Difference between revisions of "Team:HZAU-China/Design"

Line 91: Line 91:
 
             z-index: 1;
 
             z-index: 1;
 
             background-color: #EEEEEE;
 
             background-color: #EEEEEE;
−
            opacity: 0.96;
 
−
            filter: alpha(opacity=96);
 
 
             box-shadow: 0 5px 15px #CCCCCC;
 
             box-shadow: 0 5px 15px #CCCCCC;
 
             display: none;
 
             display: none;
Line 154: Line 152:
 
         }
 
         }
  
−
         .daohang a:hover span{
+
         .daohang a:hover span {
−
             transform: rotateY(180deg);          
+
             transform: rotateY(180deg);
−
             -webkit-transform: rotateY(180deg);          
+
             -webkit-transform: rotateY(180deg);
−
             -moz-transform: rotateY(180deg);          
+
             -moz-transform: rotateY(180deg);
−
             -o-transform: rotateY(180deg);          
+
             -o-transform: rotateY(180deg);
 
             -ms-transform: rotateY(180deg);
 
             -ms-transform: rotateY(180deg);
−
             transition: all 0.5s ease-in-out;          
+
             transition: all 0.5s ease-in-out;
−
             -webkit-transition: all 0.5s ease-in-out;          
+
             -webkit-transition: all 0.5s ease-in-out;
−
             -moz-transition: all 0.5s ease-in-out;          
+
             -moz-transition: all 0.5s ease-in-out;
 
             -o-transition: all 0.5s ease-in-out;
 
             -o-transition: all 0.5s ease-in-out;
 
         }
 
         }
Line 325: Line 323:
 
         .zhengwen p{
 
         .zhengwen p{
 
             text-align: justify !important;
 
             text-align: justify !important;
−
             font-family: Arial, Helvetica, sans-serif !important;
+
             font-family: 'Times New Roman', Arial, Helvetica, sans-serif !important;
−
             color: #484848;
+
             color: #3B3B3B;
 
             font-size: 22px !important;
 
             font-size: 22px !important;
 
             padding-right: 20px;
 
             padding-right: 20px;
Line 340: Line 338:
  
 
         .h1 {
 
         .h1 {
−
             height: 100px;
+
             height: 80px;
−
             line-height: 100px;
+
             line-height: 80px;
 
             font-weight: bold;
 
             font-weight: bold;
 
             font-family: 'Product Sans', sans-serif;
 
             font-family: 'Product Sans', sans-serif;
−
             font-size: 50px;
+
             font-size: 40px;
−
             color: black;
+
             color: #3B3B3B;
−
             border-bottom: 3px solid black;
+
             border-bottom: 2px solid #676767;
 
             margin-bottom: 20px;
 
             margin-bottom: 20px;
 +
        }
 +
 +
        .h2 {
 +
            height: 40px;
 +
            line-height: 40px;
 +
            /* font-weight: bold; */
 +
            font-family: 'Times New Roman', Times, serif;
 +
            font-size: 30px;
 +
            color: #484848;
 +
            /* margin-bottom: 20px; */
 
         }
 
         }
  
Line 630: Line 638:
 
                 <a class="item" href="https://2018.igem.org/Team:HZAU-China/Improve">Improve</a>
 
                 <a class="item" href="https://2018.igem.org/Team:HZAU-China/Improve">Improve</a>
 
                 <a class="item" href="https://2018.igem.org/Team:HZAU-China/InterLab">Interlab</a>
 
                 <a class="item" href="https://2018.igem.org/Team:HZAU-China/InterLab">Interlab</a>
 +
                <a class="item" href="https://2018.igem.org/Team:HZAU-China/Notebook">Notebook</a>
 
             </li>
 
             </li>
 
             <li class="hiLight shortName">
 
             <li class="hiLight shortName">
Line 670: Line 679:
 
