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

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                     therapy (<b>Figure 1</b>).
 
                     therapy (<b>Figure 1</b>).
 
                 </p>
 
                 </p>
                 <img src="https://static.igem.org/mediawiki/2018/2/20/T--HZAU-China--design1.jpg" width="100%" alt="">
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                 <div style="width: 70%; margin: 0px auto">
                 <p><b>Figure 1.</b> Overall Circuit Design.</p>
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                    <img src="https://static.igem.org/mediawiki/2018/2/20/T--HZAU-China--design1.jpg" width="100%" alt="">
 +
                </div>
 +
                 <p style="width: 100%; text-align: center !important;"><b>Figure 1.</b> Overall Circuit Design.</p>
  
 
             </div>
 
             </div>
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                 <div class="h1">Chassis</div>
 
                 <div class="h1">Chassis</div>
 
                 <p>This year, we chose <i>Salmonella enterica</i> serovar Typhimurium str. SL1344 as our chassis.
 
                 <p>This year, we chose <i>Salmonella enterica</i> serovar Typhimurium str. SL1344 as our chassis.
                     Why we chose Salmonella as our carrier is based on the following reasons. First, GSDMD-N275 can only induce pyroptosis from the inside of a cell, therefore Salmonella is a brilliant candidate as an intracellular parasite. Second, Salmonella is a widely used carrier to cancer therapy because its natural taxis to tumor (<b>Figure 2</b>).
+
                     Why we chose Salmonella as our carrier is based on the following reasons. First, GSDMD-N275 can
                     However, feedbacks from human practice suggested that we should consider more about the safety in our
+
                    only induce pyroptosis from the inside of a cell, therefore Salmonella is a brilliant candidate as
 +
                    an intracellular parasite. Second, Salmonella is a widely used carrier to cancer therapy because
 +
                    its natural taxis to tumor (<b>Figure 2</b>).
 +
                     However, feedbacks from human practice suggested that we should consider more about the safety in
 +
                    our
 
                     design and experiment. (See more details in <a href="">Human Practice</a>.)
 
                     design and experiment. (See more details in <a href="">Human Practice</a>.)
 
                     Therefore, we make efforts to improve safety of our project through knocking out <i>sifA</i> and
 
                     Therefore, we make efforts to improve safety of our project through knocking out <i>sifA</i> and
 
                     displaying RGD motif on <i>Salmonella</i>.</p><br>
 
                     displaying RGD motif on <i>Salmonella</i>.</p><br>
<img src="https://static.igem.org/mediawiki/2018/7/7a/T--HZAU-China--design2.jpg" width="100%">
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                <div style="width: 80%; margin: 0px auto">
<b>Figure 2</b>. <i>Salmonella</i> have natural taxis to tumor.
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                    <img src="https://static.igem.org/mediawiki/2018/7/7a/T--HZAU-China--design2.jpg" width="100%">
 +
                </div>
 +
                <p style="width: 100%; text-align: center !important;"><b>Figure 2</b>. <i>Salmonella</i> have natural
 +
                    taxis to tumor.</p>
 
                 <p>
 
                 <p>
 
                     <i>sifA</i> locates in <i>Salmonella</i> pathogenicity island,
 
                     <i>sifA</i> locates in <i>Salmonella</i> pathogenicity island,
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                     Thus, to reduce virulence of Salmonella, <i>sifA</i> was knocked out in our
 
                     Thus, to reduce virulence of Salmonella, <i>sifA</i> was knocked out in our
 
                     project (<b>Figure 3</b>).
 
                     project (<b>Figure 3</b>).
<img src="https://static.igem.org/mediawiki/2018/a/a3/T--HZAU-China--design3.jpg" width="100%">
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                </p>
<b>Figure 3</b>. Schematic diagram of <i>sifA</i> mutant in macrophage.  
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                <div style="width: 80%; margin: 0px auto">
                </p><br><br>
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                    <img src="https://static.igem.org/mediawiki/2018/a/a3/T--HZAU-China--design3.jpg" width="100%">
 +
                </div>
 +
                <p style="width: 100%; text-align: center !important;"><b>Figure 3</b>. Schematic diagram of <i>sifA</i>
 +
                    mutant in macrophage.</p>
 +
                <br>
 
