|
|
Line 228: |
Line 228: |
| | | |
| <div class="clear extra_space"></div><!-- 一个章节结束需要clear来留白 如果觉得内容间差异大再加上extra_space --> | | <div class="clear extra_space"></div><!-- 一个章节结束需要clear来留白 如果觉得内容间差异大再加上extra_space --> |
− | | + | <h2>Verification of expression</h2> |
− | <h2>Verification of expression</h2>
| + | |
| <div class="column full_size"> | | <div class="column full_size"> |
| <p>First, to affirm the system could work properly, we started from the very beginning by testing the expression of each gene in the cluster as well as of the polycistrons in each part. We constructed 10 plasmids(with pGEX backbone adapted to perform high level of expression), containing mcbA/ mcbB/ mcbC/ mcbD/ mcbE/ mcbF/ mcbG/ mcbABCD/ mcbBCD/ mcbBC respectively, transformed successfully them into E.coli BL21, then purified the protein products with his-tag via immobilized metal affinity chromatography to check if the basic protein-coding could all be realized correctly as the basis of our design for the system. We will make further confirmation that every part can express successfully due to the correct bands on the SDS-PAGE electrophoresis gel maps.</p> | | <p>First, to affirm the system could work properly, we started from the very beginning by testing the expression of each gene in the cluster as well as of the polycistrons in each part. We constructed 10 plasmids(with pGEX backbone adapted to perform high level of expression), containing mcbA/ mcbB/ mcbC/ mcbD/ mcbE/ mcbF/ mcbG/ mcbABCD/ mcbBCD/ mcbBC respectively, transformed successfully them into E.coli BL21, then purified the protein products with his-tag via immobilized metal affinity chromatography to check if the basic protein-coding could all be realized correctly as the basis of our design for the system. We will make further confirmation that every part can express successfully due to the correct bands on the SDS-PAGE electrophoresis gel maps.</p> |
Line 236: |
Line 235: |
| | | |
| <h2>Verification of function</h2> | | <h2>Verification of function</h2> |
| + | <div class="column full_size"> |
| + | <p>As our design page describes, mcbABCD should show a certain degree of restrain to both the growth and condition of its cocultured cell. While with mcbEFG, engineered E.coli are supposed to have better immunity themselves against the mccb17 coded by mcbABCD, so as to gain advantage, affect the surrounding WT’s metabolism(the expression of GFP), and better survive in the environment.</p> |
| + | <p>We performed 2 different kinds of experiments, rendering verification for both antimicrobial expressing part mcbABCD and immunity part mcbEFG, by testing the effect mccb17 may exert on the metabolism and survival state of microbes they cocultured with. |
| + | </div> |
| + | |
| + | <h3> influences on metabolism</h3> |
| <h4>Methods</h4> | | <h4>Methods</h4> |
| <div class="column two_thirds_size"> | | <div class="column two_thirds_size"> |
| <img src="https://static.igem.org/mediawiki/2020/d/d5/T--Fudan--img_flowchart1.svg" /> | | <img src="https://static.igem.org/mediawiki/2020/d/d5/T--Fudan--img_flowchart1.svg" /> |
− | Figure1. flowchart of this verification experiment
| + | Figure 1. flowchart of this verification experiment |
| </div> | | </div> |
− | <div class="column two_thirds_size"> | + | <div class="column third_size"> |
− | <p>As our design page describes, mcbABCD should show a certain degree of restrain to both the growth and condition of its cocultured cell. While with mcbEFG, engineered E.coli are supposed to have better immunity themselves against the antimicrobial peptide coded by mcbABCD, so as to gain advantage, affect the surrounding WT’s metabolism(the expression of GFP), and better survive in the environment.</p>
