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| − | <h1>Verification and improvement of the antimicrobial peptide expressing system</h1> | + | <h2>Improvement of the antimicrobial peptide expressing system</h2> |
| | <div class="column full_size"> | | <div class="column full_size"> |
| − | <p>Here, we tried to improve the former antimicrobial peptide( mccb17) expressing system of 2019 Fudan <a href="http://parts.igem.org/Part:BBa_K3245010 "> LINK </a>.By dividing into the peptide expressing part and the immunity part, we firstly tested whether the polycistron could work properly and separately, then tried to manipulate each of their expression levels with more efficiency an better function. | + | <p>Here, we tried to improve the former antimicrobial peptide ( Mccb17) expression, based on 2019 Fudan <a href="http://parts.igem.org/Part:BBa_K3245010"> BBa_K3245010</a>. By dividing into the peptide expressing part and the immunity part, we firstly tested whether the polycistron could work properly and separately, then tried to manipulate each of their expression levels for better results. |
| | </p> | | </p> |
| | </div> | | </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 --> | + | <div class="clear"></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>Problems of this system in the past</h2>
| |
| | <div class="column full_size"> | | <div class="column full_size"> |
| − | <p>The concept of quorum sensing system described above was proposed by Fudan 2019. Since MccB17 plays a vital role in the entire quorum sensing system, the better the MccB17 is expressed, the faster the engineered bacteria will achieve the balance of quorum sensing. However, we found that the expression level of MccB17 tested by Team Fudan 2019 last year was so low that the engineered bacteria cannot be competitive enough in the intestine.</p>
| + | <h4>Verification of the expression</h4> |
| | + | <p>First, to affirm the system could work properly, we started from the very beginning by testing the expression of each mcb gene in the cluster as well as of the polycistrons in each part. We constructed 10 plasmids (with pGEX backbone adapted to perform IPTG induced expression), containing <i>mcbA/ mcbB/ mcbC/ mcbD/ mcbE/ mcbF/ mcbG/ mcbABCD/ mcbBCD/ mcbBC</i> respectively, transformed successfully them into <i>E. coli</i> BL21. We have made further confirmation that every part can be induced successfully due to the correct bands on the SDS-PAGE gels, only shown in IPTG induced samples. If needed, we could purify these protein products via immobilized Ni affinity chromatography (GST was replaced with His-tag in our constructs). |
| | + | </p> |
| | </div> | | </div> |
| − | <div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
| |
| | | | |
| − | <h2>Improving the system</h2>
| + | <div class="column full_size"> |
| − | <div class="column full_size"> | + | <h4>Verification of the function</h4> |
| − | <p>To solve the problem, we have proposed some improvement plans. We though about using the high-copy plasmids to express MccB17, but they are more difficult to regulate and response sensitively, and will greatly increase the expression burden of engineered bacteria.
| + | <p>As <a href="/Team:Fudan/Design">our design page</a> describes, McbABCD should show a certain degree of restrain to both the growth and metabolism of its co-cultured cells. While with McbEFG, engineered <i>E. coli</i> are supposed to have better immunity themselves against the MccB17 coded by <i>mcbABCD</i>, so as to gain advantage, affect the surrounding WT’s metabolism (expressing of GFP in our experiments), and better survive in time.</p> |
| − | | + | <p>We performed 2 different kinds of experiments, verifying 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 co-cultured with.</p> |
| − | By researching on the literature and talking with Teacher Lin(iHP), who is engaged in prokaryotic expression work at the Fudan University Academy of Sciences, we decided to use low-copy plasmids to reduce the expression burden of engineered bacteria. We tried to keep the main part of MccB17 (McbABCD) a relatively low expression and high express the immune part (McbEFG) to achieve high secretion of MccB17.
| + | </div> |
| − | </p><p> | + | |
| − | The highly expressed channel proteins and immune proteins will export more MccB17 and reduce the accumulation of antimicrobial peptides in the engineered bacteria cells, thereby inducing them to produce more antimicrobial peptides.
