Our SPOT contains two main applications. One is to use the SPOT as a reaction hub to regulate various biochemical reactions. We have demonstrated that if we integrated three enzymes which could synthesize β-carotene into SPOT system, the production of β-carotene in yeasts will be enhanced if phase separation happens. In the future, if we use the yeasts to manufacture β-carotene in a large scale, we can sequester the yeasts in fermenters to prevented recombinant yeasts or recombinant DNA from flowing out into the environment. The other is to use our SPOT as a sensor. We achieved that if there are ABA or Rapamycin molecules in the environment, phase separation would happen. If we want to use the sensor in reality, we can package the yeast cells into resins. It will help us to observe the SPOT formation by microscopy, and limit yeast escape. In both two reality scenes, escape of microbes is prohibited.
In our experiments, we chose E. coli as the chassis for molecular cloning, and S. cerevisiae as SPOT formation chassis. These two species are well known and non-pathogenic. Our bioparts are constructed by collecting segments from safe plasmid by which means we avoid using unknown segments. Those parts which have potential to cause environment problem, animal and plant disease, ecosystem changing are strictly prohibited.
As the iGEM safety policy requires, we didn’t do any dangerous experiments in daily bench work or faced any unusual safety issues. The bench work followed some basic regulations as below:
1.Duplicating a key to the laboratory without permission was strictly prohibited.
2.All involved participants needed to understand the experiment completely before commencing work.
3.All experimenters had to wear rubber gloves.
4.Any steps involving potential release of live microorganisms were performed in a bio-safety cabinet.
5.Appliances such as Bunsen burners, electric heaters and microwave ovens were not left unattended while in use.
6.All liquid and solid waste potentially containing living organism was sterilized.
7.The entire lab was sterilized using UV-light every week.
8.The last person to leave the lab always made sure that water, electricity, gas, and the air conditioner were shut down, and doors and windows have been locked before leaving.