         <div class="zhengwen">
 
         <div class="zhengwen">
 
             <div id="float01" class="cur">
 
             <div id="float01" class="cur">
−
                 <div class="h1"><span class="xie">sifA</span> knock out</div>
+
                 <div class="h1">Modification of Bacteria</div>
−
                <p>SifA maintains the integrity of <i>Salmonella</i>-containing vacuole (SCV) where <i>Salmonella</i> survive and replicate1. The exsistence of SCV limits GSDMD-N275 releasing to cytoplasma. what else, growth of <span class="xie">ΔsifA</span> mutant is inhibited in macrophage2.Thus, we knock out the sifA gene in order to prevent the stability of SCV and reduce the virulence of <i>Salmonella</i>. <span class="xie">ΔsifA</span> mutant was cnstructed by using gene editing systerm based on two-step allelic exchange3.</p>
+
                <p>Gene <span class="xie">sifA</span> is a vital gene located in Salmonella pathogenicity island,
 +
                    taking the role of maintaining the stability of Salmonella-Containing Vacuole (SCV) only after
 +
                    Salmonella infection.<sup>1,2,3,4</sup>. In our experiment, a fragment including <span class="xie">sifA</span>
 +
                    promoter region and its RBS region were amplified from Salmonella SL1344 genome, with the target
 +
                    protein downstream. We also knocked out the <span class="xie">sifA</span> gene from Salmonella
 +
                    Typhimurium str. SL1344 genome by conjugational transfer. The aim is to reduce the toxicity of
 +
                    Salmonella and allow the protein, which is able to rupture the phospholipid bilayer of the cell
 +
                    membrane from the inner side purely, to release into the cytoplasm of the tumor cell.</p>
 
             </div>
 
             </div>
 
             <div id="float02">
 
             <div id="float02">
 
                 <div class="h1">Targeting</div>
 
                 <div class="h1">Targeting</div>
−
 
+
                <div class="h2">Surface Display</div>
 +
                <p>In order to enable bacteria to have the greater targeting ability towards tumor, we designed
 +
                    OmpA-RGD protein to achieve this goal.
 +
                    RGD is a well-studied tumor homing tripeptide that specifically binds to alpha v beta 3 (αvβ3)
 +
                    integrin, which is widely overexpressed on cancer cells and blood vessels during cancer
 +
                    angiogenesis. We intended to display RGD on the surface of bacteria, so as to target the αvβ3 on
 +
                    the surface of tumor cells.
 +
                </p>
 +
                <img src="" alt="Figure 1.">
 +
                <p>Thus we needed a methodto display RGD in the outer membrane. We chose Lpp signal peptide andouter
 +
                    membrane protein A (OmpA) system, which has been extensively used todisplay a diverse group of
 +
                    proteins, including Green Fluorescent Protein(GFP)<sup>5</sup>, Organophosphorus Hydrolase
 +
                    (OPH),single chain
 +
                    Fv fragments (scFv), Cellulomonas fimi exoglucanase Cexand its cellulose-binding domain(CBS<sub>cex</sub>)<sup>6</sup>.
 +
                    Therefore, we expressedLpp-OmpA-RGD fusion protein under the control of Ara promoter in oursystem.
 +
                    The fusion protein would be anchored in the outer membrane of bacteriaand display RGD on the
 +
                    surface. Then RGD would lead the Salmonella to bind withtumor cells which have αvβ3 integrin on
 +
                    their surface<sup>7</sup>.</p>
 +
                <img src="" alt="Figure 2.">
 