| + | |
| − | </p><p> | + | |
| − | Lin also recommended a series of promoters from a kit which contains combinations of specific constitutive bacterial promoters that vary in strength to us in order to have the expression of mcbABCD in better performance.</p>
| + | |
| − | </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>Verification of expression</h2>
| + | <h4>Influences on metabolism</h4> |
| − | <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 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>
| + | |
| − | </div>
| + | |
| − | <div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
| + | |
| − | | + | |
| − | <h2>Verification of function</h2>
| + | |
| − | <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 " alt="flowchart IPTG" /> |
| − | Figure1. flowchart of this verification experiment
| + | <h6>Figure 1. Flowchart of the IPTG induction experiments</h6> |
| | </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 (<i>E. coli</i> expressing GFP driven by Plac), PxAD (<i>E. coli</i> expressing <i>mcbABCD</i> driven by P1\2\9\11\12\13) and Px (<i>E. coli</i> expressing mcbABCD-mcbEFG-PtetR driven by P1\2\9\11\12\13) separately with similar OD. |
| − | <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> |
| − | 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. | + | 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 2 hours. Here, the control group would be merely WT with IPTG and WT without IPTG. |
| | </p><p> | | </p><p> |
| − | After mixture, we immediately put the engineered E.coli on ice to cease the proliferation and measure their OD later for a precise mixture ratio. Thus the ratio in the graph down below has been adjusted to be more accurate in the ratio of cell number in the mixture, different from the ratio on the left. Finally our data is measured by plate reader. | + | After mixture, we immediately put the starting cultures on ice to cease the proliferation and measure their OD for a precise mixture ratio. Thus, the ratio in the graph down below has been adjusted to be more accurate in the ratio of cell numbers in the mixture (different from the ratio on the flowchart). Finally, all samples were measured by a plate reader. |
| | </p> | | </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 --> |
| − | <h4>Result</h4>
| + | |
| | + | <h4>Results from IPTG induction experiments</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 " alt="results IPTG 5 7" /> |
| − | | + | <h6>Figure 2. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=5:7</h6> |
| − | Figure2. 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" alt="results IPTG 20 7" /> |
| − | | + | <h6>Figure 3. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=20:7</h6> |
| − | Figure3. 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" alt="results IPTG 50 7" /> |
| | + | <h6>Figure 4. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=50:7</h6> |
| | + | </div> |
| | | | |
| − | Figure4. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=50:7)
| |
| − | </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 Measurement Committee, 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 co-cultured with Px or PxAD than the control group which only contains WT, indicating that the antimicrobial peptide (MccB17) encoded by <i>mcbABCD</i> 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 unsuitable environment, proving that our part <i>mcbABCD</i> works as designed. |
| | </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 <i>mcbEFG</i> 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> | | </p> |
| | + | |
| | + | <p>Among the three graphs, it is also obvious the system works better when the McbABCD population occupies a larger percentage. When the ratio of WT:Px meets 50:7, nealy all of the groups shows very weak competitiveness or even no survival advantage at all (as in the case of P1), indicating the need of a certain amount of McbABCD population to get the system working efficiently. This result also affirms it is vital to have the antimicrobial system in our design to gain enough survival advantage for our engineered <i>E. coli</i> to colony in human intestine and further express the desired product. |
