Difference between revisions of "Team:SSTi-SZGD/Demonstrate"

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<p>
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From the <!--{cn}从-->
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<a href="https://2018.igem.org/Team:SSTi-SZGD/Human_Practices">integrated human practices<!--{cn}综合人类社会实践--></a>
 +
, we understand that there is a need for safer and more effective methods for anti-aging and wrinkle removal purpose. Therefore, the way to meet these requirements is the goal of our project. We started our project by biosynthesis of a substance, hyaluronic acid (HA), which is commonly used in preparing dermal fillers in plastic surgery via synthetic biology engineering, then we developed a safe and effective anti-wrinkle product--HA microneedle. Our methods for HA biosynthesis is via using a food grade safe strain, <!--{cn}中，我们了解到需要更安全和有效的方法达到抗老化和除皱的目的。因此，满足这些需求的方式正是我们项目的目标。我们通过合成生物学技术合成了透明质酸，在整形手术中是一种常用的真皮填充的物质，于是我们开发了一种安全有效的抗皱产品——透明质酸微针。我们使用食品级的-->
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<i>B.subtilis<!--{cn}枯草芽孢杆菌--></i>
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, which has indigenous synthetic pathway for the biosynthesis of HA precursors. By cloning a HA synthase gene from <!--{cn}生物合成透明质酸，其具有透明质酸合成途径的前体物质。通过克隆-->
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<i>Streptococcus zooepidermicus<!--{cn}兽疫链球菌--></i>
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, we obtained an engineered <!--{cn}中透明质酸合成酶基因，获得了一株能够合成透明质酸的工程菌株-->
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<i>B. subtilis<!--{cn}枯草芽孢杆菌--></i>
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strain that is able to biosynthesize HA. We managed to increase HA production level by regulating some of the precursor genes in the upstream synthetic pathway. Furthermore, we cloned a leech HA hydrolytic gene into the engineered<!--{cn}。通过调控合成通路中的一些前体基因，成功提高了透明质酸的产量。此外，我们克隆了水蛭水解酶基因到工程菌株-->
 +
<i>B.subtilis<!--{cn}枯草芽孢杆菌--></i>
 +
strain so that low molecular weight HA can be produced for the purpose of microneedle fabrication. By covalently cross-linking HA molecules, a stabilized hydrogel is obtained that serves the starting material for microneedle production. <!--{cn}以生产低分子量透明质酸用于制备微针。通过共价交联透明质酸分子，得到了一种稳定的水凝胶作为制备微针的材料。-->
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</section>
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<!--What we have achieved? -->
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<section class="achieved col-xs-12 col-sm-10">
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<p class="title"><span>What we have achieved?<!--{cn}我们实现了什么？--></span></p>
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<div class="content">
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<p>
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In summary, we demonstrate that in this project we achieved the following results<!--{cn} 综上所述，我们证明在这个项目中我们取得了以下成果-->
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</p>
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<p>
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<ul>
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<li>We successfully constructed a recombinant B.subtilis 168E strain that secretes high-molecular-weight HA. In addition, by overexpressing some of the precursors gene in the HA synthesis pathway, HA production level was elevated.<!--{cn}我们成功构建了一株分泌高分子量透明质酸的重组枯草芽孢杆菌168E菌株。此外，过表达透明质酸合成通路中的前体基因，可以提高透明质酸的产量。--></li>
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<li>We further obtained a recombinant B.subtilis 168 strain that directly secretes low-molecular-weight HA in large quantity by cloning leech HA hydrolase LHAyal gene.<!--{cn}我们进一步获得了重组枯草芽孢杆菌168，克隆水蛭水解酶LHAyal基因，直接大量分泌出低分子量透明质酸。--></li>
 +
<li>Using our purified and freeze-dried HA powder, we successfully fabricated HA microneedles that is an innovative design in dermal filler development.<!--{cn}使用我们纯化和冻干后的粉末，我们成功制备了透明质酸微针，在皮肤填充剂开发中是一种创新性的设计。--></li>
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</ul>
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</p>
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<div class="img col-xs-12 col-sm-9">
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<img src="https://static.igem.org/mediawiki/2018/b/b8/T--SSTi-SZGD--demonstrate_achieved.png"/>
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<div class="clearfix"></div>
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</div>
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</section>
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<!--note-->
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<section class="note col-xs-12 col-sm-10">
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<div class="card_title">
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<div class="col-xs-4 active">
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<span>① High-molecular weight HA<!--{cn}① 高分子量透明质酸--></span>
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</div>
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<div class="col-xs-4">
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<span>② Leech hyaluronidase<!--{cn}② 水蛭透明质酸水解酶--></span>
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</div>
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<div class="col-xs-4">
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<span>③ Low-molecular-weight HA<!--{cn}③ 低分子量透明质酸--></span>
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</div>