                 <p>
 
                 <p>
 
                     RGD motif (Arg-Gly-Asp) is a well-studied tumor homing tripeptide that specifically binds to alpha
 
                     RGD motif (Arg-Gly-Asp) is a well-studied tumor homing tripeptide that specifically binds to alpha
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                     In order to enhance targeting of bacteria to tumor, RGD motif is displayed on OmpA, an outer
 
                     In order to enhance targeting of bacteria to tumor, RGD motif is displayed on OmpA, an outer
 
                     membrane protein of bacteria (<b>Figure 4</b>).
 
                     membrane protein of bacteria (<b>Figure 4</b>).
                 </p><br><br>
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                 </p><br>
 
                 <p>Finally, the safety of our project is successfully demonstrated by a set of experiments using
 
                 <p>Finally, the safety of our project is successfully demonstrated by a set of experiments using
 
                     engineered bacteria mentioned above. (See more details in <a href="https://2018.igem.org/Team:HZAU-China/Results">Results</a>.)</p>
 
                     engineered bacteria mentioned above. (See more details in <a href="https://2018.igem.org/Team:HZAU-China/Results">Results</a>.)</p>
                 <img src="" width="100%" alt="">
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                 <div style="width: 80%; margin: 0px auto">
<img src="https://static.igem.org/mediawiki/2018/e/e1/T--HZAU-China--design4.jpg" width="100%">
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                    <img src="https://static.igem.org/mediawiki/2018/e/e1/T--HZAU-China--design4.jpg" width="100%">
                 <p><b>Figure 4.</b> Realization of tumor targeting through surface displaying RGD motif.</p>
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                </div>
 +
                 <p style="width: 100%; text-align: center !important;"><b>Figure 4.</b> Realization of tumor targeting
 +
                    through surface displaying RGD motif.</p>
  
  
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                     high response speed and great linear relation between the inducer and the expression of downstream
 
                     high response speed and great linear relation between the inducer and the expression of downstream
 
                     gene<sup>3</sup>. With the presence of anhydrotetracycline (ATc),
 
                     gene<sup>3</sup>. With the presence of anhydrotetracycline (ATc),
                     the repressor TetR which is under the control of tet promoter (P<sub>tet</sub>) will combine with ATc
+
                     the repressor TetR which is under the control of tet promoter (P<sub>tet</sub>) will combine with
 +
                    ATc
 
                     and Mg<sup>2+</sup>, resulting in expression of GSDMD-N275 (<b>Figure 5</b>).
 
                     and Mg<sup>2+</sup>, resulting in expression of GSDMD-N275 (<b>Figure 5</b>).
 
                     Finally, this system is successfully used to express GSDMD-N275 in Salmonella and induces host cell
 
                     Finally, this system is successfully used to express GSDMD-N275 in Salmonella and induces host cell
 
                     pyroptosis. (See more details in <a href="https://2018.igem.org/Team:HZAU-China/Results">Results</a>.)
 
                     pyroptosis. (See more details in <a href="https://2018.igem.org/Team:HZAU-China/Results">Results</a>.)
                 </p>              
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                 </p>
  
                        <div style="width: 80%; margin: 30px auto">
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                <div style="width: 80%; margin: 0px auto">
                            <img src="https://static.igem.org/mediawiki/2018/2/23/T--HZAU-China--tetr2.png" width="100%" alt="">
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                    <img src="https://static.igem.org/mediawiki/2018/2/23/T--HZAU-China--tetr2.png" width="100%" alt="">
                        </div>
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                </div>
<img src="https://static.igem.org/mediawiki/2018/9/9b/T--HZAU-China--design5.jpg" width="100%">
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                <div style="width: 80%; margin: 0px auto">
                        <p><b>Figure 5. </b>Schematic diagram of ATc-dependent expression of GSDMD-N275. <p>
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                    <img src="https://static.igem.org/mediawiki/2018/9/9b/T--HZAU-China--design5.jpg" width="100%">
 +
                </div>
 +
                <p style="width: 100%; text-align: center !important;"><b>Figure 5. </b>Schematic diagram of
 +
                    ATc-dependent expression of GSDMD-N275. <p>
 
             </div>
 
             </div>
 
             <div id="float04">
 
             <div id="float04">
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                     exist in <i>Salmonella</i><sup>4</sup>.
 
                     exist in <i>Salmonella</i><sup>4</sup>.
 