| + | |
| <p>We cultured WT(E.coli expressing GFP driven by plac), PxAD(E.coli expressing mcbABCD driven by P1\2\9\11\12\13)and Px(E.coli expressing mcbABCD-mcbEFG-PtetR driven by P1\2\9\11\12\13) separately at the same time. | | <p>We cultured WT(E.coli expressing GFP driven by plac), PxAD(E.coli expressing mcbABCD driven by P1\2\9\11\12\13)and Px(E.coli expressing mcbABCD-mcbEFG-PtetR driven by P1\2\9\11\12\13) separately at the same time. |
| </p><p> | | </p><p> |
Line 251: |
Line 255: |
| </div> | | </div> |
| <div class="clear"></div><!-- 一个章节结束需要clear来留白 --> | | <div class="clear"></div><!-- 一个章节结束需要clear来留白 --> |
| + | |
| <h4>Result</h4> | | <h4>Result</h4> |
| <div class="column third_size"> | | <div class="column third_size"> |
| <img src="https://static.igem.org/mediawiki/2020/1/16/T--Fudan--5mix7.jpeg" /> | | <img src="https://static.igem.org/mediawiki/2020/1/16/T--Fudan--5mix7.jpeg" /> |
| | | |
− | Figure2. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=5:7)
| + | Figure 2. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=5:7) |
| </div> | | </div> |
| <div class="column third_size"> | | <div class="column third_size"> |
| <img src="https://static.igem.org/mediawiki/2020/b/b6/T--Fudan--20mix7.jpeg" /> | | <img src="https://static.igem.org/mediawiki/2020/b/b6/T--Fudan--20mix7.jpeg" /> |
| | | |
− | Figure3. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=20:7)
| + | Figure 3. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=20:7) |
| </div> | | </div> |
| <div class="column third_size"> | | <div class="column third_size"> |
| <img src="https://static.igem.org/mediawiki/2020/8/89/T--Fudan--50mix7.jpeg" /> | | <img src="https://static.igem.org/mediawiki/2020/8/89/T--Fudan--50mix7.jpeg" /> |
| | | |
− | Figure4. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=50:7)
| + | Figure 4. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=50:7) |
| </div> | | </div> |
| <div class="column full_size"> | | <div class="column full_size"> |
| <p> Our data has been calibrated with the Excel data analysis template provided by the 2019 iGEM, where MEFL stands for the absolute units of Fluorescent Intensity for Green Fluorescent proteins and particle stands for the absolute units of cell count, so that our y axis represents fluorescent intensity per cell under iGEM standard. | | <p> Our data has been calibrated with the Excel data analysis template provided by the 2019 iGEM, where MEFL stands for the absolute units of Fluorescent Intensity for Green Fluorescent proteins and particle stands for the absolute units of cell count, so that our y axis represents fluorescent intensity per cell under iGEM standard. |
| </p> | | </p> |
− | <p>We could see an overall decrease of GFP expression in nearly all groups cocultured with Px or PxAD than the control group which only contains WT, indicating that the antimicrobial peptide(MccB17) encoded by mcbABCD does have a negative effect on over microbial. In this case, the living status of WT cells and its function in creating GFP is strongly restricted by the toxic environment, proving that our part mcbABCD has worked successfully. | + | <p>We could see an overall decrease of GFP expression in nearly all groups cocultured with Px or PxAD than the control group which only contains WT, indicating that the antimicrobial peptide(MccB17) encoded by mcbABCD does have a negative effect on microbial. In this case, the living status of WT cells and its function in creating GFP is strongly restricted by the toxic environment, proving that our part mcbABCD has worked successfully. |
