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| | <p class="title">How we design our biobrick</p> | | <p class="title">How we design our biobrick</p> |
| − | <p class="content">First, we try to find a UV promoter, and we’ve found BBa_I765001. However, it just simply doesn’t work in our experiment. So after searching on the Internet, we found a project that Rice university have done. They had found that the 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 relays this active state to a messenger protein, UirR. Active UirR is mobile, capable of binding a specific promoter called (PcsiR1), and turning on the expression of the desired gene——RFP (E1010). However, we couldn’t find the promoter sequence of PcsiR1 at first, so we use Plsir (K1725400) instead, but when we eventually found the sequence it is too late for us. So we designed this biobrick: | + | <p class="content">First, we tried to find a UV promoter, and we located <p style="color: orange">BBa_I765001</p>. However, it simply didn’t work in our experiment.</div> |
| − | Pcon RBS UirR RBS (B0034) UirS Ter Ter (B0015)
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| − | Plsir RBS RFP Ter Ter
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| − | Then, we found a way to measure the temperature, which is using the temperature regulated RBS (BBa_K115001). This RBS only allows ribosomes to bind on it under the temperature of 37 degree Celsius or above. So originally, we decided to put GFP after it, and it would be turned on 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 change reversely back into inactive state. So we then chose BFP (K592009) instead, and designed this biobrick: Pcon RBSTemp BFP Ter Ter
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| − | Nevertheless, we were afraid that the time under 37 degree Celsius or above is too short for the bacteria to produce enough amount of BFP. So we decide to use an irreversible inhibitor, and then we found the Rhl promoter. When the protein RhlI (K1541017)and RhlR (C0171) both exist, Prhl will continuously work without consuming the proteins, and thus will have enough time to produce BFP. Eventually, we designed this biobrick: Pcon RBS RhlI RBS RhlR Ter Ter + Prhl RBS BFP Ter Ter
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| − | Considering that our products might be damaged, causing the bacteria inside dead, we designed a mechanism to guarantee that our product is able to use. 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 the chromoprotein constantly, and it will be colorful when it is working. Nonetheless, when the bacteria weren’t alive anymore, the remain color will degrade fast and eventually become colorless. At the end, we designed this biobrick: Pcon RBS cj-Blue-lva Ter Ter (note that cj-Blue looks green)
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| − | Lastly, since it would be difficult to transform more than three plasmid into the bacteria, we combined two of them with one of them in the reverse direction (we are afraid of that the gene behind will express poorly), and try to make the sequence as short as possible. So the final biobrick is show as follow.</div>
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| | </section> | | </section> |
| | </h1> | | </h1> |
