Team:USTC CHINA/Project/Design

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         <div class="basic-bar">
         <div class="basic-bar">
         <h2>Introduction</h2>
         <h2>Introduction</h2>
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         <p>Our T-vaccine consists four modules. Each of them carries a gene circuit independently. The first one expresses TD1-Antigen fusion protein, as the core of our vaccine. The second one expresses TD1-Anjuvant fusion proteins, which enhance the antigenicity. The third one is reporter, it notifies users whether the status of vaccine patch is all right and when they can stick the patches to arms. The last module is a kill switch,we designed a reliable suicide system in bacillus subtilis for the very first time in iGEM. So it would be OK if our users tear patches off and throw them away. We attribute different gene circuits to different bacteria(Modularization idea), by changing the ratio of each bacteria we could make it compatible with any vaccine.</p>
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         <p align="justify">T-vaccine consists of four modules, each of which carries a gene circuit independently. With an excellent transdermal peptide TD1, three of the engineering <i>B.subtilis</i> could express a series of fusion proteins. The first one expresses TD1-Antigen fusion proteins, as the core of our vaccine. The second one expresses TD1-Anjuvant fusion proteins, which enhance the antigenicity. The third one is the reporter, which notifies users when the vaccine patch is ready to stick. The last module is a kill switch,which is designed as a reliable suicide system in <i>B.subtilis</i> to prevent it from spreading for the very first time in iGEM. We distributed different gene circuits to different bacteria(Modularization idea), and by changing the ratio of each bacteria we could make it compatible with any vaccine.</p>
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         <img src="" width="" height="" />流程图
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         <img src="https://static.igem.org/mediawiki/2013/0/0a/2013ustc-chinaWorkflow.png" width="580" height="300" style="margin-left:-30px;"/>
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<div class="atfirgure" align="center" style="width:580px;"><strong>Work Flow</strong></a></div>
         </div>
         </div>
                 <div class="basic-bar">
                 <div class="basic-bar">
         <h2>Our Design</h2>         
         <h2>Our Design</h2>         
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         <img src="https://static.igem.org/mediawiki/2013/4/4d/2013ustc-china_genecircuit.png" width="580" height="180"/>
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         <img src="https://static.igem.org/mediawiki/2013/4/4d/2013ustc-china_genecircuit.png" width="580" height="120"/>
         <h3>1. Antigen</h3>
         <h3>1. Antigen</h3>
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         <p>As the main part of T-vaccine, TD-1 is fused with different kinds of antigens, so it can penetrate the skin and provoke immune responses. We have designed three kinds of vaccine, which are against hepatitis B, tuberculosis and anthrax. Our ELISA test had proved their antigenicity and mice test will figure it out if they bave immunogenicity,thereby proving that T-vaccine can be generally used. </p>
+
         <p align="justify">As the main part of T-vaccine, TD-1 is fused with different kinds of antigens to penetrate the skin and provoke immune responses. We have designed three kinds of vaccines against hepatitis B, tuberculosis and anthrax respectively. Our ELISA test had proved their antigenicity and mice test will figure it out if they have immunogenicity, thereby proving the practicality of T-vaccine.</p>
         <br><br>
         <br><br>
-
         <h3>2. Adjuvant</h3>
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         <h3>2. Adjuvant:LTB</h3>
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         <p>Similar with traditional injection vaccine, adjuvant should be added into vaccine to ensure immunity. We fused TD-1 with LTB. LT has adjuvant activity and can assist foreign antigen to induce the body to produce systemic immune response. The B submit of LTB protein has no toxic effect and it also works.</p>
+
         <p align="justify">Similar to traditional injection vaccine, adjuvant should be added into our vaccine to enhance immunity. We fused TD-1 with LTB. LT submit has adjuvant activity and can assist foreign antigen to induce immune response, whereas the B submit of LTB protein,which has no toxic effect, also works.</p>
         <br><br>
         <br><br>
-
         <h3>3. TNFα</h3>
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         <h3>3. Adjuvant:TNFα</h3>
