Team:NEU-China/Experiments


Bacterial Strains and Growth Cultures


E. coli DH5α was used for all cloning purposes. Cultures for cloning were grown in antibiotic-supplemented Lysogeny Broth (LB) at 37°C. The E. coli expression host cultures were grown in antibiotic-supplemented Lysogeny Broth (LB) at 37°C.
Bacterial cultures were grown overnight (16-20 hours) to stationary phase before being subcultured for characterization experiments.

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.

Conclusion:In general, the iSmeller we constructed using CRISPR / Cas9 Synergistic Activation Mediator successfully amplifies the signal of the core components of the olfactory pathway and enhances its sensitivity to odor response. In addition, by stimulateing with different scent, our iSmeller's specificity of odor recognization has also been validated.