| | + | </p> |
| | + | <p>Due to the difference in strength of these promoters (P1 to P14) driven the expression of <i>mcbABCD</i>, our initial test suggested the most suitable promoter P2 for the system. |
| | + | </div> |
| | + | |
| | + | <div class="clear"></div><!-- 一个章节结束需要clear来留白 --> |
| | + | |
| | + | <h4>Influnces on survival</h4> |
| | + | <div class="column two_thirds_size"> |
| | + | <img src="https://static.igem.org/mediawiki/2020/6/61/T--Fudan--flowchart_kanamp.svg" alt="flowchart time-course" /> |
| | + | <h6>Figure 5. Flowchart of the survial (time-course) experiments</h6> |
| | + | </div> |
| | + | |
| | + | <div class="column third_size"> |
| | + | <p>We cultured WT (<i>E. coli</i> expressing RFP driven by a constitutive promoter), PxAD (<i>E. coli</i> expressing <i>mcbABCD</i> driven by P1\2\9\11\12\13) and Px (<i>E. coli</i> expressing mcbABCD-mcbEFG-PtetR driven by P1\2\9\11\12\13) separately at the same time.</p> |
| | + | <p>When the OD of WT reaches 0.6, we mixed WT with PxAD or Px, with ratio of 1:1, 4:1. Later, we collect samples from the mixture at different time points, to be measured by a plate reader.</p> |
| | + | <p>Meanwhile, we plate the mixture onto plates with different antibiotics (since WT and Px/PxAD possess different antibiotic resistance) to measure the precise ratio of mixture.</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 --> |
| − | <h4>Discussion</h4> | + | |
| | + | <h4>Results from survial experiments</h4> |
| | + | |
| | + | <div class="row"> |
| | + | <div class="col m2 l3 hide-on-small-only"> </div> |
| | + | <div class="col s12 m8 l6"> |
| | + | <img src="https://static.igem.org/mediawiki/2020/b/b9/T--Fudan--AUP21.jpeg" alt="time-course results" style="width:100%" /> |
| | + | <h6>Figure 6. Calculated Bacteria Counts with the ratio of mixure Px/PxAD:WT=1:1</h6> |
| | + | </div> |
| | + | </div> |
| | + | |
| | <div class="column full_size"> | | <div class="column full_size"> |
| − | <p>Among the three graphs, it is also obvious the system works better when the population occupies a larger percentage. When the ratio of WT:Px meets 50：7, nealy all of the groups shows very weak competitiveness or even no survival advantage at all(as in the case of P1), indicating the need of a certain amount of population to get the system working efficiently. This result also affirms it is vital to have the antimicrobial system in our design to gain enough survival advantage for our engineered E.coli to colony in human intestine and further express the desired product. | + | <p> Our data has been calibrated with the Excel data analysis template provided by the 2019 iGEM Measurement Committee. 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 to find out more detailed effect that antimicrobial peptide expressing system can exert.</p> |
| | + | <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 <i>E. coli</i>. 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, suggesting our antimicrobial peptide expressing system is working to help the engineered <i>E. coli</i> gain better survival advantage. |
| | </p> | | </p> |
| − | <p>Due to the difference in strength of these promoters, we have also screened out the most suitable promoter P2 for our system via this experiment. | + | </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>
| + | |
| | + | <h2>Reduced leakage of PtetR (improved part)</h2> |
| | + | <div class="column full_size"> |
| | + | <p>We have measured the expression level of PtetO2 (<a href="http://parts.igem.org/Part:BBa_K3606006" target="_blank">BBa_K3606006</a>), PtetO3 (<a href="http://parts.igem.org/Part:BBa_K3606007" target="_blank">BBa_K3606007</a>) along with the original PtetR (<a href="http://parts.igem.org/Part:BBa_R0040" target="_blank">BBa_R0040</a>) via plate reader by combining them with GFP, when they were induced by different concentration of anhydrotetracycline (aTc) or not induced.</p> |
| | + | </div> |
| | + | |
| | <div class="column half_size"> | | <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" /> | + | <img src="https://static.igem.org/mediawiki/2020/5/51/T--Fudan--aTc_0.jpg" alt="figure 1" /> |