             </div>
 
             </div>
 
             <div id="float03">
 
             <div id="float03">
 
                 <div class="h1">Regulation of pyroptosis</div>
 
                 <div class="h1">Regulation of pyroptosis</div>
−
                  
+
                 <div class="h2">Chemical Induction</div>
 +
                <p>We decided to useanhydrotetracycline transcriptional regulation system toexpress GSDMD-N275 protein.
 +
                    It is one of the commonest methods toexpress toxin protein because of its low expression noise,
 +
                    high response speedand the good linear relation between the inducer and the expression of
 +
                    product.Anhydrotetracycline transcriptional regulation system is a one-component signaltransduction
 +
                    system that regulates the expression of the tetracyclineresistance determinant encoded by tetA, the
 +
                    anhydrotetracycline efflux pumpingin Escherichia coli<sup>8</sup>. In this system, promoter Ptet
 +
                    expressesthe
 +
                    repression protein TetR. With the presence of ATc (anhydrotetracycline),TetR will integrate ATc
 +
                    and. This induces a conformationalchange in TetR and its dissociation from the operon of Ptetand
 +
                    abolishes the repression<sup>9</sup>.
 +
                </p>
 +
                <img src="" alt="Figure 3.">
 +
                <p> We decided to use this system to regulate the expression ofour
 +
                    GSDMD-N275 protein, which controlled by Ptet promoter.</p>
 +
                <img src="" alt="Figure 4.">
 +
                <p>However, we worried that the leakage of GSDMD-N275 will induce cytotoxicity. So we replaced
 +
                    different strength of RBS to choose the suitable RBS to control the leakage of GSDMD-N275. Owing to
 +
                    testing the linear stability of ATc induction and to transfer the expression of GSDMD-N275 to the
 +
                    concentration of ATc, we also constructed the TetR::EGFP (enhanced green fluorescent) fusion
 +
                    protein in E.coli (Fig.5) as an essay suggested<sup>10</sup>. And we induced Salmonella with
 +
                    gradients of ATc
 +
                    and measured the fluorescence accordingly. More details to our experimental method has been listed
 +
                    in our <a href="https://2018.igem.org/Team:HZAU-China/Notebook">notebook</a> and the <a href="https://2018.igem.org/Team:HZAU-China/Model">ATc
 +
                        induction model</a>.
 +
                    Finally, we constructed the EGFP::GSDMD-N275
 +
                    expressed by the anhydrotetracycline transcriptional regulation system to proof our final circuit.
 +
                </p>
 +
                <img src="" alt="Figure 5.">
 +
                <div class="h2">The Intracellular Specific Induction</div>
 +
                <p>Once delivered into the host cell, Gram-negative bacteria Salmonella always express its virulence
 +
                    genes to alter host cell functions with the advantage of the pathogen. These genes are in
 +
                    Salmonella Pathogenicity Island and enterobactin gene cluster<sup>1,11</sup>. Owing to the feature
 +
                    of
 +
                    expressing intracellularly, we used the promoters of these genes to express GSDMD-N275 after its
 +
                    entrance to tumor cells. To achieve this goal, we constructed PsifA::GSDMD-N275 and transferred it
 +
                    via electroporation (see details in our Methods). The figure below shows their functions (Fig 6.).
 +
                    Promoters in the circuits won't work outside cells but only express GSDMD-N275 after the entry to
 +
                    tumor cells.
 +
                </p>
 
             </div>
 
             </div>
 
             <div id="float04">
 
             <div id="float04">
 
                 <div class="h1">Reference</div>
 
                 <div class="h1">Reference</div>
 +
                <p> 1. Garmendia, J., Beuzón, C. R., Ruiz-Albert, J. & Holden, D. W. The roles of SsrA-SsrB and
 +
                    OmpR-EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion
 +
                    system. Microbiology 149, 2385–2396 (2003).</p>
 +
                <p>2. Beuzon, C. R. Salmonella maintains the integrity of its intracellular vacuole through the action
 +
                        of SifA. EMBO J. 19, 3235–3249 (2000).</p>
 +
                <p>3. Steele-Mortimer, O. The Salmonella-containing vacuole-Moving with the times. Curr. Opin.
 +
                        Microbiol. 11, 38–45 (2008).</p>
 +
                <p>4. Nevozhay, D., Adams, R. M., Murphy, K. F., Josic, K. & Balazsi, G. Negative autoregulation
 +
                        linearizes the dose-response and suppresses the heterogeneity of gene expression. Proc. Natl. Acad.
 +
                        Sci. 106, 5123–5128 (2009).</p>
 +
                <p>5. Shi, H. & Wen Su, W. Display of green fluorescent protein on Escherichia coli cell surface.
 +
                        Enzyme Microb. Technol. 28, 25–34 (2001).</p>
 +
                <p>6. Earhart, C. F. Use of a n Lpp-OmpA Fusion Vehicle for Bacterial Surface Display. Methods
 +
                        Enzymol. 326, 506–516 (2000).</p>
 +
                <p>7. Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer: Biological implications and
 +
                        therapeutic opportunities. Nat. Rev. Cancer 10, 9–22 (2010).</p>
 +
                <p>8. Cuthbertson, L. & Nodwell, J. R. The TetR Family of Regulators. Microbiol. Mol. Biol. Rev. 77,
 +
                        440–475 (2013).</p>
 +
                <p>9. Kisker, C., Hinrichs, W., Tovar, K., Hillen, W. & Saenger, W. The complex formed between Tet
 +
                        repressor and tetracycline-Mg2+ reveals mechanism of antibiotic resistance. J. Mol. Biol.
 +
                        247,260–280 (1995).</p>
 +
                <p>10. Nevozhay, D., Adams, R. M., Murphy, K. F., Josic, K. & Balazsi, G. Negative autoregulation
 +
                        linearizes the dose-response and suppresses the heterogeneity of gene expression. Proc. Natl. Acad.
 +
                        Sci. 106, 5123–5128 (2009).</p>
 +
                <p>11. Crouch, M. L. V, Castor, M., Karlinsey, J. E., Kalhorn, T. & Fang, F. C. Biosynthesis and
 +
                        IroC-dependent export of the siderophore salmochelin are essential for virulence of Salmonella
 +
                        enterica serovar Typhimurium. Mol. Microbiol. 67, 971–983 (2008).</p>
 
