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− | <div class="topic"><p class="text_color">Expression | + | <div class="topic"><p class="text_color">Expression Result</p></div> |
<div class="active-circle"> | <div class="active-circle"> | ||
<div class="splash"></div> | <div class="splash"></div> | ||
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− | <img src="https://static.igem.org/mediawiki/2016/6/6b/NCTU_table1.png" class="picture" style="left: | + | <img src="https://static.igem.org/mediawiki/2016/6/6b/NCTU_table1.png" class="picture" style="left:12vw;width:50%;"> |
</div> | </div> | ||
− | <p class="content">After amplification with PCR, the PCE products of Hv1a, Sf1a, and OAIP have a length of around 500bp; and Hv1a-Lectin, Sf1a-Lectin, and OAIP-Lectin has a length of 750bp~1000bp; and Hv1a-Lectin with | + | <p class="content">After amplification with PCR, the PCE products of Hv1a, Sf1a, and OAIP have a length of around 500bp; and Hv1a-Lectin, Sf1a-Lectin, and OAIP-Lectin has a length of 750bp~1000bp; and Hv1a-Lectin with GS linker has a length of 893bp. (Figure 1a, 1b, 1c, 1d, 1e, 1f, 1g)</p> |
<div> | <div> | ||
− | <img src=" | + | <img src="https://static.igem.org/mediawiki/2016/8/81/NCTU_TTTT.png" class="picture"style="left:0vw;width:100%;"> |
− | <p class="content-image" style="text-align:justify !important;">Figure 1. Polymerase chain reaction (PCR) amplification analysis of electrophoresis of seven PCR products with Marker labeled length on the left hand (a)Hv1a (BBa_K1974011, 495 base pairs). (b)Sf1a (BBa_K1974012, 522 base pairs). (c)OAIP (BBa_K1974013, 489 base pairs). (d)Hv1a-Lectin (BBa_K1974021, 819 base pairs). (e)Sf1a-Lectin (BBa_K1974022, 846 base pairs). (f)OAIP-Lectin (BBa_K1974023, 813 base pairs). (g)Hv1a-Lectin with | + | <p class="content-image" style="text-align:justify !important;">Figure 1. Polymerase chain reaction (PCR) amplification analysis of electrophoresis of seven PCR products with Marker labeled length on the left hand (a)Hv1a (BBa_K1974011, 495 base pairs). (b)Sf1a (BBa_K1974012, 522 base pairs). (c)OAIP (BBa_K1974013, 489 base pairs). (d)Hv1a-Lectin (BBa_K1974021, 819 base pairs). (e)Sf1a-Lectin (BBa_K1974022, 846 base pairs). (f)OAIP-Lectin (BBa_K1974023, 813 base pairs). (g)Hv1a-Lectin with GS linker(BBa_K1974033, 883 base pairs).</p> |
</div> | </div> | ||
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− | <img src="https://static.igem.org/mediawiki/2016/3/3d/NCTU_Table2.png" class="picture" style="left: | + | <img src="https://static.igem.org/mediawiki/2016/3/3d/NCTU_Table2.png" class="picture" style="left:12vw;width:50%;"> |
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<div> | <div> | ||
− | <img src="" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/6/63/NCTU_FIG2_1.png" class="picture" style="left:0vw;width:100%;"> |
− | <p class="content-image"style="text-align:justify !important;">Figure 2. SDS-PAGE result of the | + | <p class="content-image"style="text-align:justify !important;">Figure 2. SDS-PAGE result of the six Pantide-expressed sonicated products compared with the unexpressed sonicated one as a control and with Marker labeled length on the left hand. A: add β-mercaptoethanol and sample buffer, B: add sample buffer. (a)Hv1a (5.3 kDa) (b)Sf1a (6.2 kDa) (c)OAIP (5.3 kDa) (d)Hv1a-Lectin (17.1 kDa) (e)Sf1a-Lectin (18.1 kDa) (f)OAIP-Lectin (17.2 kDa) </p> |
</div> | </div> | ||
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<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/8/8c/NCTU_table3.png" class="picture" style="left: | + | <img src="https://static.igem.org/mediawiki/2016/8/8c/NCTU_table3.png" class="picture" style="left:12vw;width:50%;"> |
</div> | </div> | ||