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         <p>TNFα can recruit Langerhans cell(LC) which work as antigen-presenting cells around epidermis, and improved LC could transmit into adjacent lymph node provoking the immune response. Because this circuit is quite similar with 1# and 2# ,while its effect might be difficult to certified, so we delayed the schedule of this part. 3# is the only circuit which has not been built or tested during the whole summer experiment.
+
         <p align="justify">TNFα can recruit Langerhans cells(LC), which work as antigen-presenting cells around epidermis, and improve LC could transmit into adjacent lymph node to provoke immune responses. This circuit is quite similar to 1# and 2# , yet its effect might be difficult to certify, which put off the schedule of this part. 3# is the only circuit which has not been built or tested during the whole summer experiment.
         </div>
         </div>
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         <div class="basic-bar">
         <div class="basic-bar">
         <h2>4. Reporter System</h2>
         <h2>4. Reporter System</h2>
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         <p>To be more user-friendly, 4# contains a reporting system. After melting in water, the spores will germinate and express blue pigment protein (amilCP) to report the best using time. Meanwhile, 4# could also ensure biosafety, as 4# engineering bacteria can kill all the engineered bacteria after use.</p>
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         <p align="justify">To be more user-friendly, 4# contains a reporting system. Melting in water, the spores will germinate and express blue pigment protein (amilCP) to report the optimal using time. Meanwhile, 4# could also ensure biosafety by killing all the engineered bacteria after usage.</p>
         <h3>4.1 Reporter</h3>
         <h3>4.1 Reporter</h3>
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         <img src="https://static.igem.org/mediawiki/igem.org/a/aa/2013ustc-china_design111reporter.png" width="580" height="160"/>
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         <img src="https://static.igem.org/mediawiki/igem.org/a/aa/2013ustc-china_design111reporter.png" width="580" height="130"/>
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         <p>T-vaccine is also a user-friendly product. Our potential users are not medical professionals, they activate T-vaccine by exposing it to water, and the patch gives visualized signal to notify users whether the status of engineered bacteria is all right and when to stick the patch to arms. We achieve this simply with the regulation of promoter 43, which is recognized by sigma factor A. The activity of promoter 43 is maximal during the exponential growth phase. In other words, we expect engineering Bacillus subtilis to express blue pigment a few hours before expressing massive antigens and adjuvants. It is the perfect time.</p>
+
         <p align="justify">T-vaccine is also a user-friendly product. Our potential users are not medical professionals, they activate T-vaccine by exposing it to water, and the patch gives visualized signal to notify users whether the status of engineered bacteria is all right and when to stick the patch to arms. We achieve this simply with the regulation of promoter 43, which is recognized by sigma factor A. The activity of promoter 43 is maximal during the exponential growth phase. In other words, we expect engineering <i>B.subtilis</i> to express blue pigment a few hours before expressing massive antigens and adjuvants.</p>
         <br><br>
         <br><br>
         <h3>4.2 Kill Switch</h3>
         <h3>4.2 Kill Switch</h3>
         <img src="https://static.igem.org/mediawiki/2013/f/f0/2013ustc-china_sdpABC.png" width="580" height="450"/>
         <img src="https://static.igem.org/mediawiki/2013/f/f0/2013ustc-china_sdpABC.png" width="580" height="450"/>
<br><br>
<br><br>
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         <p>In B.subtilis, when it comes to the stationary phase, the environmental pressure increases and nutrition becomes limited, so B.subtilis begins to produce spore. Now the community will be divided into two different parts. One of them are trying to kill others to get enough nutrient, delaying the production of spores and achieving a competitive advantage. Killing is mediated by the exported toxic protein SdpC. SdpI will appear on the membrane surface to avoid itself from being damaged. SdpI could bind free SdpC and autopressor SdpR, to remove inhibition of SdpR against I and R, to produce more SdpI to offset SdpC, finally guaranteeing the subgroup alive, thereby delaying the spores production.We transfer SdpC which is fused by promoter SdpI/R into high copy plasmids in order to damage the balance of the system, thereby killing whole colony. When SdpC appears, SdpI on the membrane will bind free SdpC and adsorb SdpR to cease its inhibition against SdpI P/R, trying to produce more SdpI. At the same time, it will activate the promoter SdpR/I in our circuits and generate more SdpC. The system would fall into an infinite loop, and according to our modeling,the amount of SdpC increases beyond the ability of SdpI. Thus, the cells with protection mechanism will crack and die because of too much SdpC. All of them formed the killing device.</p>