| | + | <h6>Figure 7. When aTc=0, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.</h6> |
| | + | |
| | + | <img src="https://static.igem.org/mediawiki/2020/d/d3/T--Fudan--aTc_200.jpg" alt="figure 3" /> |
| | + | <h6>Figure 9. When aTc=200, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.</h6> |
| | </div> | | </div> |
| | + | |
| | <div class="column half_size"> | | <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 | + | <img src="https://static.igem.org/mediawiki/2020/0/06/T--Fudan--aTc_100.jpg" alt="figure 2" /> |
| − | porosis patien China has an estimated 70 million osteoporosis patien China has an estimated 70 million osteoporosis patienChina has an estimated 70 million oste
| + | <h6>Figure 8. When aTc=100, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.</h6> |
| − | oporosis patienChina has an estimated 70 million osteoporosis patien</p>
| + | <img src="https://static.igem.org/mediawiki/2020/4/46/T--Fudan--aTc_400.jpg" alt="figure 4" /> |
| | + | <h6>Figure 10. When aTc=400, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.</h6> |
| | </div> | | </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 full_size"> |
| − | <div class="column third_size"> | + | <p>Under the induction of various concentrations of aTc, the expression level of PtetO2 is very low, but the expression level of PtetO3 is moderate. Although the expression level of PtetO3 is lower than PtetR, it is very suitable for our project design. Since PtetO3 is used to drive McbEFG, if McbEFG translates too much channel protein, then when McbEFG is turned off, the expressed channel protein will take a long time to degrade completely, reducing the sensitivity of quorum sensing.</p> |
| − | <img src="https://static.igem.org/mediawiki/2020/8/8b/T--Fudan--map_2.svg" alt="map of calcium intake" /> | + | |
| | </div> | | </div> |
| − | <div class="column two_thirds_size">
| |
| − | <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
| |
| − | 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 -->
| |
| | | | |
| − | <div class=" column full_size"> <!-- 全幅面的文字内容，上面是否留白可选 --> | + | <div class="clear"></div> |
| − | <p>However, for the Chinese population, the bottlenecks are
| + | |
| − | as follows:
| + | |
| − | </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>
| + | |
| | | | |
| | + | <div class="column third_size"> |
| | + | <img src="https://static.igem.org/mediawiki/parts/7/7a/T--Fudan--rate_100.jpg" alt="figure 5"> |
| | + | <h6>Figure 11. When aTc=100, fold of increase of promoters</h6> |
| | + | </div> |
| | + | <div class="column third_size"> |
| | + | <img src="https://static.igem.org/mediawiki/2020/a/a4/T--Fudan--rate_200.jpg" alt="figure 6"> |
| | + | <h6>Figure 12. When aTc=200, fold of increase of promoters</h6> |
| | + | </div> |
| | + | <div class="column third_size"> |
| | + | <img src="https://static.igem.org/mediawiki/2020/0/0f/T--Fudan--rate_400.jpg" alt="figure 7"> |
| | + | <h6>Figure 13. When aTc=400, fold of increase of promoters</h6> |
| | + | </div> |
| | | | |
| − | <div class="highlight decoration_background decoration_A_top"> | + | <div class="clear"></div> |
| − | <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 --> | + | <div class="column full_size"> |
| | + | <p>The results show that among the three promoters, PtetO3 has the highest fold of increase. This means that it is the most sensitive to the induction of aTc, which can improve the sensitivity of quorum sensing. |
| | + | </p> |
| | + | </div> |
| | | | |
| − | <h2>又来个章节 sis</h2>
| + | <div class="clear"></div><!-- 一个章节结束需要clear来留白 --> |
| − | <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>
| + | <h2>Demonstration of CaAP and signal-peptide guided CaAP secretion system</h2> |
| − | 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
| + | <div class="column full_size"> |
| − | oporosis patienChina has an estimated 70 million osteoporosis pat
| + | <h4>CaAP (Calcium Absorption Peptide)</h4> |
| − | </p>