− | <p class="content">The gel of SDS-PAGE result of Hv1a, Sf1a, OAIP have a length of 5kDa~7 kDa;and Hv1a-Lectin, Sf1a-Lectin, OAIP-Lectin, and Hv1a- | + | <p class="content">The gel of SDS-PAGE result of Hv1a, Sf1a, OAIP have a length of 5kDa~7 kDa;and Hv1a-Lectin, Sf1a-Lectin, OAIP-Lectin, and Hv1a-GS linker-Lectin have a length of 17kDa~19kDa.(Figure 3a, 3b, 3c, 3d, 3e, 3f, 3g) As a result, the constructions of BioBrick were correct.</p> |
<div> | <div> | ||
− | <img src="" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/c/c7/NCTU_tttt.png" class="picture" style="left:0vw;width:100%;"> |
− | <p class="content-image"style="text-align:justify !important;">Figure 3. SDS-PAGE result of the seven purified Pantide products compared with the unpurified one | + | <p class="content-image"style="text-align:justify !important;">Figure 3. SDS-PAGE result of the seven purified Pantide products compared with the unpurified one, and with Marker labeled length on the left hand. A is the sonication product. B is the elution product of purification. (a) Hv1a (5.3 kDa) (b) Sf1a (6.2 kDa) (c) OAIP (5.3 kDa) (d) Hv1a-Lectin (17.1 kDa) (e) Sf1a-Lectin (18.1 kDa) (f) OAIP-Lectin (17.2 kDa) (g) Hv1a-GS linker-Lectin (18.2 kDa)</p> |
</div> | </div> | ||
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<p class="title">Insect Test Result</p> | <p class="title">Insect Test Result</p> | ||
<p class="content-1">Feeding Assay Pre-test</p> | <p class="content-1">Feeding Assay Pre-test</p> | ||
− | <p class="content"> To know the qualitative toxicity effect of Pantide, we prepared the samples with the sonicated LB solution lysate containing Pantide-expressed E. coli Rosetta-Gami strain and diluted it with the three concentration. This experiment is the pre-test that shows us whether the amount of Pantide is sufficient enough to perform the toxicity against the larvae. We applied the sample onto the leaf disks and put five cutworms into the separate cabinets for feeding assays. The positive control in the experiment was to apply Bacillus thuringiensis on the leaf disks, which is nowadays the most widely-used bioinsecticide, and the negative control group in the test was DD water. We preserved all the result of the remained leaves sealing with the glass paper and calculated the percentage of the remained area on the leaves. The collected data were analyzed by t–test and calculated with the p-value. The p-value is a function that measures how extreme the observation is. The widespread use of “statistical significance” (interpreted as “p ≤ 0.05”) is a license for making a claim of a scientific finding that leads to considerable distortion of the scientific process. In the picture, it shows as a star. The more the stars are, the more significant difference is. Here are the feeding assay results.</p> | + | <p class="content"> To know the qualitative toxicity effect of Pantide, we prepared the samples with the sonicated LB solution lysate containing Pantide-expressed <i>E. coli</i> Rosetta-Gami strain and diluted it with the three concentration. This experiment is the pre-test that shows us whether the amount of Pantide is sufficient enough to perform the toxicity against the larvae. We applied the sample onto the leaf disks and put five cutworms into the separate cabinets for feeding assays. The positive control in the experiment was to apply Bacillus thuringiensis on the leaf disks, which is nowadays the most widely-used bioinsecticide, and the negative control group in the test was DD water. We preserved all the result of the remained leaves sealing with the glass paper and calculated the percentage of the remained area on the leaves. The collected data were analyzed by t–test and calculated with the p-value. The p-value is a function that measures how extreme the observation is. The widespread use of “statistical significance” (interpreted as “p ≤ 0.05”) is a license for making a claim of a scientific finding that leads to considerable distortion of the scientific process. In the picture, it shows as a star. The more the stars are, the more significant difference is. Here are the feeding assay results.</p> |
