Plasmid Construction & Gene Cloning
Cloning Strategy:
1. PCR-amplify OR1A1 sequences from genomic DNA
2. Add Rho-tag sequencing to N-terminal during PCR
3. Ligate the PCR fragment to pcDNA3.1+ plasmid
| Parts | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| OR1A1 | pcDNA3.1+ | E.coli.DH5α |
| OR1D2 | pcDNA3.1+ | E.coli.DH5α |
OR1A1 and pcDNA3.1+ fragments gel electrophoresis
Sanger sequencing of OR1A1
Sanger sequencing of OR1D2
Test the sensitivity and specificity of this basic odor biosensors with their cognate odors β-citronellol or bourgeonal.
In order to further verify the specificity of OR1A1, we also used some different odors to stimulate it.
Not surprisingly, only when we stimulated with β-citronellol could we detect an enhance in luminescence. Judging from it we can consider that OR1A1 only shows a specific role in β-citronellol recognition.
CRE-luciferase reporter does tranduce cAMP signaling in HEK293 cells
As for the cAMP-activated reporter gene system, we choose Forskolin to induce expression of the reporter gene, and expression of the reporter protein luciferase was confirmed by treatment with 10 millimole（mM）forskolin.
After a 24 h incubation, cracked the cell and added the substrate reacting with luciferase, then detected it by fluorescent microplate reader. Here is the data we got. Cells expressing luciferase treated only with 0.1%DMSO was used as a negative control. We can see that the luminescence data gets significantly improved, which proves that the reporter gene we choose is available.
Cloning into lenti sgRNA(MS2)-puro backbone vector for CRISPRa（gRNA）
Referencing to some papers, we knew that there are three genes which can help the olfactory receptors anchored in the membrane. So we aimed to activate these three key accessory protein to enhance the express of odor receptors.
| Parts | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| GNAL | Lenti MS2 | E.coli.DH5α |
| RIC8B | Lenti MS2 | E.coli.DH5α |
| RTP1 | Lenti MS2 | E.coli.DH5α |
Here shows multiple sgRNAs we designed to target the core genes, GNAL, RIC8B and RTP1 in the signal pathway.
The Sanger sequencing results shows we’ve got correct sequence and successfully cloned them into the sgRNA-expressing vector.
Virus infection experiment
First, we transfected HEK293 with lenti dCas9-VP64-Blast, lenti MS2-P65-HSF1-Hygro
and lenti sgRNA to produce virus.
Then, we coinfected HEK293 with the virus of dCas9-VP64-Blast and MS2-P65-HSF1-Hygro
to construct stable cell lines. Based on these cell lines, we infected different
sgRNAs of GNAL,RIC8B and RTP1 respectively.
This are the results of real-time PCR.
These three graphs show that our sgRNAs has successfully activated the three genes that mentioned before.
OR1A1 biosensor evaluation with its cognate odor molecule
Here comes the most essential experiment. We infected the stable cell lines with all virus to construct a super cell line, which has a high expression of the three genes. Then we transfected OR1A1 and OR1D2 respectively in the super cell, and stimulated with β-citronellol and bourgeonal, which can recognized by OR1A1 and OR1D2 respectively.
This is the luminescence result of OR1A1.