+
         <p align="justify">Cells of <i>B. subtilis</i> enter the pathway to sporulate under conditions of nutrient limitation but delay becoming committed to spore formation by killing nonsporulating siblings and feeding on the dead cells. Killing is mediated by the exported toxic protein SdpC. Extracellular SdpC induces the synthesis of an immunity protein, SdpI, which protects toxin-producing cells from being killed. SdpI, a polytopic membrane protein, is encoded by a two-gene operon under sporulation control that contains the gene for an autorepressor SdpR. The autorepressor binds to and blocks the promoter for the operon. Evidence indicates that SdpI is also a signal-transduction protein that responds to the SdpC toxin by sequestering the SdpR autorepressor at the membrane. Sequestration relieves repression and stimulates synthesis of immunity protein.</br>
 +
The kill switch is based on a high-copy vector fused with promoter for operon sdpIR and coding sequence for protein SdpC. When SdpC toxins are sensed,they will be captured by Immunity Protein SdpI at the membrane, enabling SdpI to sequester SdpR. As a result, repression on promoter SdpIR is released and more SdpC will be produced. Trapped in this endless loop, the SdpC producing cells fail to cope with enormous toxin SdpC and doomed after eliminating their siblings. Eventually, the group of engineered <i>B.subtilis</i> is destroyed instead of sporulating.</p>
         <img src="https://static.igem.org/mediawiki/igem.org/5/5d/2013ustc-china_design11sdpABC.png" width="580" height="160"/>
         <img src="https://static.igem.org/mediawiki/igem.org/5/5d/2013ustc-china_design11sdpABC.png" width="580" height="160"/>
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<p>We Also designed a test circuit, which contains promotor grac and sdpABC only, aiming to determine the ability of SdpC.</p>
+
<p align="justify">We also designed a test circuit, which contains promotor grac and sdpABC only, aiming to determine the ability of SdpC.</p></br></br>
 +
<h1>References<h1>
 +
<p align="justify" style="font-size:xx-small;">
 +
1.Kang, T.J., et al., Expression of the B subunit of <i>E. coli</i> heat-labile enterotoxin in the chloroplasts of plants and its characterization. Transgenic Res, 2003. 12(6): p. 683-91.<br>
 +
2.Mrsny, R.J., et al., Bacterial toxins as tools for mucosal vaccination. Drug Discovery Today, 2002. 7(4): p. 247-258.<br>
 +
3."Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis."Belisle J.T., Vissa V.D., Sievert T., Takayama K., Brennan P.J., Besra G.S. Science 276:1420-1422(1997)<br>
 +
4."Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis." Puech V., Guilhot C., Perez E., Tropis M., Armitige L.Y., Gicquel B., Daffe M.Mol. Microbiol. 44:1109-1122(2002)<br>
 +
5."The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity." Singh Y., Klimpel K.R., Quinn C.P., Chaudhary V.K., Leppla S.H.J. Biol. Chem. 266:15493-15497(1991) <br>
 +
6."Identification of a receptor-binding region within domain 4 of the protective antigen component of anthrax toxin." Varughese M., Teixeira A.V., Liu S., Leppla S.H.
 +
Infect. Immun. 67:1860-1865(1999)<br>
 +
7.Blumberg B, Alter H (1965). "A "new" antigen in leukemia sera". JAMA 191: 101–106.<br>
 +
8.Tiollais P, Christine P, Dejean A. The hepatitis B virus.Nature, 1985,371: 489.<br></p></div>
         </div>
         </div>
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<div id="t1"><a href="https://2013.igem.org/Team:USTC_CHINA/Project/Results">Results</a></div>
<div id="t1"><a href="https://2013.igem.org/Team:USTC_CHINA/Project/Results">Results</a></div>
<div id="t2"><a href="https://2013.igem.org/Team:USTC_CHINA/Project/Results">Basic Experiment</a></div>
<div id="t2"><a href="https://2013.igem.org/Team:USTC_CHINA/Project/Results">Basic Experiment</a></div>
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<div id="t2"><a href="https://2013.igem.org/Team:USTC_CHINA/Project/Results/FurtherWork">Further Work</a></div>
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<div id="t2"><a href="https://2013.igem.org/Team:USTC_CHINA/Project/AdvancingWork">Advancing Work</a></div>
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<div id="t2"><a href="https://2013.igem.org/Team:USTC_CHINA/Project/FutureWork">Future Work</a></div>
<div id="t1"><a href="https://2013.igem.org/Team:USTC_CHINA/Parts">Parts</a></div>
<div id="t1"><a href="https://2013.igem.org/Team:USTC_CHINA/Parts">Parts</a></div>
</div>
</div>