<div> | <div> | ||
<p style="text-align:center;padding-top:50px;font-size:22pt;color:#F3F7F7;padding-left:2px;">Feeding Assay Pre-test of Hv1a and Hv1a-lectin</p> | <p style="text-align:center;padding-top:50px;font-size:22pt;color:#F3F7F7;padding-left:2px;">Feeding Assay Pre-test of Hv1a and Hv1a-lectin</p> | ||
<img src="https://static.igem.org/mediawiki/2016/8/81/NCTU_result_H.jpg" class="picture"> | <img src="https://static.igem.org/mediawiki/2016/8/81/NCTU_result_H.jpg" class="picture"> | ||
− | <p class="content-image" style="text-align:center !important;"> | + | <p class="content-image" style="text-align:center !important;">Figur4. Remained leaf disks in the pre-test with the Hv1a and Hv1a-lectin.</p> |
</div> | </div> | ||
<div style="margin-top:20px;"> | <div style="margin-top:20px;"> | ||
<img src="https://static.igem.org/mediawiki/2016/f/fb/NCTU_result_dos_H.jpg" class="picture"> | <img src="https://static.igem.org/mediawiki/2016/f/fb/NCTU_result_dos_H.jpg" class="picture"> | ||
− | <p class="content-image" style="text-align:center !important;"> | + | <p class="content-image" style="text-align:center !important;">Figure5. The dose response analysis of Hv1a/Hv1a-lectin.</p> |
</div> | </div> | ||
<div style="margin-top:20px;"> | <div style="margin-top:20px;"> | ||
<img src="https://static.igem.org/mediawiki/2016/b/be/Fig3-HHL.jpeg" class="picture"> | <img src="https://static.igem.org/mediawiki/2016/b/be/Fig3-HHL.jpeg" class="picture"> | ||
− | <p class="content-image" style="text-align:center !important;"> | + | <p class="content-image" style="text-align:center !important;">Figure6. The T-test analysis in different dose of Hv1a/Hv1a-lectin.</p> |
</div> | </div> | ||
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− | <p class="content" style="padding-top:40px !important;padding-bottom:30px; !important"> | + | <p class="content" style="padding-top:40px !important;padding-bottom:30px; !important">Figure4 shows the pictures of the remained leaf disks after twelve hours of feeding assays. After we had done the feeding assays on tobacco cutworms with the three dilution ratios, we measured the area with the software imageJ. Figure5 shows the percentage of the remained area on the leaf disks. The higher the bar is, the larger the remained leaves area is. Figure6 shows the p-value with the star attached to indicate P < 0.05 and two stars to indicate P < 0.01. The p-value of the T-test can be used to determine if two sets of data are significantly different from each other. The smaller the p-value is, the more significant the differences are between the two groups. The observed phenomenon can be analyzed below.</p> |
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<ul style="list-style-image:none;list-style-type:disc;"> | <ul style="list-style-image:none;list-style-type:disc;"> | ||
− | <li class="list">With the dilution of Hv1a/Hv1a-lectin, the remained leaves area decreased, which indicates the dose response. The dose response is shown in | + | <li class="list">With the dilution of Hv1a/Hv1a-lectin, the remained leaves area decreased, which indicates the dose response. The dose response is shown in figure5.</li> |
− | <li class="list">Compared Hv1a with Hv1a-lectin, the remained leaves area in the Hv1a-lectin is higher than that of Hv1a, which means the repellent efficiency of Hv1a-lectin is higher than that of Hv1a. It shows in | + | <li class="list">Compared Hv1a with Hv1a-lectin, the remained leaves area in the Hv1a-lectin is higher than that of Hv1a, which means the repellent efficiency of Hv1a-lectin is higher than that of Hv1a. It shows in figure5.</li> |
