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.
In the process of shipment, the DNA parts are absolutely safe because they encode non-hazardous proteins like HOtags and enzymes for carotene synthesis. The DNA parts were safely confined within 96-well plate as the Parts Registry requires.
Since SPOT can form in the cell and be controlled, we go further to consider the functions of SPOT. The functions of SPOT can be descripted in three catalogs: Spatial segmentation, Sensor and metabolic regulation. We verified the spatial segmentation with the condensation of substrates, also we can load the protein we want by fusing it with nanobody. We then verified the sensor with detecting rapamycin and ABA, which shows strong relativity between the concentration and the proportion of yeasts with SPOT. To find the law behind metabolism in the SPOT, we fuse the enzymes that can produce β-carotene into SPOT and measure the difference between with or without SPOT in produce of β-carotene.
SPOT has been well verified and has various functions. And in the future, this modular system will have great potential in science and practice using. SPOT can change the modules to gain more different properties like diverse inducing method, we can also use it as a platform and then load other protein with some interactions like the interaction between nanobody and GFP. What’s more, we might have the ability to form differernt SPOTs in the cell and regulate them respectively. The functions of SPOT can also diverse. We can build a real time sensor for molecule in living cells to monitoring the concentration changing in environment or in cells. More metabolism pathway can be test in SPOT and we will find some laws of the function of regulate the metabolism. To be summary, more achievement is coming true with SPOT.