Latest revision as of 12:28, 28 October 2013

Gene Circuit

Introduction

T-vaccine consists of four modules, each of which carries a gene circuit independently. With an excellent transdermal peptide TD1, three of the engineering B.subtilis could express a series of fusion proteins. The first one expresses TD1-Antigen fusion proteins, as the core of our vaccine. The second one expresses TD1-Anjuvant fusion proteins, which enhance the antigenicity. The third one is the reporter, which notifies users when the vaccine patch is ready to stick. The last module is a kill switch,which is designed as a reliable suicide system in B.subtilis to prevent it from spreading for the very first time in iGEM. We distributed different gene circuits to different bacteria(Modularization idea), and by changing the ratio of each bacteria we could make it compatible with any vaccine.

Work Flow

Our Design

1. Antigen

As the main part of T-vaccine, TD-1 is fused with different kinds of antigens to penetrate the skin and provoke immune responses. We have designed three kinds of vaccines against hepatitis B, tuberculosis and anthrax respectively. Our ELISA test had proved their antigenicity and mice test will figure it out if they have immunogenicity, thereby proving the practicality of T-vaccine.



2. Adjuvant:LTB

Similar to traditional injection vaccine, adjuvant should be added into our vaccine to enhance immunity. We fused TD-1 with LTB. LT submit has adjuvant activity and can assist foreign antigen to induce immune response, whereas the B submit of LTB protein,which has no toxic effect, also works.



3. Adjuvant:TNFα

TNFα can recruit Langerhans cells(LC), which work as antigen-presenting cells around epidermis, and improve LC could transmit into adjacent lymph node to provoke immune responses. This circuit is quite similar to 1# and 2# , yet its effect might be difficult to certify, which put off the schedule of this part. 3# is the only circuit which has not been built or tested during the whole summer experiment.

4. Reporter System

To be more user-friendly, 4# contains a reporting system. Melting in water, the spores will germinate and express blue pigment protein (amilCP) to report the optimal using time. Meanwhile, 4# could also ensure biosafety by killing all the engineered bacteria after usage.

4.1 Reporter

T-vaccine is also a user-friendly product. Our potential users are not medical professionals, they activate T-vaccine by exposing it to water, and the patch gives visualized signal to notify users whether the status of engineered bacteria is all right and when to stick the patch to arms. We achieve this simply with the regulation of promoter 43, which is recognized by sigma factor A. The activity of promoter 43 is maximal during the exponential growth phase. In other words, we expect engineering B.subtilis to express blue pigment a few hours before expressing massive antigens and adjuvants.



4.2 Kill Switch



Cells of B. subtilis enter the pathway to sporulate under conditions of nutrient limitation but delay becoming committed to spore formation by killing nonsporulating siblings and feeding on the dead cells. Killing is mediated by the exported toxic protein SdpC. Extracellular SdpC induces the synthesis of an immunity protein, SdpI, which protects toxin-producing cells from being killed. SdpI, a polytopic membrane protein, is encoded by a two-gene operon under sporulation control that contains the gene for an autorepressor SdpR. The autorepressor binds to and blocks the promoter for the operon. Evidence indicates that SdpI is also a signal-transduction protein that responds to the SdpC toxin by sequestering the SdpR autorepressor at the membrane. Sequestration relieves repression and stimulates synthesis of immunity protein.
The kill switch is based on a high-copy vector fused with promoter for operon sdpIR and coding sequence for protein SdpC. When SdpC toxins are sensed,they will be captured by Immunity Protein SdpI at the membrane, enabling SdpI to sequester SdpR. As a result, repression on promoter SdpIR is released and more SdpC will be produced. Trapped in this endless loop, the SdpC producing cells fail to cope with enormous toxin SdpC and doomed after eliminating their siblings. Eventually, the group of engineered B.subtilis is destroyed instead of sporulating.

We also designed a test circuit, which contains promotor grac and sdpABC only, aiming to determine the ability of SdpC.



References

1.Kang, T.J., et al., Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization. Transgenic Res, 2003. 12(6): p. 683-91.
2.Mrsny, R.J., et al., Bacterial toxins as tools for mucosal vaccination. Drug Discovery Today, 2002. 7(4): p. 247-258.
3."Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis."Belisle J.T., Vissa V.D., Sievert T., Takayama K., Brennan P.J., Besra G.S. Science 276:1420-1422(1997)
4."Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis." Puech V., Guilhot C., Perez E., Tropis M., Armitige L.Y., Gicquel B., Daffe M.Mol. Microbiol. 44:1109-1122(2002)
5."The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity." Singh Y., Klimpel K.R., Quinn C.P., Chaudhary V.K., Leppla S.H.J. Biol. Chem. 266:15493-15497(1991)
6."Identification of a receptor-binding region within domain 4 of the protective antigen component of anthrax toxin." Varughese M., Teixeira A.V., Liu S., Leppla S.H. Infect. Immun. 67:1860-1865(1999)
7.Blumberg B, Alter H (1965). "A "new" antigen in leukemia sera". JAMA 191: 101–106.
8.Tiollais P, Christine P, Dejean A. The hepatitis B virus.Nature, 1985,371: 489.