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<div class="clearfix"></div>
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</div>
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<div class="content card_content">
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<p>
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We successfully constructed a recombinant<!--{cn}我们成功构建了-->
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<i>B.subtilis<!--{cn}枯草芽孢杆菌--></i>
 +
168E strain, containing a HA synthase (<!--{cn}包含了透明质酸合成酶（-->
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<i>hasA</i>
 +
) gene, to produce HMW-HA, and overexpressed some of the precursor genes (<!--{cn}）基因生产高分子量的透明质酸，并成功过表达了透明质酸合成通路中的前体基因（-->
 +
<i>tuaD, gtaB, glmU</i>
 +
) obtained from the HA synthesis pathway. CTAB methods verified that overexpression of the precursor genes further helped increasing HA production. Molecular weight analysis showed that the increase of HA yield did not affect the molecular weight. We also purified and freeze-dried our HA product!<!--{cn}）。CTAB方法验证了前体基因的过表达进一步提高了透明质酸的产量。分子量分析表明，透明质酸的产量增加并不影响分子量。我们还净化和冻干了我们的透明质酸产品！-->
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</p>
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<div class="img">
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<div class="col-xs-12 col-sm-4">
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<img src="https://static.igem.org/mediawiki/2018/0/08/T--SSTi-SZGD--demonstrate_note.jpeg"/>
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<p>
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Effects of
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<i>hasA</i>
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and precursors genes on HA production
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</p>
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</div>
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<div class="col-xs-12 col-sm-4">
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<img src="https://static.igem.org/mediawiki/2018/9/9b/T--SSTi-SZGD--demonstrate_note2.jpeg"/>
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<p>
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Molecular weight analysis of HA products
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</p>
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</div>
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<div class="col-xs-12 col-sm-4">
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<img src="https://static.igem.org/mediawiki/2018/e/ed/T--SSTi-SZGD--demonstrate_note3.jpeg"/>
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<p>
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Our purified and freeze-dried HA powder
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</p>
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</div>
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<div class="clearfix"></div>
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</div>
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</div>
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<div class="content card_content">
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<p>
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We successfully expressed recombinant leech hyaluronidase (<!--{cn}我们成功表达了水蛭透明质酸酶（-->
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<i>LHAyal</i>
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) in<!--{cn}）在重组-->
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<i>B. subtilis<!--{cn}枯草芽孢杆菌--></i>
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168. Enzyme activity of LHAase was visualized using a HA agarose plate assay and verified by DNS reducing sugar assay. With its confirmed hydrolase activity, we were one step closer to in vivo hydrolysis of high molecular weight HA.<!--{cn}168。用透明质酸琼脂糖平板法观察透明质酸水蛭水解酶活性，用DNS还原糖法验证。通过证实其水解酶活性，我们离高分子量透明质酸在体内的水解又近了一步。-->
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</p>
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<div class="img">
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<div class="col-xs-12 col-sm-4">
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<img src="https://static.igem.org/mediawiki/2018/a/a2/T--SSTi-SZGD--demonstrate_note4.jpeg"/>
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<p>
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LHAase enzyme activity analysis (DNS reducing sugar)
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</p>
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</div>
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<div class="col-xs-12 col-sm-4">
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<img src="https://static.igem.org/mediawiki/2018/4/4e/T--SSTi-SZGD--demonstrate_note5.jpeg"/>
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<p>
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LHAase enzyme activity vs. Bacterial growth
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</p>
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</div>
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<div class="col-xs-12 col-sm-4">
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<img src="https://static.igem.org/mediawiki/2018/0/00/T--SSTi-SZGD--demonstrate_note6.jpeg"/>
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<p>
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LHAase enzyme activity visualized by HA agarose plate
