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| + | <h5>Contents</h5> |
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| + | <article> |
| + | <h1>Introduction</h1> |
| + | <p>We have made a lot of innovations in multiple aspects, including software, hardware and |
| + | experience for wet lab. Numerous basic and composite parts were also constructed, |
| + | contributing to the proliferation of synthetic biology. We hope our project can give some |
| + | help and guidance to the future iGEM teams.</p> |
| + | <h1>Parts</h1> |
| + | <p>Biology is different from other engineering fields due to a lack of modular nature of the |
| + | structure and function, which means the expression of gene will change when transferred from |
| + | one species to another. What synthetic biology is dedicated to is the modularization and |
| + | standardization of functional genes. Our project has registered 19 basic parts and 56 |
| + | composite parts, all qualified for standardized requirements of RFC[10]. We have also tested |
| + | these parts. And we hope that everyone can utilize our parts based on our research through |
| + | registration and further improve the information of our parts to make more people can use |
| + | them. For more details please refer to <a |
| + | href="https://2021.igem.org/Team:HUST-China/Parts">parts</a>.</p> |
| + | <h1>Experience of wet lab</h1> |
| + | <p>During our experiment, we summarized the experience in genetic engineering, heterologous |
| + | expression of yeast, hair dyeing and perm and so on. In addition we have compiled a <a |
| + | href="https://2021.igem.org/Team:HUST-China/Protocol">protocol</a> by our |
| + | team and a <a |
| + | href="https://static.igem.org/mediawiki/2021/9/9b/T--HUST-China--doc--YeastHandbook7.0.pdf">yeast |
| + | handbook</a> by collaborate with the Yeast alliance.</p> |
| + | <p><strong>genetic engineering:</strong></p> |
| + | <p>In the process of constructing the whole pathway, We first replaced the promoter of this |
| + | component by restriction endonuclease digestion and T4 ligase ligation. However, when we |
| + | carried out enzyme digestion, we found that the efficiency of double digestion of the two |
| + | adjacent sites was very low, so we improved the double enzyme digestion system, increased |
| + | the amount of endonuclease, prolonged the enzyme digestion time (did overnight enzyme |
| + | digestion).</p> |
| + | <p>When we were doing enzyme digestion and enzyme linking, we used the method of plasmid enzyme |
| + | digestion. But we met the situation in which the concentration of the plasmid vector was |
| + | very low. Therefore, we suggest that the products of multiple enzyme digestion should be |
| + | recovered into the same system after gel electrophoresis to increase the concentration of |
| + | the vector. The fragments amplified by PCR need to be further purified otherwise salt ions |
| + | will affect the efficiency of enzyme digestion. In addition, the fragments also need to be |
| + | further purified after enzymatic digestion to avoid affecting the efficiency of ligation. |
| + | </p> |
| + | <p>Secondly, when we were going to carry out seamless cloning and splicing, we had never been |
| + | able to succeed. The fragments we connected have long repetitions. We speculate that the |
| + | principle of seamless cloning enzymes of some companies may be different from that of Gibson |
| + | assembly. The operation and result according to the instructions provided by these companies |
| + | failed to meet expectations. Therefore, it is recommended that if seamless cloning is needed |
| + | in the design of the project, try to ensure that there are no repetitions in fragments or |
| + | use the seamless cloning kit of Gibson assembly.</p> |
| + | <p><strong>genetic engineering of yeast:</strong> In the process of expression, we used α-factor |
| + | as a signal peptide for extracellular expression, but some proteins could not be expressed |
| + | successfully. Extracellular expression of eukaryotic system is a very complex process, the |
| + | adaptations between signal peptide and protein, signal peptide and chassis, protein and |
| + | chassis are very important, but there is no accurate method for the mutual selection of the |
| + | three. After removing the signal peptide for intracellular expression, we successfully |
| + | expressed most of the proteins that could not be expressed with the signal peptide.</p> |
| + | <p><strong>The Best Condition of hair dye:</strong></p> |
| + | <table style="float:center"> |
| + | <thead> |
| + | <tr> |
| + | <th>Dye/Condition</th> |
| + | <th>Time</th> |
| + | <th>temperature</th> |
| + | <th>Dyeing aid ingredients</th> |
| + | <th>concentration(g/L)</th> |
| + | <th>comment</th> |
| + | </tr> |
| + | </thead> |
| + | <tbody> |
| + | <tr> |
| + | <td>indigo</td> |
| + | <td>2min</td> |
| + | <td>Room temperature</td> |
| + | <td>none</td> |
| + | <td>2</td> |
| + | <td>The color deepens significantly while dyeing for multiple times</td> |
| + | </tr> |
| + | <tr> |
| + | <td>curcumin</td> |
| + | <td>30min</td> |
| + | <td>50℃</td> |
| + | <td>Ethyl alcohol</td> |
| + | <td>0.5</td> |
| + | <td></td> |
| + | </tr> |
| + | <tr> |
| + | <td>lycopene</td> |
| + | <td>30min</td> |
| + | <td>Room temperature</td> |
| + | <td>alum</td> |
| + | <td>2</td> |
| + | <td></td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| + | <p>And we found that as for the three colors selected for the experiment, bleach the hair to 8 |
