Team:KOREA/Description

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Inspiration and Description

Inspiration

Team Korea originates from the Highschool Science Camp of KIST Glia Research Group. Our members with different interests and backgrounds have come up with a variety of ideas. Though ideas came from a wide range of fields such as brain science, molecular biology, biochemistry, informatics, and humanities, we all found common ground in the interests of therapeutics, especially in the treatment of neural diseases. So we decided to use synthetic biological methods to improve the current treatments.

While studying tendencies and research previously done, we were enthralled by "the chimeric protein". Making our protein that doesn't exist in nature, it's gorgeous!. We MUST do that. So we decided to make a chimeric protein. To remedy the problems of existing drawbacks; tolerance, irreversibility, slow response and heavy burden, we needed a protein that enabled precise targeting and easy isomerization.

At that time, the first thing we came up with was the animal's eye and the phytochrome of the plants. They all have in common that they are controlled by a light signal. As the eyes are isomerized from cis to trans, phytochromes are freely and reversibly isomerized according to the ratio of red light and near-infrared light, which both can lead to a rapid reaction. So, we've determined to apply the light property to the molecular level, and we decided on a topic "Optogenetics with chimeric proteins". This is how the scheme of Track 1 was created.

However, if gene therapy was carried out by transfecting chimeric proteins, we thought that a permanent cure would be possible, but a shallower step without the introduction of external genes was also needed. Track 2, which started like this, uses CRISPR, the recent hot topic of molecular biology. It's not the typical CRISPR-Cas9 system, but dCas9. dCas9 is a kind of Cas9 enzyme in which the exonuclease domain is dead. Therefore, unlike Cas9, which irreversibly cuts DNA, not only reversible inhibition is possible, but also reversible promotion is possible by making a fusion protein of dCas9 and transcription factors. We introduced a protein system to dimerize by light(photodimerization) into dCas9.

The planning and realization of these two track schemes required the role of members with diverse talents Three members of the biological field oversaw the WetLAB experiment, while members I the chemical field were responsible for making reagents and modeling. The three members of computer science were team wikis and modeling, and the other members were human practices and design. In the future, we plan to extend this project to the in vivo area. For the mousse, a technique is known for transmitting blue light signals to the deep brain through the skull behind the ear. By conducting LED photostimulation on mice transformed with virus injection, we will analyze changes in the behavior of the mice and ensure that they are safe and effective for higher animals like a human.

Description

Our goal is to cure Parkinson's disease by optogenetics.
We want to initiate the dopamine pathway by light. In the preceding paper, CXCR4 and Rhodopsin were fused. Both are the GPCR family. Therefore, we thought we can make a chimera protein of rhodopsin and dopamine receptor because dopamine receptor is also GPCR.

For our experiments, we choose a U-87 MG cell. We used the membrane potential to check that our fusion protein worked. U-87 MG has an ion channel that is downstream of the dopamine signaling pathway. First, we transfected our fusion protein into a U-87 MG cell. If our protein works well, membrane potential will change at the light condition. Next, We check our protein's expression by GFP. We used membrane potential dye to compare our control with our cells, light and dark conditions to assess whether our fusion protein worked properly.

As Track 2, we linked the photodimerizable proteins CRYII and CIBN to the dCas9 system. dCas9 is a Cas9 enzyme with dead exonuclease activity, and thus can reversibly bind and dissociate DNA. When the sgRNA of dCas9 is located in the target gene, the blue light stimulus causes the CIBN attached to dCas9 to form a dimer with CRYII bound to the transcription factor. We applied it to ABAT and tried to fix Parkinson's disease by adjusting the concentration of GABA at synapses. Because GABA is the significant inhibitory neurotransmitter, this method could be applied broadly in our central nerve system.

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