| </p> | | </p> |
| <p>While inside each paired group, there clearly are a better inhibition effect in Px group than PxAD group, as the MEFL/particle is much lower in the Px group than the PxAD group when driven by certain promoters. This shows that the immunity function of mcbEFG is working successfully, as the E.coli expressing mcbEFG can help the engineered strain to survive with efflux exporting the toxic peptide, as well as better killing off other strains to gain survival advantage. | | <p>While inside each paired group, there clearly are a better inhibition effect in Px group than PxAD group, as the MEFL/particle is much lower in the Px group than the PxAD group when driven by certain promoters. This shows that the immunity function of mcbEFG is working successfully, as the E.coli expressing mcbEFG can help the engineered strain to survive with efflux exporting the toxic peptide, as well as better killing off other strains to gain survival advantage. |
Line 283: |
Line 288: |
| </div> | | </div> |
| <div class="clear"></div><!-- 一个章节结束需要clear来留白 --> | | <div class="clear"></div><!-- 一个章节结束需要clear来留白 --> |
− | <h2>每个章节标题用h2 为了搜索引擎全页面只能一个h1标签</h2>
| + | |
− | <div class="column half_size">
| + | |
− | <img src="https://static.igem.org/mediawiki/2020/8/8b/T--Fudan--map_2.svg" alt="map of calcium intake" />
| + | <h3>influnces on survival states</h3> |
| + | <h4>Methods</h4> |
| + | <div class="column two_thirds_size"> |
| + | <img src="https://static.igem.org/mediawiki/2020/6/61/T--Fudan--flowchart_kanamp.svg" /> |
| + | Figure 5. flowchart of this verification experiment |
| </div> | | </div> |
− | <div class="column half_size">
| |
− | <p>在宽屏,这部分内容占页面1/2;窄屏就是全幅。添加了column同时 图1/2 + 字1/2 正好宽屏的一幅。这个代码不在 https://2020.igem.org/Resources/Template_Documentation 但是是支持的 China has an estimated 70 million osteo
| |
− | porosis patien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| |
− | oporosis patienChina has an estimated 70 million osteoporosis patien</p>
| |
− | </div>
| |
− | <div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
| |
− |
| |
− | <h2>每个章节标题用h2 为了搜索引擎全页面只能一个h1标签</h2>
| |
| <div class="column third_size"> | | <div class="column third_size"> |
− | <img src="https://static.igem.org/mediawiki/2020/8/8b/T--Fudan--map_2.svg" alt="map of calcium intake" /> | + | <p>We cultured WT(E.coli expressing rFP driven by constitutive promoter), PxAD(E.coli expressing mcbABCD driven by P1\2\9\11\12\13)and Px(E.coli expressing mcbABCD-mcbEFG-PtetR driven by P1\2\9\11\12\13) separately at the same time. |
− | </div>
| + | </p><p> |
− | <div class="column two_thirds_size">
| + | When the OD of WT reaches 0.6, we mixed WT with PxAD or Px, with ratio of 1:1, 4:1, 10:1, then induce them with IPTG for 2h. Here, the control group would be merely WT with IPTG and WT without IPTG. |
− | <p>在宽屏,这部分内容占页面1/3;窄屏就是全幅。添加了column同时 图1/3 + 字2/3 正好宽屏的一幅。 China has an estimated 70 million osteo
| + | </p><p> |
− | porosis patien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| + | Meanwhile, we culture plates with different antibiotics(since WT and Px/PxAD possess different resistance) to measure the precise ratio of mixture. |
− | oporosis patienChina has an estimated 70 million osteoporosis patien</p>
| + | </p><p> |
| + | Finally our data is measured by plate reader. |
| + | </p> |
| </div> | | </div> |
| <div class="clear"></div><!-- 一个章节结束需要clear来留白 --> | | <div class="clear"></div><!-- 一个章节结束需要clear来留白 --> |