| + | degrees could achieve a bright coloring effect.</p> |
| + | <p><strong>The best condition of perming:</strong></p> |
| + | <p>1)immerse 10 hairs in SDS 3 times to remove charges on hair surface.</p> |
| + | <p>2)temperature:50℃</p> |
| + | <p>3)time: 1 hour</p> |
| + | <p>For more information please refer to <a |
| + | href="https://2021.igem.org/Team:HUST-China/Engineering">engineering</a> |
| + | and <a href="https://2021.igem.org/Team:HUST-China/Results">result</a>.</p> |
| + | <h1>Software</h1> |
| + | <h2>1)MCN Counter:</h2> |
| + | <div class="image"><img alt="MCN Counter" |
| + | src="https://static.igem.org/mediawiki/2021/0/0c/T--HUST-China--img--contribution--contribution-particles.png" |
| + | style="width: 50%" /> |
| + | <p>Figure 1: MCN Counter</p> |
| + | </div> |
| + | <p>We customized a pair of data bases and data tags about the micronucleus counting of broad |
| + | bean root tips. This data base includes images of hundreds of typical chromosomes and |
| + | micronuclei, and a model was trained based on this to conduct the counting of micronuclei in |
| + | root tips of broad beans, which was used to determine the toxicity of multiple dyes.</p> |
| + | <p>This tool has greatly liberated manpower and gives the heavy work of processing images to the |
| + | computer. We have opened the access of this model on GITHUB and detailed the customization |
| + | process in the MODEL section (if you are only using micronuclei identification) to ensure |
| + | that each user can train a counting model that is appropriate for their individual |
| + | experimental conditions.</p> |
| + | <h2>2)Staining predictor</h2> |
| + | <div class="image"><img alt="Staining predictor" |
| + | src="https://static.igem.org/mediawiki/2021/4/4a/T--HUST-China--img--contribution--contribution-Color_Challenge_APP.png" |
| + | style="width: 50%" /> |
| + | <p>Figure 2: Staining predictor</p> |
| + | </div> |
| + | <p>We set up several BP neural networks in the environment of python3+tensorflow+keras, which |
| + | can be used to predict hair dyeing results according to the type, concentration and time of |
| + | pigments. This network applies to virtually all molecular attachment systems. Therefore, it |
| + | is possible for later groups to customize their own predictive models according to this |
| + | network structure, thus enriching the predictive models including fiber staining, |
| + | macromolecular surface adsorption, etc. The contribution of this model lies more in the |
| + | mathematical part, which is determining the data labels and the layer activation functions. |
| + | You can learn more details in the MODEL section. For more information please refer to <a |
| + | href="https://2021.igem.org/Team:HUST-China/Model">Modeling</a>.</p> |
| + | <h1>Hardware:</h1> |
| + | <div class="image"><img alt="Fermentation tank" |
| + | src="https://static.igem.org/mediawiki/2021/a/ac/T--HUST-China--img--contribution--contribution-Fermentation_tank.png" |
| + | style="width: 50%" /> |
| + | <p>Figure 3: Fermentation tank</p> |
| + | </div> |
| + | <p>Hardware includes the fermentation bottle and the dyeing comb. Together, they make up a |
| + | product of Mr.Tony, providing the customers with a powerful device that allows them to dye |
| + | their hair entirely on their own. What's more fascinating is that our fermentation bottle is |
| + | not limited to producing our own product. Theoretically, any fermentation work that can be |
| + | carried out in our fermentation flask at room temperature and oxygen. The bottle owns the |
| + | basic needed for a fermentation device: the air pump, the agitator, and the dam-boards. |
| + | Additionally, what makes the bottle amazing is that as long as you turn the bottle upside |
| + | down, you can quickly get a pure (yeast-free) product. The specially designed filter could |
| + | filter out the yeasts and let the product into the storage chamber. This bottle provides a |
| + | possible solution to conveniently purify fermentation products. Also, the bottle is |
| + | portable, making it possible for scientists and customers to bring it to anywhere they like. |
| + | For more information please refer to <a |
| + | href="https://2021.igem.org/Team:HUST-China/Hardware">hardware</a>.</p> |
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| + | <div class="col-4 girl"><img |
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| + | <div class="col-5"> |
| + | <div class="dialogbox"> |
| + | <p>Wow! The team contributes to synthetic biology in multiple ways! I was told that |
| + | their achievement is not only related to the dedication of the team members, but |
| + | also can be attributed to <a |
| + | href="https://2021.igem.org/Team:HUST-China/Human Practices">human |
| + | practice </a>from which they drew expertise and inspiration.</p> |
| + | </div> |
| + | </div> |
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| + | <p><span>CONTACT US: </span><a href="mailto:iGEMHUSTChina@163.com">iGEMHUSTChina@163.com</a></p> |
| + | <p>Huazhong University of Sci. & Tech., Wuhan, China</p> |
| + | <p>1037# Luoyu Rd, Wuhan, P.R.China 430074</p> |
| + | <p>Copyright © <span>HUST-China </span>iGEM 2021</p> |
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− | Bronze Medal Criterion #4
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− | <p>Make a useful contribution for future iGEM teams. Use this page to document that contribution.
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