|
|
| Line 381: |
Line 381: |
| | | | |
| | <div class="node"> | | <div class="node"> |
| − | <div class="topic"><p class="text_color">Overview</p></div> | + | <div class="topic"><p class="text_color">Background</p></div> |
| | <div class="active-circle"> | | <div class="active-circle"> |
| | <div class="splash"></div> | | <div class="splash"></div> |
| Line 395: |
Line 395: |
| | | | |
| | <div class="node"> | | <div class="node"> |
| − | <div class="topic"><p class="text_color">How we design our biobrick</p></div> | + | <div class="topic"><p class="text_color">Overview</p></div> |
| | <div class="active-circle"> | | <div class="active-circle"> |
| | <div class="splash"></div> | | <div class="splash"></div> |
| Line 429: |
Line 429: |
| | </p> | | </p> |
| | | | |
| | + | |
| | + | <p class="title">Background</p> |
| | + | <p class="content">The popularity of internet has been increasing considerably in the 21st century, and purchasing in non-physical store has thus become the mainstream of consumption pattern. By investigating through our online questionnaire, we’ve found that at least 95.5% of customers do care what happened with the items they bought during the transportation process. The change of purchasing habit make transporting very crucial. Buyer and seller usually do not know what kind of environment had the cargoes gone through. As a result, a portion of defectives were damaged or deteriorated through the transporting process, but others already had flaws as soon as they were produced. And this lead to a controversial situation: how can we determine who’s responsible for this? We thus aim on three aspects, which are the factory, the transporting company, and the consumers, to carry out our questionnaire survey. The results show that there are three primary reasons that give rise to product damaged or deteriorated, which are external impacts, high temperature, and sunlight (UV light). Since whether the product is damaged by external force is obviously to be seen just by looking at its outlook, we chose to design a sticker to sense temperature and UV light. The sticker sticks on the boxes or products that need to be transport, and when the sticker changes its color, it means that the process of transportation is not adequate and so needs to be changed. </p> |
| | | | |
| | | | |
| Line 435: |
Line 438: |
| | | | |
| | | | |
| − | <p class="title">How we design our biobrick</p>
| |
| − | <p class="content">First, we tried to find a UV promoter, and we located<font style="color: orange">BBa_I765001</font>. 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 (<font style="color:orange">K1725420</font>) and UirS (<font style="color: orange">K1725410</font>) 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 (<font style="color: orange">E1010</font>). However, we couldn’t find the promoter sequence of PcsiR1 at first, so we used Plsir (<font style="color: orange">K1725400</font>) instead. But when we eventually found the sequence, it was too late for us. So we designed this biobrick:</p>
| |
| − |
| |
| − | <p class="content"><font style="color: lightblue">Pcon RBS UirR RBS</font> (<font style="color: orange">B0034</font>) <font style="color: lightblue">UirS Ter Ter</font> (<font style="color: orange">B0015</font>)</p>
| |
| − |
| |
| − | <p class="content"><font style="color: lightblue">Plsir RBS RFP Ter Ter</font></p>
| |
| − |
| |
| − | <p class="content">Then, we needed to find a way to measure the temperature, which is using the temperature regulated RBS (<font style="color: orange">BBa_K115001</font>). 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 (<font style="color: orange">K592009</font>) instead, and designed this biobrick:</p>
| |
| − |
| |
| − | <p class="content"><font style="color: lightgreen">Pcon RBSTemp BFP Ter Ter</font></p>
| |
| − |
| |
| − | <p class="content">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:</p>
| |
| − | <p class="content"><font style="color: lightblue">Pcon RBS RhlI RBS RhlR Ter Ter + Prhl RBS BFP Ter Ter</font></p>
| |
| − |
| |
| − | <p class="content">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:</p>
| |
| − |
| |
| − | <p class="content"><font style="color: lightblue">Pcon RBS cj-Blue-lva Ter Ter</font> <font style="color: red">(note that cj-Blue looks green)</font></p>
| |
| | | | |
| − | <p class="content">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:</p>
| |
| | | | |
| − | <img src="https://static.igem.org/mediawiki/2017/3/38/Composite3.jpeg" class="bigphoto" width="70%">
| |
| | </div> | | </div> |
| | </section> | | </section> |