| + | <p>We hope that our project can solve the problem of calcium deficiency in the elderly. According to our investigation, due to the diet structure, the calcium absorption of Chinese people is not enough, so we <a href="/Team:Fudan/Design">design</a> CaAP from the perspective of improving the calcium absorption rate of the elderly.</p> |
| | + | |
| | + | <p>We have successfully cloned our designed CaAP (Calcium Absorption Peptide) into pFAB plasmid and transformed the construct into <i>E. coli</i> BL21 strain to demonstrate its expression.</p> |
| | + | </div> |
| | | | |
| − | <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>
| |
| | | | |
| | + | <div class="column half_size"> |
| | + | <h4> Secretion Peptides guided CaAP</h4> |
| | + | <p>To promote calcium bioavailability, CaAP needs to be expressed in our probiotics and subsequently secreted into the extracellular space. Therefore, we linked 5 signal peptides which belong to the Type II secretion system in <i>E. coli</i>, to the N terminal of CaAP, respectively. |
| | + | The 5 secretion peptides are listed below:</p> |
| | <table> | | <table> |
| | <tr> | | <tr> |
| − | <th> Header 1 这是igem hq的表格的写法 </th> <th> Header 2 </th> | + | <td> NSP4 </td> <td> <a href="http://parts.igem.org/Part:BBa_K3606042" target="_blank">BBa_K3606042</a> </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| − | <td> Content A 1 </td> <td> Content B 1 </td> | + | <td> OmpA </td> <td> <a href="http://parts.igem.org/Part:BBa_K3606043" target="_blank">BBa_K3606043</a> </td> |
| | </tr> | | </tr> |
| | <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> | + | <td> DsbA </td> <td> <a href="http://parts.igem.org/Part:BBa_K3606030" target="_blank">BBa_K3606030</a> </td> |
| | </tr> | | </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> | | <tr> |
| − | <th>Name</th> <th>Item Name</th> <th>Item Price</th> | + | <td> PelB </td> <td> <a href="http://parts.igem.org/Part:BBa_K208004" target="_blank">BBa_K208004</a> </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| − | <td>Alvin</td> <td>Eclair</td> <td>$0.87</td> | + | <td> PhoA </td> <td> <a href="http://parts.igem.org/Part:BBa_K808028" target="_blank">BBa_K808028</a> </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> | | </tr> |
| | </table> | | </table> |
| | + | </div> |
| | | | |
| − | <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 | + | <div class="column half_size"> |
| − | oporosis patienChina has an estima
| + | <p> To test the secretion efficiency of our 5 secretion peptides and compare their secretion capabilities, we also fused them to MBP (Maltose Binding Protein) that normally expressed in the cytoplasm of <i>E. coli</i> cells. Driven by an IPTG inducible promoter Ptac, we can achieve quantified characterization of these secretion peptides through differences of the amount of signal-peptide-MBP fusion protein in bacterial lysate and culture medium.</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 have successfully cloned OmpA guided MBP (Maltose Binding Protein) and expressed it in <i>E. coli</i> BL21. As shown in Figure 14,the amount of MBP in the lysate shows no difference with or without IPTG induction, suggesting that OmpA has the strong ability to secrete MBP into the extracellular space. As for the relatively low amount of MBP in the culture medium concentrated by TCA than in the cell lysate (shown in Figure 15), it may be caused by other interference factors during the preparation of protein samples.</p> |
| − | oporosis patienChina has an estimated 70 mill
| + | </div> |
| − | 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 class="column half_size"> |
| − | </div>
| + | <div class="row"> |
| | + | <div class="col s2 m3 l2"></div> |
| | + | <div class="col s8 m6 l6"> |
| | + | <img src="https://static.igem.org/mediawiki/2020/0/05/T--Fudan--denoted_gel2_OmpA_MBP.png" alt="OmpA MBP lysate" style="width:75%"/> |
| | + | <h6>Figure 14. OmpA-MBP expression with and without IPTG induction in the bacterial lysate.</h6> |
| | + | </div> |
| | + | </div> |
| | + | </div> |
| | + | <div class="column half_size"> |
| | + | <div class="row"> |
| | + | <div class="col s2 m3 l2"></div> |
| | + | <div class="col s8 m6 l6"> |
