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| | <h1>Design</h1> | | <h1>Design</h1> |
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| + | <p> |
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| + | Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project. |
| − | <div class="top_text">Scientists have long noticed that the extracellular environment of colon cancer is usually oxygen-insufficient, since the rapid growth of cancer cell consume too much oxygen, so this could be an important clue for us to research cancer cells. For achieving our aim, we disigned three plasmids described as bolow:</div>
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| − | <h2>1. The first plasmid construction:</h2>
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| − | <p>The plasmid pSB1C3 was used for the first plasmid skeleton which information is showing as Fig.1.</p> | + | <p> |
| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/5/51/JNFLS-Design-1.png" />
| + | This page is different to the " Applied Design Award" page. Please see the <a href="https:// 2017.igem.org/ Team:JNFLS/ Applied_Design"> Applied Design</ a> page for more information on how to compete for that award. |
| − | <em style="text-align:center">Figure 1. The information of pSB1C3 plasmid serving as the skeleton for the first plasmid construction.</em>
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| − | <p>Based on the pSB1C3, the first plasmid was designed as following:</p>
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| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/6/67/JNFLS-Design-2.png" />
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| − | <em style="text-align:center">Figure 2. The first plasmid construction.</em>
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| − | <p>In the first plasmid, fdHF promoter will start to express when the extracellular environment lacks of oxygen. In our project, it indicates that the bacteria are close to cancer cells, then the invasion gene will express a kind of membrane protein, helping the bacteria get into the cancer cells (unfortunately, the protein expressed here is not specifically targeted on cancer cell, which means if it express in a wrong place, the bacteria will invade into a normal cell) and Hyl gene can help the bacteria escape from the lysosome, which facilitates the expression of the second plasmid.</p>
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| − | <p>However, we also face a serious problem: We do not know when actually the plasmid will start to express, because in colon, there is also little oxygen. What if the promoter is activated by the environment in the colon, but not where the cancer grow? To avoid this we need to design the second plasmid.</p>
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| − | <h2>2. The second plasmid construction:</h2>
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| − | <p>In the second plasmid, we use hTERT promoter to start the whole expression. Since 90% cancer cells show increase in the expression of human telomerase( this can prolong the end of telomere, enable the cancer cell to reproduct themselves without limitation on the number of times), but it can hardly be detected in normal cells. Thus, it can largely insure the expression of TAT apoptin fusion protein will only be operated in cancer cells. TAT apoptin fusion protein is a special protein that can induce apoptosis of cancer cells. In other word, it force cancer cells to kill themselves. TetR can help bacteria to defend Tetracycline (TET), yet here its function is different-------it is used to repress the promoter in the third plasmid.</p> | + | |
| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/6/64/JNFLS-Design-3.png" />
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| − | <em style="text-align:center">Figure 3. The second plasmid construction.</em>
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| − | <h2>3. The third plasmid construction:</h2>
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| − | <p>The third plasmid is a security insurance. Whether it will express or not depends on the expression of tetR in second plasmid. U6 is the promoter of sgRNA which can guide Cas9 protein to a certain place on a plasmid and cut it. The key component of this plasmid is the tetR repressible promoter. If the second plasmid express successfully, which means it actually invade in a cancer cell, with tetR being synthesized. Then tetR repressible promoter will not start the subsequent transcription of Cas9 gene, preventing the plasmid being damaged by mistake. However, if the second plasmid do not express eventually, indicating that the bacteria accidentally invade into a normal cell. In this case, the Cas9 protein will be produced, and cut the common sequence of the plasmid, such as original replication site, avoiding the risk of killing normal cells. Here, the efficiency of the tetR repressible promoter and hTERT promoter will need to be tested separately and together.</p>
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| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/f/f6/JNFLS-Design-4.png" />
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| − | <em style="text-align:center">Figure 4. The third plasmid construction.</em>
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| − | <h2>4. Two plasmids work coordinately:</h2>
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| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/3/35/JNFLS-Design-5.png"> | + | |
| − | <em style="text-align:center">Figure 5. Two plasmids work coordinately.</em>
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| | </div> | | </div> |
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| | + | <div class="column half_size"> |
| | + | <h5>What should this page contain?</h5> |
| | + | <ul> |
| | + | <li>Explanation of the engineering principles your team used in your design</li> |
| | + | <li>Discussion of the design iterations your team went through</li> |
| | + | <li>Experimental plan to test your designs</li> |
| | + | </ul> |
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| | </div> | | </div> |
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| | + | <div class="column half_size"> |
| | + | <h5>Inspiration</h5> |
| | + | <ul> |
| | + | <li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li> |
| | + | <li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li> |
| | + | <li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li> |
| | + | </ul> |
| | + | </div> |
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| | </html> | | </html> |
JNFLS
Design
Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
This page is different to the "Applied Design Award" page. Please see the Applied Design page for more information on how to compete for that award.
What should this page contain?
- Explanation of the engineering principles your team used in your design
- Discussion of the design iterations your team went through
- Experimental plan to test your designs