| | | |
− | <div class="column full_size"><!-- 全幅面的文字内容,上面是否留白可选 --> | + | <h4>Result</h4> |
− | <p>However, for the Chinese population, the bottlenecks are
| + | <div class="column half_size"> |
− | as follows:
| + | <img src="https://static.igem.org/mediawiki/2020/b/b9/T--Fudan--AUP21.jpeg" /> |
− | </p>
| + | |
− | <ol>
| + | |
− | <li>注意这里列表的写法 页面字会变深 突出内容 ol是编数字的 nts</li>
| + | |
− | <li>calcium-supplementing pills daste</li>
| + | |
− | <li>The body's absorptioes as calcium intake increases</li>
| + | |
− | <li>Vegetables conta列表的写法 页面字会变深 突出内容 ol是编数字的 ul是没有标数字的列表 </li>
| + | |
− | </ol>
| + | |
− | <p>
| + | |
− | However, the currently popular calcium supplement method on the market is either to adjust diet or to consume a lot of calcium supplementing pills. For the elderly, they suffer from poor memory or neglect the importance of calcium pills, often forgetting to consume the supplement; for children, they often refuse to take calcium pills or to maintain some diet high in calcium, thus requiring the "liquid calcium" method to ensure sufficient calcium intake. Ho可以 继续大段内容
| + | |
− | </p>
| + | |
| | | |
− | | + | Figure 6. Calculated Bacteria Counts when the ratio of mixure(Px:WT=1:1) |
− | <div class="highlight decoration_background decoration_A_top">
| + | |
− | <p>
| + | |
− | 章节内容的分段就用p开p关,额外需要关注的可以套一个highlight的框,有多种版本
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | <p>
| + | |
− | 章节内容的分段就用p开p关
| + | |
− | </p>
| + | |
− | <div class="highlight decoration_B_full">
| + | |
− | <p>
| + | |
− | 章节内容的分段就用p开p关,额外需要关注的可以套一个highlight的框,细看 https://2020.igem.org/Resources/Template_Documentation#highlight
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | <p>
| + | |
− | 章节内容的分段就用p开p关
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | 章节内容的分段就用p开p关 <a href="https://URL"> EXAM 外部链接 href尽量使用https </a>
| + | |
− | </p>
| + | |
− | <ul>
| + | |
− | <li>注意这里列表的写法 页面字会变深 突出内容 ul是没有标数字的列表 nts</li>
| + | |
− | <li>calcium-supple menting pills don’t haat taste</li>
| + | |
− | <li>Vegetables conta列表的写法 页面字会变深 突出内容 ol是编数字的 ul是没有标数字的列表 </li>
| + | |
− | </ul>
| + | |
− | <p>
| + | |
− | 章节内容的分段就用p开p关
| + | |
− | </p>
| + | |
− | </div><!-- 每个幅面结束都需要 关div -->
| + | |
− | | + | |
− | <div class="clear extra_space"></div><!-- 一个章节结束需要clear来留白 如果觉得内容间差异大再加上extra_space -->
| + | |
− | | + | |
− | <h2>又来个章节 sis</h2>
| + | |
− | <div class="column full_size">
| + | |
− | <p>引入文字 ion.</p>
| + | |
− | <p>Firstly, the product is designed for oral use and our target user is the elderly. 英文分项不需要ol或ul</p>
| + | |
− | <ol>
| + | |
− | <li>The produc</li>
| + | |
− | <li>The short pep</li>
| + | |
− | <li>The expression level of short peptides has a threshold, preventing any excessive calcium absorption that might lead to hypercalcemia 注意句子超长后的回折</li>
| + | |
− | <li>There is a kill swit.</li>
| + | |
− | </ol>
| + | |
− | | + | |
− | <p>
| + | |
− | Based on relevant research, the product is exp 章节内分段 ina. tien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| + | |
− | oporosis patienChina has an estimated 70 million osteoporosis pat
| + | |
− | </p>
| + | |
− | | + | |
− | <p>Compared w 章节内分段 ique advantages tien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| + | |
− | oporosis patienChina has an estimated 70 million osteoporosis pat tien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| + | |
− | oporosis patienChina has an estimated 70 million osteoporosis pat tien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| + | |
− | oporosis patienChina has an estimated 70 million osteoporosis pat.