| | + | <img src="https://static.igem.org/mediawiki/2020/3/3b/T--Fudan--denoted_gel3_OmpA_MBP.png" alt="1_1" style="width:100%"/> |
| | + | <h6>Figure 15. OmpA-MBP expression with and without IPTG induction in the culture medium concentrated by TCA.</h6> |
| | + | </div> |
| | + | </div> |
| | + | </div> |
| | + | <div class="column full_size"> |
| | + | <p> To test the secretion efficiency of our 5 secretion peptides fused CaAP (Calcium Absorption Peptide) guided by 5 secretion peptides, we cloned the Secretion peptide-CaAP segment into the pGEX vector respectively and transformed into <i>E. coli</i> BL21 strain. Further, quantified characterization of these secretion peptides fused with CaAP is expected to be achieved through the differences of calcium ion concentration in bacterial lysate and culture medium.</p> |
| | + | </div> |
| | | | |
| − | <div class="clear"></div><!-- 一个章节结束需要clear来留白 --> | + | <div class="row"> |
| | + | <div class="col m2 l3 hide-on-small-only"> </div> |
| | + | <div class="col s12 m8 l6"> |
| | + | <img src="https://static.igem.org/mediawiki/parts/6/67/T--Fudan--denoted_gel2_signalpeptides.png" alt="2_6" style="width:100%"> |
| | + | <h6>Figure 16. NSP4/PhoA/OmpA-CaAP expression with and without IPTG induction in the bacterial lysate.</h6> |
| | + | </div> |
| | + | </div> |
| | | | |
| − | <h2>再来例子 Market Analysis</h2> | + | <div class="row"> |
| − | <div class="column half_size"> | + | <div class="col m2 l3 hide-on-small-only"> </div> |
| − | <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> | + | <div class="col s12 m8 l6"> |
| | + | <img src="https://static.igem.org/mediawiki/parts/0/0d/T--Fudan--File-T--Fudan--denoted_gel3_signalpeptides.png" alt="2_6" style="width:100%"> |
| | + | <h6>Figure 17. NSP4/PhoA/OmpA-CaAP expression expression with and without IPTG induction in the culture medium concentrated by trichloroacetic acid (TCA) precipitation.</h6> |
| | + | </div> |
| | + | </div> |
| | | | |
| − | <table> | + | <div class="column full_size"> |
| − | <tr> | + | <p>Please note that our modeling results are at <a href="/Team:Fudan/Model">/Team:Fudan/Model</a>, and our online survey results are at <a href="/Team:Fudan/Human_Practices">/Team:Fudan/Human_Practices</a>.</p> |
| − | <th> 这是igem hq的表格的写法 </th> <th> Header 2 </th> <th> Header 3 </th>
| + | </div> |
| − | </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="clear"></div><!-- 一个章节结束需要clear来留白 --> |
| − | <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 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">
| + | |
| − | <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
| + | |
| − | 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 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>
| + | |
| − | </div>
| + | |
| | | | |
| | <div class="column full_size"><div id="FudanSignature"> | | <div class="column full_size"><div id="FudanSignature"> |
| − | <p>Signature: <a href="/Team:Fudan/Attributions#LiMW" class="z-depth-2">Mingwei</a> | + | <p>Signature: |
| | + | <a href="/Team:Fudan/Attributions# ZhangZR" class="z-depth-2"> Zhenru</a> |
| | + | <a href="/Team:Fudan/Attributions#ZhangGC" class="z-depth-2">Gaochen</a> |
| | </p> | | </p> |
| | </div></div> | | </div></div> |
| Line 480: |
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| | <div class="container"><div class="row"> | | <div class="container"><div class="row"> |
| | <div class="col m3 s12 row" | | <div class="col m3 s12 row" |
| − | style="margin-top:4rem" > | + | style="margin-top:4rem" tabIndex="0" > |
| | <a href="http://www.yfc.cn/" target="_blank"><img class="col s3 m6 l4 FudanSponsors" alt="Yunfeng Capital" src="https://static.igem.org/mediawiki/2020/b/ba/T--Fudan--YFcapital.svg"> </a> | | <a href="http://www.yfc.cn/" target="_blank"><img class="col s3 m6 l4 FudanSponsors" alt="Yunfeng Capital" src="https://static.igem.org/mediawiki/2020/b/ba/T--Fudan--YFcapital.svg"> </a> |
| | <a href="http://www.fudan.edu.cn/en/" target="_blank"><img alt="Fudan University" class="col s3 m6 l4 FudanSponsors" src="https://static.igem.org/mediawiki/2020/9/9d/T--Fudan--sponsor2.svg"> </a> | | <a href="http://www.fudan.edu.cn/en/" target="_blank"><img alt="Fudan University" class="col s3 m6 l4 FudanSponsors" src="https://static.igem.org/mediawiki/2020/9/9d/T--Fudan--sponsor2.svg"> </a> |
