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| | <h1>Results</h1> | | <h1>Results</h1> |
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| − | <div class="container_in_content">
| + | <p>Here you can describe the results of your project and your future plans. </p> |
| − | <div class="top_text">Section 1: Sensitivity detection of fdhF promoter</div>
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| − | <p>fdhF is hypoxia-inducible promoter. In our project, it was used for initiation the expression of invasion gene and hyl gene. Under hypoxia environment it has a much higher efficiency of starting transcription than under normal 20% oxygen pressure. The starting efficiency of fdhF promoter is the key matter in our project. For detecting the starting efficiency of fdhF promoter, reporter gene GFP was connected to fdhF promote plasmid. And then the fluorescence intensity of GFP was detected under hypoxia environment.</p> | + | <h5>What should this page contain?</h5> |
| − | <h2>1.1 Construction of fdhF induced GFP expression device</h2>
| + | <ul> |
| − | <p>fdhF promoter (99bp) was synthesized by Genscript, nanjing, China. It was inserted into psB1C3 directly when synthesized. In order to detect the efficiency of this promoter at different oxygen concentration, we constructed the fdhF promoter induced GFP expression device. fdHF plasmid was digested by SpeI and PstI, while GFP geneator (BBa_E0840) was digested by XbaI and PstI. After purification the fragments, GFP was connected to the fdhF plasmid. The identification result was shown as below.</p> | + | <li> Clearly and objectively describe the results of your work.</li> |
| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/f/fe/JNFLS-Results-1.png" /> | + | <li> Future plans for the project. </li> |
| − | <em style="text-align:center">Figure 1. Construction and identification of fdhF induced GFP device.</em> | + | <li> Considerations for replicating the experiments. </li> |
| − | <h2>1.2 Sensitivity detection of fdhF promoter</h2> | + | </ul> |
| − | <p>After construction of fdhF induced GFP expression device, it was transformed into the E.coli. Under the hypoxia environment, we detected the fluorescence intensity of GFP. The O2 was limited to provide for the culture medium, so the pressure of O2 decreased down to the zero very quickly. Since the instrument can not detect the O2 below zero, we used the increasing of CO2 to stand for decreasing of O2. Some results are shown as below.</p> | + | |
| − | <img src="https://static.igem.org/mediawiki/2017/6/6d/JNFLS-Results-2.png" /> | + | <h5>You should also describe what your results mean: </h5> |
| − | <em style="text-align:center">Table 1 Data of the corelation of GFP expression to the pressure of CO2.</em>
| + | |
| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/6/64/JNFLS-Results-3.png" /> | + | <ul> |
| − | <em style="text-align:center">Fig.2 The corelation of GFP expression to the pressure of CO2</em>
| + | <li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li> |
| − | <p>From the table 1, we can find that the pressure of O2 can not be detected when the E.coli were cultured after 4 hours. So we analyzed the corelation of GFP expression with the pressure of CO2. These data indicated that with the pressure of CO2 inscreasing, the starting efficiency of fdhF is increasing significantly, which means that fdhF promoter is very sensitive and efficient.</p>
| + | <li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li> |
| − | <div class="top_text">Section 2: Sensitivity detection of TetR and tetR repressible promoter:</div> | + | <li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li> |
| − | <p>For detecting the sensitivity of the tetR repressible promoter, we built two expression vectors: the first one contains tetR repressor tagged with yBFP (J04500 + K642000 + B0015); another one is tetR repressible GFP expression device (R0040 + E0840) containing tetR repressible promoter and green fluorescent protain as a reporter.</p> | + | </ul> |
| − | <h2>2.1.Sensitivity of TetR for tetracycline concentration</h2>
| + | |
| − | <p>For constructing of tetR repressor tagged with yBFP device, we connected three parts J04500, K642000 and B0015.</p>
| + | |
| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/8/84/JNFLS-Results-4.png" />
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| − | <em style="text-align:center;">Fig.3 Identification of tetR tagged with yBFP expression device.</em>
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| − | <p>After construction, this plasmid was transferred into the competent cells (trans 5α). According to the BFP fluorescence would be affected when tetR combining with tetracycline, we added tetracycline with a series of concentration gradient to some of the tubes respectively with the same initial fluorescence intensity. Then we detected the changes of their fluorescence intensity of BFP at different time points. Consequently, we got the curve which shows the influence of the tetracycline concentration on the fluorescence intensity of tetR fusion protein. From the results, we can concluded that 20ug/ml tetracycline is the best concentration for inhibitiing of tetR expression.</p>
| + | |
| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/7/77/JNFLS-Results-5.png" /> | + | |
| − | <em style="text-align:center;">Fig.4 The influence of tetracycline concentration on the fluorescence intensity of tetR fusion protein.</em>
| + | |
| − | <h2>2.2.Influence of tetracycline on the tetR inhibition to the tetR repressible promoter</h2> | + | |
| − | <p>For detecting the influence of tetracycline on the tetR inhibition to the tetR repressible promoter, we construct the tetR repressible GFP expression device (R0040 + E0840) which contains tetR repressible promoter and GFP as a reporter.</p>