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</p>
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</div>
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<div class="clearfix"></div>
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</div>
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</div>
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<div class="content card_content">
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<p>
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We cloned leech LHAyal gene into the recombinant<!--{cn}我们将水蛭-->
 +
<i>B.subtilis<!--{cn}枯草芽孢杆菌--></i>
 +
168E strain and successfully made it hydrolyzing HMW-HA to become LMW-HA. Reduction in the molecular weight of HA was confirmed by gel chromatography (GPC-RI-MALS). CTAB results showed an increasing LMW-HA concentrations, reflecting a decrease of the viscosity of the fermentation liquid. Therefore, we successfully produced LMW-HA in<!--{cn}基因克隆到重组枯草芽孢杆菌168E并且成功水解高分子量透明质酸变成低分子量透明质酸。凝胶色谱法(GPC-RI-MALS)证实了透明质酸分子量的降低。CTAB法的结果显示低分子量透明质酸浓度增加，反映发酵液粘度降低。因此，我们在-->
 +
<i>B.subtilis<!--{cn}枯草芽孢杆菌--></i>
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!<!--{cn}中成功生产了低分子量透明质酸!-->
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</p>
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<div class="img">
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<div class="col-xs-12 col-sm-6">
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<img src="https://static.igem.org/mediawiki/2018/b/b8/T--SSTi-SZGD--demonstrate_note7.jpeg"/>
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<p>
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Molecular weight analysis of hydrolyzing HA
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</p>
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</div>
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<div class="col-xs-12 col-sm-6">
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<img src="https://static.igem.org/mediawiki/2018/8/82/T--SSTi-SZGD--demonstrate_note8.jpeg"/>
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<p>
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Analysis of HMW-HA and LMW-HA concentrations
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</p>
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</div>
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<div class="clearfix"></div>
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</div>
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</div>
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<div class="content card_content">
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    <p>
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    We covalently cross-linked HA molecules (cHA) to make hydrogel powder, and designed a specially crafted micromold. By decoding the optimal weight ratio of HA-cHA (5:1) to make a hydrogel mixture, we successfully fabricated a HA microneedle patch with good mechanical strength for skin epidermal penetration.<!--{cn}我们通过共价交联透明质酸分子(cHA)制备水凝胶粉末，并精心设计了一种特殊的微模板。通过调试HA-cHA(5:1)的最佳比例制备水凝胶混合物，我们成功制备了一种具有良好机械强度的透明质酸微针贴片以渗透皮肤表皮层。-->
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</p>
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<div class="img">
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<div class="col-xs-12 col-sm-4">
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<img src="https://static.igem.org/mediawiki/2018/9/96/T--SSTi-SZGD--demonstrate_note9.jpeg"/>
 +
<p>
 +
The cross-linked HA hydrogel powder
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</p>
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</div>
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<div class="col-xs-12 col-sm-4">
 +
<img src="https://static.igem.org/mediawiki/2018/7/71/T--SSTi-SZGD--demonstrate_note10.jpeg"/>
 +
<p>
 +
Demoulding of HA microneedle patch after overnight curing
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</p>
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</div>
 +
<div class="col-xs-12 col-sm-4">
 +
<img src="https://static.igem.org/mediawiki/2018/7/77/T--SSTi-SZGD--demonstrate_note11.jpeg"/>
 +
<p>
 +
    Display of our HA microneedles!
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</p>
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</div>
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<div class="clearfix"></div>
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</div>
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</div>
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</section>
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 +
<!--Summary-->
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<section class="col-xs-12 col-sm-10">
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<p class="title"><span>Summary<!--{cn}总结--></span></p>
 +
<div class="content">
 +
<p>
 +
To conclude, we successfully obtained three HA products with commercial values via synthetic engineering approach, and managed to obtain a miniature of the innovated cosmetic product--HA microneedles--that has a great potential in future beauty industry!<!--{cn}综上所述，我们通过生物合成工程的方法成功获得了三种具有商业价值的HA产品，并成功获得了一款创新的化妆产品——HA微针，这款产品在未来的美容行业有着巨大的潜力!-->
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</p>
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</div>
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</section>
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</div>
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Revision as of 00:50, 18 October 2018