− | <li class="list">To use the p-value as an indicator of statistical significance, we compared Hv1a/Hv1a-lectin with the negative control; it shows that the remained leaf disk area of Hv1a and Hv1a-lectin are significantly different from negative control, shown in | + | <li class="list">To use the p-value as an indicator of statistical significance, we compared Hv1a/Hv1a-lectin with the negative control; it shows that the remained leaf disk area of Hv1a and Hv1a-lectin are significantly different from negative control, shown in figure6.</li> |
− | <p class="content">To sum up, the Hv1a and Hv1a-lectin work well in vivo. The effect can be roughly ranked as:<br>Hv1a-lectin > Hv1a > E. coli > Negative control</p> | + | <p class="content">To sum up, the Hv1a and Hv1a-lectin work well <i>in vivo</i>. The effect can be roughly ranked as:<br>Hv1a-lectin > Hv1a > <i>E. coli</i> > Negative control</p> |
<p class="content">We got the similar result in the Sf1a/Sf1a-lectin and OAIP/OAIP-lectin.</p> | <p class="content">We got the similar result in the Sf1a/Sf1a-lectin and OAIP/OAIP-lectin.</p> | ||
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<p style="text-align:center;padding-top:50px;font-size:22pt;color:#F3F7F7;padding-left:2px;">Feeding Assay Pre-test of Sf1a and Sf1a-lectin</p> | <p style="text-align:center;padding-top:50px;font-size:22pt;color:#F3F7F7;padding-left:2px;">Feeding Assay Pre-test of Sf1a and Sf1a-lectin</p> | ||
<img src="https://static.igem.org/mediawiki/2016/c/c4/NCTU_result_S.jpg" class="picture"> | <img src="https://static.igem.org/mediawiki/2016/c/c4/NCTU_result_S.jpg" class="picture"> | ||
− | <p class="content-image" style="text-align:center !important;"> | + | <p class="content-image" style="text-align:center !important;">Figure7. Remained leaf disks in the pre-test with the Sf1a and Sf1a-lectin.</p> |
</div> | </div> | ||
<div style="margin-top:20px;"> | <div style="margin-top:20px;"> | ||
<img src="https://static.igem.org/mediawiki/2016/a/a7/NCTU_result_dos_S.jpg" class="picture"> | <img src="https://static.igem.org/mediawiki/2016/a/a7/NCTU_result_dos_S.jpg" class="picture"> | ||
− | <p class="content-image" style="text-align:center !important;"> | + | <p class="content-image" style="text-align:center !important;">Figure8. The dose response analysis of Sf1a/Sf1a-lectin.</p> |
</div> | </div> | ||
<div style="margin-top:20px;"> | <div style="margin-top:20px;"> | ||
<img src="https://static.igem.org/mediawiki/2016/2/29/Fig6-S.jpeg" class="picture"> | <img src="https://static.igem.org/mediawiki/2016/2/29/Fig6-S.jpeg" class="picture"> | ||
− | <p class="content-image" style="text-align:center !important;"> | + | <p class="content-image" style="text-align:center !important;">Figure9. The T-test analysis in different dose of Sf1a/Sf1a-lectin.</p> |
</div> | </div> | ||
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<p style="text-align:center;padding-top:50px;font-size:22pt;color:#F3F7F7;padding-left:2px;">Feeding Assay Pre-test of OAIP and OAIP-lectin</p> | <p style="text-align:center;padding-top:50px;font-size:22pt;color:#F3F7F7;padding-left:2px;">Feeding Assay Pre-test of OAIP and OAIP-lectin</p> | ||
<img src="https://static.igem.org/mediawiki/2016/a/a0/NCTU_result_O.jpg" class="picture"> | <img src="https://static.igem.org/mediawiki/2016/a/a0/NCTU_result_O.jpg" class="picture"> | ||
− | <p class="content-image" style="text-align:center!important;"> | + | <p class="content-image" style="text-align:center!important;">Figure10. Remained leaf disks in the pre-test with the OAIP and OAIP-lectin.</p> |
</div> | </div> | ||
<div style="margin-top:20px;"> | <div style="margin-top:20px;"> | ||
