="http:// https://parts.igem.org/Part:BBa_K3924031"> miniSOG (Part:BBa_K3924031)</ a>, 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 labelling ability exposed in blue light, trace amount of miRNA can be highly effectively labeled in specific way. Then the labelled miRNA can be detected combined with lateral flow assay, and by it, 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, which means our fusion protein is not limited to one option. Instead, by using a combination of different RNA binding proteins, we can construct a fusion protein library that share the same effector protein miniSOG while having different RNA binding protein. In this way, we might be able to fine-tune the system properties to achieve better labelling effect.
| FUNCTION | PROTEIN1 | PROTEIN2 | PROTEIN3 |
|---|---|---|---|
| RNA HAIRPIN | TBP | SLBP | -- |
| SSRNA BINDING | ORF5 | HBEGF | β-defensin 3 |
In our part, 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.
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 showed positive result. For the Coomassie dyeing, the results not only showed the expression of Pro-LAC but also prove the purified efficiency. For the Western Blotting result, it showeds the reliability of induction and there was a little leaking without the inducer.
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.
Worrying that the fusion of miniSOG to TBP and ORF5 could possibly cause the loss of its labelling function, we first tested the non-specific labelling using fusion protein (Fig. 4). The non-specific result showed that fusion protein can effectively label RNA under blue light illumination as well as miniSOG alone can do, which supported our fusion strategy.
Our hypothesis is that gRNA will facilitate the formation of target RNA-fusion protein-gRNA complex and specific label target RNA. But to test this, we need to make sure the non-specific labelling efficiency is low to minimize background noise. We first tested the non-specific labelling effect using different protein concentration (Fig. 5). Our results indicated that if the protein concentration in the reaction system is higher than 200 uM, the non-specific labelling of RNA will generate very high background noises, which will be a big concern when testing the specific labelling effect. Based on this result, we would use the concentration of 200 uM to test Pro-LAC specific labelling ability.
Due to limited time, we were unable to gain any solid conclusion currently. We plan to optimize the reaction condition and to test the specific labelling ability of Pro-LAC in the future.
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
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