From the integrated human practices , we understand that there is a need for safer and more effective methods for anti-aging and wrinkle removal purpose. Therefore, the way to meet these requirements is the goal of our project. We started our project by biosynthesis of a substance, hyaluronic acid (HA), which is commonly used in preparing dermal fillers in plastic surgery via synthetic biology engineering, then we developed a safe and effective anti-wrinkle product--HA microneedle. Our methods for HA biosynthesis is via using a food grade safe strain, B.subtilis , which has indigenous synthetic pathway for the biosynthesis of HA precursors. By cloning a HA synthase gene from Streptococcus zooepidermicus , we obtained an engineered B. subtilis strain that is able to biosynthesize HA. We managed to increase HA production level by regulating some of the precursor genes in the upstream synthetic pathway. Furthermore, we cloned a leech HA hydrolytic gene into the engineered B.subtilis strain so that low molecular weight HA can be produced for the purpose of microneedle fabrication. By covalently cross-linking HA molecules, a stabilized hydrogel is obtained that serves the starting material for microneedle production.

What we have achieved?

In summary, we demonstrate that in this project we achieved the following results

  • We successfully constructed a recombinant B.subtilis 168E strain that secretes high-molecular-weight HA. In addition, by overexpressing some of the precursors gene in the HA synthesis pathway, HA production level was elevated.
  • We further obtained a recombinant B.subtilis 168 strain that directly secretes low-molecular-weight HA in large quantity by cloning leech HA hydrolase LHAyal gene.
  • Using our purified and freeze-dried HA powder, we successfully fabricated HA microneedles that is an innovative design in dermal filler development.

① High-molecular weight HA
② Leech hyaluronidase
③ Low-molecular-weight HA

We successfully constructed a recombinant B.subtilis 168E strain, containing a HA synthase ( hasA ) gene, to produce HMW-HA, and overexpressed some of the precursor genes ( tuaD, gtaB, glmU ) obtained from the HA synthesis pathway. CTAB methods verified that overexpression of the precursor genes further helped increasing HA production. Molecular weight analysis showed that the increase of HA yield did not affect the molecular weight. We also purified and freeze-dried our HA product!

Effects of hasA and precursors genes on HA production

Molecular weight analysis of HA products

Our purified and freeze-dried HA powder

We successfully expressed recombinant leech hyaluronidase ( LHAyal ) in B. subtilis 168. Enzyme activity of LHAase was visualized using a HA agarose plate assay and verified by DNS reducing sugar assay. With its confirmed hydrolase activity, we were one step closer to in vivo hydrolysis of high molecular weight HA.

LHAase enzyme activity analysis (DNS reducing sugar)

LHAase enzyme activity vs. Bacterial growth

LHAase enzyme activity visualized by HA agarose plate

We cloned leech LHAyal gene into the recombinant B.subtilis 168E strain and successfully made it hydrolyzing HMW-HA to become LMW-HA. Reduction in the molecular weight of HA was confirmed by gel chromatography (GPC-RI-MALS). CTAB results showed an increasing LMW-HA concentrations, reflecting a decrease of the viscosity of the fermentation liquid. Therefore, we successfully produced LMW-HA in B.subtilis !

Molecular weight analysis of hydrolyzing HA

Analysis of HMW-HA and LMW-HA concentrations

We covalently cross-linked HA molecules (cHA) to make hydrogel powder, and designed a specially crafted micromold. By decoding the optimal weight ratio of HA-cHA (5:1) to make a hydrogel mixture, we successfully fabricated a HA microneedle patch with good mechanical strength for skin epidermal penetration.

The cross-linked HA hydrogel powder

Demoulding of HA microneedle patch after overnight curing

Display of our HA microneedles!

Summary

To conclude, we successfully obtained three HA products with commercial values via synthetic engineering approach, and managed to obtain a miniature of the innovated cosmetic product--HA microneedles--that has a great potential in future beauty industry!

Copyright © 2018 SSTi-SZGD All Rights Reserved.
Copyright © 2018 SSTi-SZGD All Rights Reserved.