<img src="https://static.igem.org/mediawiki/2016/2/2f/NCTU_result_dos_O.jpg" class="picture"> | <img src="https://static.igem.org/mediawiki/2016/2/2f/NCTU_result_dos_O.jpg" class="picture"> | ||
− | <p class="content-image" style="text-align:center !important;"> | + | <p class="content-image" style="text-align:center !important;">Figure11. The dose response analysis of OAIP/OAIP-lectin.</p> |
</div> | </div> | ||
<div style="margin-top:20px;"> | <div style="margin-top:20px;"> | ||
<img src="https://static.igem.org/mediawiki/2016/0/03/Fig9-O.jpeg" class="picture"> | <img src="https://static.igem.org/mediawiki/2016/0/03/Fig9-O.jpeg" class="picture"> | ||
− | <p class="content-image" style="text-align:center !important;"> | + | <p class="content-image" style="text-align:center !important;">Figure12. The T-test analysis in different dose of OAIP/OAIP-lectin.</p> |
</div> | </div> | ||
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+ | <div style="width:1px;height:60px;"id="GS_Linker"> | ||
+ | </div> | ||
<p class="content-1">The Fusion Protein Improvement Test</p> | <p class="content-1">The Fusion Protein Improvement Test</p> | ||
− | <p class="content">In this experiment, we compared the toxicity effect of the three toxin designs including Hv1a/Hv1a-lectin/Hv1a-lectin with GS linker to test the toxicity effect. (For more about the improved Pantide with GS linker, see the <a href="http://parts.igem.org/wiki/index.php?title=Part: | + | <p class="content">In this experiment, we compared the toxicity effect of the three toxin designs including Hv1a/Hv1a-lectin/Hv1a-lectin with GS linker to test the toxicity effect. (For more about the improved Pantide with GS linker, see the <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1974033" style="color:#44E287;">link</a>)</p> |
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/ | + | <img src="https://static.igem.org/mediawiki/2016/c/c8/Fig10-pro.jpeg" class="picture" style="width:100%;left:0.5vw !important;"> |
− | <p class="content-image" style="text-align:justify !important;"> | + | <p class="content-image" style="text-align:justify !important;">Figure13. Remained leaves in the GS-linker improvement test with the Hv1a, Hv1a-lectin, and Hv1a-lectin GS-linker improved.</p> |
</div> | </div> | ||
<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/b/b8/Fig11-pro.jpeg" class="picture"> | <img src="https://static.igem.org/mediawiki/2016/b/b8/Fig11-pro.jpeg" class="picture"> | ||
− | <p class="content-image" style="text-align:center !important;"> | + | <p class="content-image" style="text-align:center !important;">Figure14. The T-test analysis in different dose of GS-linker improvement test.</p> |
</div> | </div> | ||
− | <p class="content">To know how the length of the linker affect the function of the fusion protein, we constructed Hv1a-lectin with the original three-alanine linker and a longer GS linker. Besides, there is also a Hv1a without the linker between the repellent peptide and the lectin served as a comparative sample. | + | <p class="content">To know how the length of the linker affect the function of the fusion protein, we constructed Hv1a-lectin with the original three-alanine linker and a longer GS linker. Besides, there is also a Hv1a without the linker between the repellent peptide and the lectin served as a comparative sample. Figure13 shows the pictures of the remained leaf disks after twelve hours of feeding assays. After we had done the feeding assays on tobacco cutworms with the three dilution ratio, we measured the area with the software imageJ. Figure11 shows the ratio of the remained area on the leaf disks. The higher the bar is, the larger the remained leaves area is. The observed phenomenon can be analyzed below.</p> |
Latest revision as of 03:09, 4 November 2016