| Line 490: |
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| | <div class="col s12 l6 row"> | | <div class="col s12 l6 row"> |
| | <div class="col s12 m4"> | | <div class="col s12 m4"> |
| − | <span><a href="/Team:Fudan/Description#Project">Project</a></span> | + | <span tabIndex="2"><a href="/Team:Fudan/Description#Project">Project</a></span> |
| | <ul><li><a href="/Team:Fudan/Description">Description</a></li> | | <ul><li><a href="/Team:Fudan/Description">Description</a></li> |
| | <li><a href="/Team:Fudan/Sustainable">Sustainable</a></li> | | <li><a href="/Team:Fudan/Sustainable">Sustainable</a></li> |
| Line 498: |
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| | </ul></div> | | </ul></div> |
| | <div class="col s12 m4"> | | <div class="col s12 m4"> |
| − | <span><a href="/Team:Fudan/Notebook">Notebook</a></span> | + | <span tabIndex="3"><a href="/Team:Fudan/Notebook">Notebook</a></span> |
| | <ul><li><a href="/Team:Fudan/Safety">Safety</a></li> | | <ul><li><a href="/Team:Fudan/Safety">Safety</a></li> |
| | <li><a href="/Team:Fudan/Poster">Poster</a></li> | | <li><a href="/Team:Fudan/Poster">Poster</a></li> |
| Line 506: |
Line 491: |
| | </ul></div> | | </ul></div> |
| | <div class="col s12 m4"> | | <div class="col s12 m4"> |
| − | <span><a href="/Team:Fudan/Results">Results</a></span> | + | <span tabIndex="4"><a href="/Team:Fudan/Results">Results</a></span> |
| | <ul><li><a href="/Team:Fudan/Contribution">Contribution</a></li><!-- replace Judging --> | | <ul><li><a href="/Team:Fudan/Contribution">Contribution</a></li><!-- replace Judging --> |
| | <li><a href="/Team:Fudan/Implementation">Implementation</a></li> | | <li><a href="/Team:Fudan/Implementation">Implementation</a></li> |
| Line 516: |
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| | <div class="col s12 l6 row"> | | <div class="col s12 l6 row"> |
| | <div class="col s12 m4"> | | <div class="col s12 m4"> |
| − | <span><a href="/Team:Fudan/Parts">Parts</a></span> | + | <span tabIndex="5"><a href="/Team:Fudan/Parts">Parts</a></span> |
| | <ul><li><a href="/Team:Fudan/Parts#Basic">Basic part</a></li> | | <ul><li><a href="/Team:Fudan/Parts#Basic">Basic part</a></li> |
| | <li><a href="/Team:Fudan/Parts#Composite">Composite part</a></li> | | <li><a href="/Team:Fudan/Parts#Composite">Composite part</a></li> |
| Line 523: |
Line 508: |
| | </ul></div> | | </ul></div> |
| | <div class="col s12 m4"> | | <div class="col s12 m4"> |
| − | <span><a href="/Team:Fudan/Human_Practices">Outreach</a></span> | + | <span tabIndex="6"><a href="/Team:Fudan/Human_Practices">Outreach</a></span> |
| | <ul><li><a href="/Team:Fudan/Human_Practices#Integrated">Integrated HP</a></li> | | <ul><li><a href="/Team:Fudan/Human_Practices#Integrated">Integrated HP</a></li> |
| | <li><a href="/Team:Fudan/Education">Education</a></li> | | <li><a href="/Team:Fudan/Education">Education</a></li> |
| Line 530: |
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| | </ul></div> | | </ul></div> |
| | <div class="col s12 m4"> | | <div class="col s12 m4"> |
| − | <span><a href="/Team:Fudan/Team">Team</a></span> | + | <span tabIndex="7"><a href="/Team:Fudan/Team">Team</a></span> |
| | <ul><li><a href="/Team:Fudan/Team">Members</a></li> | | <ul><li><a href="/Team:Fudan/Team">Members</a></li> |
| | <li><a href="/Team:Fudan/Attributions">Attributions</a></li> | | <li><a href="/Team:Fudan/Attributions">Attributions</a></li> |
| | <li><a href="/Team:Fudan/Partnership">Partnership</a></li> | | <li><a href="/Team:Fudan/Partnership">Partnership</a></li> |
| | <li><a href="/Team:Fudan/Acknowledgement">Acknowledgement</a></li> | | <li><a href="/Team:Fudan/Acknowledgement">Acknowledgement</a></li> |
| − | <li><a href="https://2019.igem.org/Team:Fudan-TSI/Heritage" target="_blank">Heritage</a></li> | + | <li><a href="/Team:Fudan/Heritage">Heritage</a></li> |
| | </ul></div> | | </ul></div> |
| | <div class="col s12 m4"> </div> | | <div class="col s12 m4"> </div> |