             </div>
 
             </div>
 
         </div>
 
         </div>
Line 693: Line 793:
 
             </a>
 
             </a>
 
             <div class="daohangyi">
 
             <div class="daohangyi">
−
                    <span class="biaoti">Design</span>
+
                <span class="biaoti">Design</span>
−
                    <span class="xsjPic"><img src="https://static.igem.org/mediawiki/2018/8/8d/T--HZAU-China--xjt.svg" alt=""></span>
+
                <span class="xsjPic"><img src="https://static.igem.org/mediawiki/2018/8/8d/T--HZAU-China--xjt.svg" alt=""></span>
 
             </div>
 
             </div>
 
             <div class="floatCtro">
 
             <div class="floatCtro">
−
                 <p class="daohanger">sifA knock out</p>
+
                 <p class="daohanger">Modification of Bacteria</p>
 
                 <p class="daohanger">Targeting</p>
 
                 <p class="daohanger">Targeting</p>
 
                 <p class="daohanger">Regulation of pyroptosis</p>
 
                 <p class="daohanger">Regulation of pyroptosis</p>
Line 764: Line 864:
 
                 }
 
                 }
 
                 if (sTop >= f4 - 100) {
 
                 if (sTop >= f4 - 100) {
−
fixRight.eq(3).addClass('cur').siblings().removeClass('cur');
+
                    fixRight.eq(3).addClass('cur').siblings().removeClass('cur');
−
}
+
                }
 
             }
 
             }
 
         })
 
         })

Revision as of 19:22, 11 October 2018

Modification of Bacteria

Gene sifA is a vital gene located in Salmonella pathogenicity island, taking the role of maintaining the stability of Salmonella-Containing Vacuole (SCV) only after Salmonella infection.1,2,3,4. In our experiment, a fragment including sifA promoter region and its RBS region were amplified from Salmonella SL1344 genome, with the target protein downstream. We also knocked out the sifA gene from Salmonella Typhimurium str. SL1344 genome by conjugational transfer. The aim is to reduce the toxicity of Salmonella and allow the protein, which is able to rupture the phospholipid bilayer of the cell membrane from the inner side purely, to release into the cytoplasm of the tumor cell.

Targeting
Surface Display

In order to enable bacteria to have the greater targeting ability towards tumor, we designed OmpA-RGD protein to achieve this goal. RGD is a well-studied tumor homing tripeptide that specifically binds to alpha v beta 3 (αvβ3) integrin, which is widely overexpressed on cancer cells and blood vessels during cancer angiogenesis. We intended to display RGD on the surface of bacteria, so as to target the αvβ3 on the surface of tumor cells.

Figure 1.

Thus we needed a methodto display RGD in the outer membrane. We chose Lpp signal peptide andouter membrane protein A (OmpA) system, which has been extensively used todisplay a diverse group of proteins, including Green Fluorescent Protein(GFP)5, Organophosphorus Hydrolase (OPH),single chain Fv fragments (scFv), Cellulomonas fimi exoglucanase Cexand its cellulose-binding domain(CBScex)6. Therefore, we expressedLpp-OmpA-RGD fusion protein under the control of Ara promoter in oursystem. The fusion protein would be anchored in the outer membrane of bacteriaand display RGD on the surface. Then RGD would lead the Salmonella to bind withtumor cells which have αvβ3 integrin on their surface7.

