What is “Detecoli”?
We intend to invent a STICKER called “Detecoli”—a word we coin by combining “detect” and “Ecoli”—which changes color in environments of excess sunlight or inadequate temperatures. It will be attached to the product during manufacture and thus is able to monitor the whole process of transportation. Detecoli alerts consumers to possible deterioration or contamination by changing color, and serves as a guarantee of quality.
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 knew 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)
Also, to avoid the color mixture and the overconsuming of the amino acid, we designed a negative control promoter. We use LacI at the end, since it is the most popular one.
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:
Operation Model
We designed a device to detect harmful UV lights and high temperature, and we are able to confirm if it is working. Our design of the device will prevent e-coli from leaking out, and the e-coli in it will be annihilated after use. In the end, it can be freely disposed of without causing any potential health concern.
Mechanism
UV receptor
Rice University found that protein UirR and UirS 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.
Temperature thermometer
The RBSTemp only allows ribosomes to bind on it at the temperature of 37 degree Celsius or above. The main feature of all RNA thermometers is that they function through conformational shifts in structure. These shifts cause conformational changes to expose the Shine-Dalgarno sequence, which acts as a binding site to allow translation.3 For translation to occur, the ribosome must have the aforementioned SD sequence. The structural differences are caused by the transcription regions, but the SD sequence is common.
Color changing
The chromoprotein cj-blue, and the fluorescent proteins BFP and RFP can perform different colors.
Chromoprotein
The LVA tag, served as a degradation peptide sequence, is one of the most effective of them. If we put LVA tags on our desired gene, we can make them degrade faster.
Target
The principal application for our sticker is to monitor the WHOLE transporting process. This is, from the second that the product was made by the factory, to the moment that the customer received the product (even until the customer finishes using the product), every moment was monitored. This not only guarantee the quality of the products, but helps to solve the conflict between the factory and the transport company. When the customers received the products with the stickers on it color changed, it indicates that the transporting condition is not acceptable. Likely, the transport company can also use this kind of stickers to prove to the factory that their employees do treat every cargo properly. This way, we can avoid consumer dispute and the problems of compensation, and add more goodwill for the company.
In addition, what we emphasize is to ensure EVERY product that have our stickers on and to maximize the accuracy of quality control. We extend the monitoring process from initially only in the factory to eventually the customers’ hands, making customers no longer buy, or use, the damaged product caused by inappropriately transporting or storing. The most considerable benefits of this bio-sticker are “long-term monitor” and “cumulative”. The factors of the harmful environments accumulate, and as soon as they surpass the limitation, it activates the color changing process. Still more, the basic material of this bio-sticker is e-coli. As the fact that e-coli replicates itself extremely rapidly, the cost of the sticker will substantially decrease.
