Difference between revisions of "Team:SSTi-SZGD/Demonstrate"
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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--> | 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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| − | + | <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> | |
| − | + | <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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<img src="https://static.igem.org/mediawiki/2018/b/b8/T--SSTi-SZGD--demonstrate_achieved.png"/> | <img src="https://static.igem.org/mediawiki/2018/b/b8/T--SSTi-SZGD--demonstrate_achieved.png"/> | ||
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Latest revision as of 15:45, 7 November 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.
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!
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- Application
- Applied Design
- Entrepreneurship
- Demonstrate
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- Human Practices
- Integrated Human Practices
- Public Engagement & Out Reach
- Awards
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- Acknowledgements
- Team
- Collaborations
- Attributions