Motivation
A bottle of cold drink is a fantastic idea on a scorching summer day, but what if the drink is contaminated with plasticizers? Many people know that plastic bottles will release harmful plasticizers into the drink at high temperatures, so they sensibly avoid leaving bottled drinks or water in hot places, like their cars. However, what if the drink has been contaminated during transportation from the factory to the retail store? Besides bottled beverages, many other products, such as cosmetics or medicine, may pose potential threats to human health if they are stored at inadequate temperatures or exposed to sunlight for too long during the delivery process. The danger is real and immediate, but it is humanly impossible to monitor every delivery procedure. Therefore, we plan to invent a biosensor which can help monitor and ensure the cargo quality in transit.
Overview
We transplant a modified gene into e-coli, and after drying them up, we put these special e-coli into a specialized sticker made by ourselves, and manufactured a device that can do long-term detection. The sticker monitors the temperature and UV light in the whole transportation process, and will change the color on its outlook. By simply take a glance on it, the customers can directly confirm the condition of the merchandise.(see Design & Device)
How we design our biobrick
First, we tried to find a UV promoter, and we locatedBBa_I765001. However, it simply didn’t work in our experiment. So after searching on the Internet, we found a project that had been conducted by Rice university. They found that protein UirR (K1725420) and UirS (K1725410) can be used as a photo receptor. The UirS protein is anchored in the bacterial membrane where it “sees” the color illuminating the bacterium. If the illumination is UV, UirS activates itself and releases the protein, UirR. UirR will then be phosphorylated, and become active. Active UirR is mobile, capable of binding a specific promoter called (PcsiR1), and triggering the expression of the desired gene—RFP (E1010). However, we couldn’t find the promoter sequence of PcsiR1 at first, so we used Plsir (K1725400) instead. But when we eventually found the sequence, it was too late for us. So we designed this biobrick:
Pcon RBS UirR RBS (B0034) UirS Ter Ter (B0015)
Plsir RBS RFP Ter Ter
Then, we needed to find a way to measure the temperature, which is using the temperature regulated RBS (BBa_K115001). This RBS only allows ribosomes to bind on it at the temperature of 37 degree Celsius or above. Originally, we decided to put GFP after it, and the GFP would be activated if it reaches the target temperature. But we then noticed that GFP would produce green light, and green light would cause the protein UirS to reverse back into an inactive state. So we then chose BFP (K592009) instead, and designed this biobrick:
Pcon RBSTemp BFP Ter Ter
Nevertheless, we were afraid that the length of exposure time to 37 degrees Celsius or above is too short for the bacteria to produce enough amounts of BFP. So we decided to use an irreversible inhibitor, and then we came across the Rhl promoter. When the product of RhlI (K1541017) C4-HSR and protein RhlR (C0171) bind together, Prhl will continuously work without consuming the proteins, and thus will have enough time to produce BFP. Late after, we found that the team iGEM14_ETH_Zurich had improved this gene to prevent the “leakiness”. But unfortunately, it’s again too late for us to change. Eventually, we designed this biobrick:
Pcon RBS RhlI RBS RhlR Ter Ter + Prhl RBS BFP Ter Ter
For fear that our products might be damaged, causing the bacteria inside to die, we designed a mechanism to guarantee that our product will remain effective. We found that if we put an LVA tag behind the chromoprotein, it will degrade much faster. So our concept is to make the bacteria produce chromoprotein constantly, and it will be colorful when it is working. Nonetheless, when the bacteria aren’t alive anymore, the color will degrade fast and eventually become colorless. In the end, we designed this biobrick:
Pcon RBS cj-Blue-lva Ter Ter (note that cj-Blue looks green)
Lastly, since it would be difficult to transform more than three plasmid into the bacteria, we combined two of them with one in the reverse direction (we are afraid that the gene behind will express poorly), and try to make the sequence as short as possible. So the final biobrick is:
