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Team:USTC CHINA/Project/Results/FurtherWork
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<h1>Introduction<h1> | <h1>Introduction<h1> | ||
| - | <p>According to our experiment result, we have proved the secretion and expression possibility of TD1-antigen, TD1-adjuvant, | + | <p>According to our experiment result, we have proved the secretion and expression possibility of TD1-antigen, TD1-adjuvant, and the antigenicity of TD1-antigen after transdermal process. So in the following experiments, we decided to utilize Bacillus subtilis WB800N as engineered bacteria, plus shuttle vector pHT43 as secretion vector to build the Bacillus subtilis secretory expression system. On top of this, we have taken advantage of different kinds of TD1-antigen, testing HBsAg, PA, Ag85b that have been applied into the market to check the universal property of TD1-antigen. Besides, reporters essential during real application have been found to realize the final circuit.<p> |
<img src="https://static.igem.org/mediawiki/2013/c/cb/Further_work.png"width="580" height="250" /> | <img src="https://static.igem.org/mediawiki/2013/c/cb/Further_work.png"width="580" height="250" /> | ||
| - | <div>< | + | <div><h2>ONE: Expression of TD1-"X" in WB800N</h2></div> |
| - | <div><p>In order to realize the secretory expression in Bacillus | + | <div><h3>1.Expression of TD1-GFP</h3></div> |
| + | <img src="https://static.igem.org/mediawiki/2013/c/c3/2013ustc-chinaWB800N-GFP.png" width="580" height="420" /> | ||
| + | <div class="atfigure" align="center" style="width:580px;font-size:14px;">Fig4. fluorescent microscope shows the expression of GFP in WB800N</div> | ||
| + | <div><h3>2.Expression of TD1-antigen/adjuvant</h3></div> | ||
| + | <div><p>In order to realize the secretory expression in Bacillus subtilis, we inserted signal peptide between promoter and the TD1-Antigen/adjuvant sequence to secrete our recombinant protein. GFP was chosen to check the reliability of this circuit. After large amounts of experiments, GFP has finally been found via fluorescence microscope.</p></div> | ||
<img src="https://static.igem.org/mediawiki/2013/f/f0/Pctc-sp-td1-antigenadjuvent.png"width="580" height="120" /> | <img src="https://static.igem.org/mediawiki/2013/f/f0/Pctc-sp-td1-antigenadjuvent.png"width="580" height="120" /> | ||
| - | <div><p>Then, three kinds of recombinant antigen, TD1-HBsAg, TD1-PA, TD1-Ag85b, and recombinant immunologic adjuvant were designed. The earliest successful | + | <div><p>Then, three kinds of recombinant antigen, TD1-HBsAg, TD1-PA, TD1-Ag85b, and recombinant immunologic adjuvant were designed. The earliest successful protein TD1-LTB can not only be observed clear stripes in SDS-PAGE, but also be proved according to HPLC-MS.</p></div> |
<img src="https://static.igem.org/mediawiki/igem.org/f/fd/20-%E6%9E%AF%E8%8D%89%E8%9B%8B%E7%99%BD%E8%A1%A8%E8%BE%BE.jpg" width="580" height="300"> | <img src="https://static.igem.org/mediawiki/igem.org/f/fd/20-%E6%9E%AF%E8%8D%89%E8%9B%8B%E7%99%BD%E8%A1%A8%E8%BE%BE.jpg" width="580" height="300"> | ||
| - | <div class="atfigure" align="center" style="width:580px"> | + | <div class="atfigure" align="center" style="width:580px;font-size:14px;">Fig5. SDS PAGE shows the expression of LTB in WB800N</div> |
| + | <div><h2>TWO: Neomycin resistance of WB800N</h2></div> | ||
| + | <div><h3>Selection on the of resistance of Bacillus subtilis WB800N against neomycin</h3> | ||
| + | <p>As WB800N is a novel secretory expression system, most of whose parameters remain unclear, among which the chief problem is that its unique resistance against neomycin has never been quantitatively analyzed, and our work made up for this blank.</p></div> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/f/f7/Xinmeisu.png" width="580" height="300"> | ||
| + | <div class="atfigure" align="center" style="width:580px;font-size:14px;">Fig6. Selection on the of resistance of Bacillus subtilis WB800N against neomycin</div> | ||
| + | <div><p>We have figured it out that the best concentration of neomycin for selection use is 128mg/ml.</p></div> | ||
</div> | </div> | ||
</div> | </div> | ||
Latest revision as of 04:44, 19 October 2013
Introduction
According to our experiment result, we have proved the secretion and expression possibility of TD1-antigen, TD1-adjuvant, and the antigenicity of TD1-antigen after transdermal process. So in the following experiments, we decided to utilize Bacillus subtilis WB800N as engineered bacteria, plus shuttle vector pHT43 as secretion vector to build the Bacillus subtilis secretory expression system. On top of this, we have taken advantage of different kinds of TD1-antigen, testing HBsAg, PA, Ag85b that have been applied into the market to check the universal property of TD1-antigen. Besides, reporters essential during real application have been found to realize the final circuit.
ONE: Expression of TD1-"X" in WB800N
1.Expression of TD1-GFP
2.Expression of TD1-antigen/adjuvant
In order to realize the secretory expression in Bacillus subtilis, we inserted signal peptide between promoter and the TD1-Antigen/adjuvant sequence to secrete our recombinant protein. GFP was chosen to check the reliability of this circuit. After large amounts of experiments, GFP has finally been found via fluorescence microscope.
Then, three kinds of recombinant antigen, TD1-HBsAg, TD1-PA, TD1-Ag85b, and recombinant immunologic adjuvant were designed. The earliest successful protein TD1-LTB can not only be observed clear stripes in SDS-PAGE, but also be proved according to HPLC-MS.
TWO: Neomycin resistance of WB800N
Selection on the of resistance of Bacillus subtilis WB800N against neomycin
As WB800N is a novel secretory expression system, most of whose parameters remain unclear, among which the chief problem is that its unique resistance against neomycin has never been quantitatively analyzed, and our work made up for this blank.
We have figured it out that the best concentration of neomycin for selection use is 128mg/ml.