Figure 2.
Regulation of pyroptosis
Chemical Induction

We decided to useanhydrotetracycline transcriptional regulation system toexpress GSDMD-N275 protein. It is one of the commonest methods toexpress toxin protein because of its low expression noise, high response speedand the good linear relation between the inducer and the expression of product.Anhydrotetracycline transcriptional regulation system is a one-component signaltransduction system that regulates the expression of the tetracyclineresistance determinant encoded by tetA, the anhydrotetracycline efflux pumpingin Escherichia coli8. In this system, promoter Ptet expressesthe repression protein TetR. With the presence of ATc (anhydrotetracycline),TetR will integrate ATc and. This induces a conformationalchange in TetR and its dissociation from the operon of Ptetand abolishes the repression9.

Figure 3.

We decided to use this system to regulate the expression ofour GSDMD-N275 protein, which controlled by Ptet promoter.

Figure 4.

However, we worried that the leakage of GSDMD-N275 will induce cytotoxicity. So we replaced different strength of RBS to choose the suitable RBS to control the leakage of GSDMD-N275. Owing to testing the linear stability of ATc induction and to transfer the expression of GSDMD-N275 to the concentration of ATc, we also constructed the TetR::EGFP (enhanced green fluorescent) fusion protein in E.coli (Fig.5) as an essay suggested10. And we induced Salmonella with gradients of ATc and measured the fluorescence accordingly. More details to our experimental method has been listed in our notebook and the ATc induction model. Finally, we constructed the EGFP::GSDMD-N275 expressed by the anhydrotetracycline transcriptional regulation system to proof our final circuit.

Figure 5.
The Intracellular Specific Induction

Once delivered into the host cell, Gram-negative bacteria Salmonella always express its virulence genes to alter host cell functions with the advantage of the pathogen. These genes are in Salmonella Pathogenicity Island and enterobactin gene cluster1,11. Owing to the feature of expressing intracellularly, we used the promoters of these genes to express GSDMD-N275 after its entrance to tumor cells. To achieve this goal, we constructed PsifA::GSDMD-N275 and transferred it via electroporation (see details in our Methods). The figure below shows their functions (Fig 6.). Promoters in the circuits won't work outside cells but only express GSDMD-N275 after the entry to tumor cells.

Reference

1. Garmendia, J., Beuzón, C. R., Ruiz-Albert, J. & Holden, D. W. The roles of SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system. Microbiology 149, 2385–2396 (2003).

2. Beuzon, C. R. Salmonella maintains the integrity of its intracellular vacuole through the action of SifA. EMBO J. 19, 3235–3249 (2000).

3. Steele-Mortimer, O. The Salmonella-containing vacuole-Moving with the times. Curr. Opin. Microbiol. 11, 38–45 (2008).

4. Nevozhay, D., Adams, R. M., Murphy, K. F., Josic, K. & Balazsi, G. Negative autoregulation linearizes the dose-response and suppresses the heterogeneity of gene expression. Proc. Natl. Acad. Sci. 106, 5123–5128 (2009).

5. Shi, H. & Wen Su, W. Display of green fluorescent protein on Escherichia coli cell surface. Enzyme Microb. Technol. 28, 25–34 (2001).

6. Earhart, C. F. Use of a n Lpp-OmpA Fusion Vehicle for Bacterial Surface Display. Methods Enzymol. 326, 506–516 (2000).

7. Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer: Biological implications and therapeutic opportunities. Nat. Rev. Cancer 10, 9–22 (2010).

8. Cuthbertson, L. & Nodwell, J. R. The TetR Family of Regulators. Microbiol. Mol. Biol. Rev. 77, 440–475 (2013).

9. Kisker, C., Hinrichs, W., Tovar, K., Hillen, W. & Saenger, W. The complex formed between Tet repressor and tetracycline-Mg2+ reveals mechanism of antibiotic resistance. J. Mol. Biol. 247,260–280 (1995).

10. Nevozhay, D., Adams, R. M., Murphy, K. F., Josic, K. & Balazsi, G. Negative autoregulation linearizes the dose-response and suppresses the heterogeneity of gene expression. Proc. Natl. Acad. Sci. 106, 5123–5128 (2009).

11. Crouch, M. L. V, Castor, M., Karlinsey, J. E., Kalhorn, T. & Fang, F. C. Biosynthesis and IroC-dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium. Mol. Microbiol. 67, 971–983 (2008).

Description
Design

Modification of Bacteria

Targeting

Regulation of pyroptosis

Reference

Back to Top
Results Demonstrate