| + | |
− | </p>
| + | |
− | | + | |
− | <table>
| + | |
− | <tr>
| + | |
− | <th> Header 1 这是igem hq的表格的写法 </th> <th> Header 2 </th>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td> Content A 1 </td> <td> Content B 1 </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td> Content A 2 </td> <td> Content B 2 这是igem hq的表格的写法。表格都需要说明,之前之后都会有p来文字 </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | | + | |
− | <p></p><!-- 无文字p是换行隔开,在关闭的html元素间回车是没有用的 -->
| + | |
− | | + | |
− | <table class="striped"><!-- 这是另一个版本的表格,间隔行有阴影。一个页面不建议交叉两种格式。太宽表格可以加 responsive-table 方便窄屏 -->
| + | |
− | <tr>
| + | |
− | <th>Name</th> <th>Item Name</th> <th>Item Price</th>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>Alvin</td> <td>Eclair</td> <td>$0.87</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>Alan</td> <td>Jellybean</td> <td>$3.76</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>Jonathan</td> <td>Lollipop</td> <td>$7.00</td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | | + | |
− | <p>Calciu 章节内分段 fety. tien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| + | |
− | oporosis patienChina has an estima
| + | |
− | ted 70 million osteoporosis pat tien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| + | |
− | oporosis patienChina has an estimated 70 mill
| + | |
− | ion osteoporosis pattien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| + | |
− | oporosis patienChina has an estimated 70 million osteoporosis pat
| + | |
− | </p>
| + | |
− | | + | |
− | <p>From the us 章节内分段 etition.</p>
| + | |
| </div> | | </div> |
| + | <div class="column half_size"> |
| + | <img src="https://static.igem.org/mediawiki/2020/f/f9/T--Fudan--AUP24.jpeg" /> |
| | | |
− | <div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
| + | Figure 7. Calculated Bacteria Counts when the ratio of mixure(Px:WT=1:4) |
− | | + | |
− | <h2>再来例子 Market Analysis</h2>
| + | |
− | <div class="column half_size">
| + | |
− | <p>文本在左侧一半,宽屏 According to the Chinese Dietetic Sociietary Nutrient Intakes of Chinese Residents", the calcium requirement for infants and young children increases with age, from 300mg/day to 800mg/day, and the calcium requirement for adolescents reaches 1000mg/day. The amof the required intake, with the small exception of some coastal and pastoral areas 图片在右侧一半</p>
| + | |
− | | + | |
− | <table>
| + | |
− | <tr>
| + | |
− | <th> 这是igem hq的表格的写法 </th> <th> Header 2 </th> <th> Header 3 </th>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td> Content A 1 </td> <td> Content B 1 </td> <td> Content C 1 </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td colspan=2> 列融合 </td> <td> Content C 2 这是igem hq的表格的写法。半幅或者2/3幅可以用。<br/>行融合<br/>就<br/>用br了 </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | | + | |
− | </div>
| + | |
− | <div class="column half_size">
| + | |
− | <img src="https://static.igem.org/mediawiki/2020/0/00/T--Fudan--calcium_intake_graph.svg" alt="calcium intake graph">
| + | |
| </div> | | </div> |
− |
| |
− | <div class="clear extra_space"></div><!-- 一个章节结束需要clear来留白 因为马上最后一幅,建议extra_space -->
| |
| | | |
| <div class="column full_size"> | | <div class="column full_size"> |
− | <p>每页结束需要小结,可以 ul ol 列表highlight,可以是每页第一段的另一种写法fety. tien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| + | <p> Our data has been calibrated with the Excel data analysis template provided by the 2019 iGEM. In this chart, Calculated Bacteria Counts stands for the relative increase of OD, which represents the total number of cells, and MERL, which represents the total number of WT cells. As we have screened out P2 in front, here we used the mixture ratio of P2(P2AD):WT=1:1/1:4 to find out more detailed effect that antimicrobial peptide expressing system can exert. |
− | oporosis patienChina has an estima
| + | </p> |
− | ted 70 million osteoporosis pat tien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| + | <p>We could see the population increase are not significant in total over time, yet WT grows rather fast at first in the P2AD group, indicating its lack of immunity part mcbEFG can lead to the death of engineered E.coli. more, thus shows less restrain on the WT cells. Yet, as mccb17 accumulates, a clear decrease can be observed when time extends in both P2 and P2AD, proving our antimicrobial peptide expressing system are working to help the engineered E.coli. gain better survival advantage. |
− | oporosis patienChina has an estimated 70 mi 每页结束需要小结,可以 ul ol 列表highlight,可以是每页第一段的另一种写法</p>
| + | </p> |
| </div> | | </div> |
| + | <div class="clear"></div><!-- 一个章节结束需要clear来留白 --> |
| | | |
− | <div class="column full_size"><div id="FudanSignature">
| |
− | <p>Signature: <a href="/Team:Fudan/Attributions#LiMW" class="z-depth-2">Mingwei</a>
| |
− | </p>
| |
− | </div></div>
| |
| | | |
| <!-- 此处以上可更改 | | <!-- 此处以上可更改 |