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| − | <div class="imgs"> | + | |
| − | <img class="small" src="https://static.igem.org/mediawiki/2017/a/aa/JNFLS-Results-6.png" />
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| − | <em>Fig.5 Identification of tetR repressible GFP device (R0040 + E00840).</em>
| + | |
| | </div> | | </div> |
| − | <p>After construction of these two plasmids, we transferred them into competent cells(trans 5α). According to their different resistances, we selected the clone containing these two plasmids. We added tetracycline with different concentration into LB medium to combine with tetR in order to remove the inhibition of tetR on the tetR repressible promoter, which indicates the more tetracycline was added, the stronger fluorescence intensity of GFP as well as the weaker fluorescence intensity of BFP. The sensitivity of tetR repressible promoter was detected by measuring the fluorescence intensity of GFP at different tetracycline concentrations and different time points.</p>
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| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/0/09/JNFLS-Results-7.png" /> | + | <div class="clear"></div> |
| − | <em style="text-align:center;">Fig.6 The influence of tetracycline concentration on the fluorescence intensity of tetR fusion protein in the double plasmids containing E.coli.</em>
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| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/1/1f/JNFLS-Results-8.png" /> | + | <div class="column half_size" > |
| − | <em style="text-align:center;">Fig.7 The influence of tetracycline concentration on the fluorescence intensity of tetR repressible GFP expression in the double plasmids containing E.coli.</em> | + | |
| − | <p>The rsults showed that 20ug/ml tetracycline is the best concentration for inhibitiing of tetR expression in the double plasmids containing E.coli.</p> | + | |
| − | <div class="top_text">Section 3: Construction and function analysis of TAT plasmid in Eukaryote</div>
| + | <h5> Project Achievements </h5> |
| − | <p>TAT serves as an inducing factor in our project. TAT stands for "Trans-Activator of Transcription", serving as a trans-activating factor in HIV-1. TAT protein plays an important role in the HIV disease process. The protein is released by infected cells in culture, and is found in the blood of HIV-1 infected patients. It also can be absorbed by cells that are not infected with HIV, and act directly as a toxin producing cell death via apoptosis in uninfected. So, we constructed the TAT plasmid which can be expressed in Eukaryote to induce apoptosis of colon cancer in our project.</p>
| + | |
| − | <h2>2.1 Construction and identification of TAT expression vector</h2> | + | <p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p> |
| − | <p>TAT is synthesized by Genscript, Nanjing, China. The length of TAT is 309bp which was inserted into pUC57 plasmid. We amplied the TAT gene using PCR method, and the PCR product and pcDNA 3.1 were digested by the same double enzymes (EcoRI and PastI). After purification, TAT gene was connected with pCDNA3.1, forming TAT plasmid. Then we redigested the TAT plasmid for identification. The result is shown in Figure 8.</p> | + | |
| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/9/91/JNFLS-Results-9.png" /> | + | <ul> |
| − | <em style="text-align:center;">Figure 8. Identification of TAT plasmid.</em>
| + | <li>A list of linked bullet points of the successful results during your project</li> |
| − | <h2>2.1 Function analysis of TAT plasmid in Eukaryote</h2>
| + | <li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li> |
| − | <p>For analyzing the function of TAT plasmid in Eukaryote, we extracted TAT plasmid from E.coli, and then transfected them into the colon cancer cell line LOVO, using Lipofectamine 3000. After transfection 48h, we used DAPI staining method to detect the apoptosis of cells. From the result we can see that the transfected cells showed apoptosis obviously. The results is shown in Figure 9.</p>
| + | </ul> |
| − | <img class="medium" src="https://static.igem.org/mediawiki/2017/3/39/JNFLS-Results-10.png" /> | + | |
| − | <em style="text-align:center;">Figure 9. Apoptosis inducing function of TAT plasmid in LOVO cells. 1 and 4 are negative control which are transfected with empty vector; 2, 3, 5 and 6 are cells transfected with TAT plasmid.</em>
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| | + | <div class="column half_size" > |
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| | + | <h5>Inspiration</h5> |
| | + | <p>See how other teams presented their results.</p> |
| | + | <ul> |
| | + | <li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li> |
| | + | <li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li> |
| | + | <li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li> |
| | + | </ul> |
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| | </div> | | </div> |
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| | </html> | | </html> |
JNFLS
Results
Here you can describe the results of your project and your future plans.
What should this page contain?
- Clearly and objectively describe the results of your work.
- Future plans for the project.
- Considerations for replicating the experiments.
You should also describe what your results mean:
- Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
- Show data, but remember all measurement and characterization data must be on part pages in the Registry.
- Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.
Project Achievements
You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.
- A list of linked bullet points of the successful results during your project
- A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.
Inspiration
See how other teams presented their results.
