Difference between revisions of "Team:HZAU-China/Design"
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<div id="float01" class="cur"> | <div id="float01" class="cur"> | ||
<div class="h1">Modification of Bacteria</div> | <div class="h1">Modification of Bacteria</div> | ||
| − | <p>Gene <span class="xie">sifA</span> is a vital gene located in Salmonella pathogenicity island, | + | <p>Gene <span class="xie">sifA</span> is a vital gene located in <span class="xie">Salmonella</span> pathogenicity island, |
| − | taking the role of maintaining the stability of Salmonella-Containing Vacuole (SCV) only after | + | taking the role of maintaining the stability of <span class="xie">Salmonella</span>-Containing Vacuole (SCV) only after |
| − | Salmonella infection.<sup>1,2,3,4</sup>. We also knocked out the <span class="xie">sifA</span> gene from Salmonella | + | <span class="xie">Salmonella</span> infection.<sup>1,2,3,4</sup>. We also knocked out the <span class="xie">sifA</span> gene from <span class="xie">Salmonella</span> |
Typhimurium str. SL1344 genome by conjugational transfer. The aim is to reduce the toxicity of | 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 | + | <span class="xie">Salmonella</span> 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 cytoplasma of the tumor cell.</p> | membrane from the inner side purely, to release into the cytoplasma of the tumor cell.</p> | ||
</div> | </div> | ||
| Line 705: | Line 705: | ||
Therefore, we expressedLpp-OmpA-RGD fusion protein under the control of Ara promoter in oursystem. | 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 | 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 | + | surface. Then RGD would lead the <span class="xie">Salmonella</span> to bind withtumor cells which have αvβ3 integrin on |
their surface<sup>7</sup>.</p> | their surface<sup>7</sup>.</p> | ||
<img src="" alt="Figure 2."> | <img src="" alt="Figure 2."> | ||
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testing the linear stability of ATc induction and to transfer the expression of GSDMD-N275 to the | 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 | 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 | + | protein in E.coli (Fig.5) as an essay suggested<sup>10</sup>. And we induced <span class="xie">Salmonella</span> with |
gradients of ATc | gradients of ATc | ||
and measured the fluorescence accordingly. More details to our experimental method has been listed | and measured the fluorescence accordingly. More details to our experimental method has been listed | ||
| Line 741: | Line 741: | ||
<img src="" alt="Figure 5."> | <img src="" alt="Figure 5."> | ||
<div class="h2">The Intracellular Specific Induction</div> | <div class="h2">The Intracellular Specific Induction</div> | ||
| − | <p>Once delivered into the host cell, Gram-negative bacteria Salmonella always express its virulence | + | <p>Once delivered into the host cell, Gram-negative bacteria <span class="xie">Salmonella</span> always express its virulence |
genes to alter host cell functions with the advantage of the pathogen. These genes are in | 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 | + | <span class="xie">Salmonella</span> Pathogenicity Island and enterobactin gene cluster<sup>1,11</sup>. Owing to the feature |
of | of | ||
expressing intracellularly, we used the promoters of these genes to express GSDMD-N275 after its | expressing intracellularly, we used the promoters of these genes to express GSDMD-N275 after its | ||
| Line 755: | Line 755: | ||
<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 | <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 | + | OmpR-EnvZ in the regulation of genes encoding the <span class="xie">Salmonella</span> typhimurium SPI-2 type III secretion |
system. Microbiology 149, 2385–2396 (2003).</p> | system. Microbiology 149, 2385–2396 (2003).</p> | ||
| − | <p>2. Beuzon, C. R. Salmonella maintains the integrity of its intracellular vacuole through the action | + | <p>2. Beuzon, C. R. <span class="xie">Salmonella</span> maintains the integrity of its intracellular vacuole through the action |
of SifA. EMBO J. 19, 3235–3249 (2000).</p> | of SifA. EMBO J. 19, 3235–3249 (2000).</p> | ||
| − | <p>3. Steele-Mortimer, O. The Salmonella-containing vacuole-Moving with the times. Curr. Opin. | + | <p>3. Steele-Mortimer, O. The <span class="xie">Salmonella</span>-containing vacuole-Moving with the times. Curr. Opin. |
Microbiol. 11, 38–45 (2008).</p> | Microbiol. 11, 38–45 (2008).</p> | ||
<p>4. Nevozhay, D., Adams, R. M., Murphy, K. F., Josic, K. & Balazsi, G. Negative autoregulation | <p>4. Nevozhay, D., Adams, R. M., Murphy, K. F., Josic, K. & Balazsi, G. Negative autoregulation | ||
| Line 779: | Line 779: | ||
Sci. 106, 5123–5128 (2009).</p> | Sci. 106, 5123–5128 (2009).</p> | ||
<p>11. Crouch, M. L. V, Castor, M., Karlinsey, J. E., Kalhorn, T. & Fang, F. C. Biosynthesis and | <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 | + | IroC-dependent export of the siderophore salmochelin are essential for virulence of <span class="xie">Salmonella</span> |
enterica serovar Typhimurium. Mol. Microbiol. 67, 971–983 (2008).</p> | enterica serovar Typhimurium. Mol. Microbiol. 67, 971–983 (2008).</p> | ||
</div> | </div> | ||
Revision as of 12:28, 15 October 2018
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. 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 cytoplasma of the tumor cell.
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.
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.
We decided to use anhydrotetracycline transcriptional regulation system to express GSDMD-N275 protein. It is one of the commonest methods to express toxin protein because of its low expression noise, high response speed and the good linear relation between the inducer and the expression of product. Anhydrotetracycline transcriptional regulation system is a one-component signal transduction system that regulates the expression of the tetracycline resistance determinant encoded by tetA, the anhydrotetracycline efflux pumping in Escherichia coli8. In this system, promoter Ptet expresses the repression protein TetR. With the presence of ATc (anhydrotetracycline), TetR will integrate ATc and. This induces a conformational change in TetR and its dissociation from the operon of Ptet and abolishes the repression9.
We decided to use this system to regulate the expression ofour GSDMD-N275 protein, which controlled by Ptet promoter.
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.
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.
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).