                     Therefore, this feature gives an approach for us to implement specific expression of
 
                     Therefore, this feature gives an approach for us to implement specific expression of
                     GSDMD-N275. We utilized the regulatory part from the upstream of <i>sifA</i> (P<sub>sifA</sub>) to control the expression of GSDMD-N275 (<b>Figure 6.</b>)
+
                     GSDMD-N275. We utilized the regulatory part from the upstream of <i>sifA</i> (P<sub>sifA</sub>) to
 +
                    control the expression of GSDMD-N275 (<b>Figure 6.</b>)
 
                     Ultimately, we successfully demonstrated the intracellular specificty of P<sub>sifA</sub>
 
                     Ultimately, we successfully demonstrated the intracellular specificty of P<sub>sifA</sub>
 
                     .(See more details in <a href="https://2018.igem.org/Team:HZAU-China/Results">Results</a>.)
 
                     .(See more details in <a href="https://2018.igem.org/Team:HZAU-China/Results">Results</a>.)
 
                 </p>
 
                 </p>
                 <img src="https://static.igem.org/mediawiki/2018/5/54/T--HZAU-China--sifA.png" width="100%" alt="">
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                 <div style="width: 80%; margin: 0px auto">
<img src="https://static.igem.org/mediawiki/2018/1/14/T--HZAU-China--design6.jpg" width="100%">
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                    <img src="https://static.igem.org/mediawiki/2018/5/54/T--HZAU-China--sifA.png" width="100%" alt="">
                 <p><b>Figure 6.</b> Schematic diagram of intracellular environment-dependent expression of
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                </div>
 +
                <div style="width: 80%; margin: 0px auto">
 +
                    <img src="https://static.igem.org/mediawiki/2018/1/14/T--HZAU-China--design6.jpg" width="100%">
 +
                </div>
 +
                 <p style="width: 100%; text-align: center !important;"><b>Figure 6.</b> Schematic diagram of
 +
                    intracellular environment-dependent expression of
 
                     GSDMD-N275.</p>
 
                     GSDMD-N275.</p>
 
             </div>
 
             </div>
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                 <p>2. Danhier, F., Le Breton, A. & Preat, V. RGD-based strategies to target alpha(v) beta(3) integrin
 
                 <p>2. Danhier, F., Le Breton, A. & Preat, V. RGD-based strategies to target alpha(v) beta(3) integrin
 
                     in cancer therapy and diagnosis. Mol Pharm 9, 2961-2973, doi:10.1021/mp3002733 (2012).</p>
 
                     in cancer therapy and diagnosis. Mol Pharm 9, 2961-2973, doi:10.1021/mp3002733 (2012).</p>
                 <p>3.Nevozhay, D. Negative autoregulation linearizes the dose–response and suppresses the heterogeneity
+
                 <p>3. Nevozhay, D. Negative autoregulation linearizes the dose–response and suppresses the
 +
                    heterogeneity
 
                     of gene expression. PNAS 106
 
                     of gene expression. PNAS 106
 
                     5123-5128, doi:10.1073/pnas.0809901106 (2008).
 
                     5123-5128, doi:10.1073/pnas.0809901106 (2008).

Revision as of 20:30, 17 October 2018

Overview

In our project, we redesigned Salmonella to act as a delivery vehicle that can target tumor cells and replicate in their cytoplasm. By inducing the bacterial expression of the N-terminal domain of Gasdermin D (GSDMD-N275), bacteria are led to lysis and release this protein into the cytoplasm of tumor cell and then induce pyroptosis to the tumor cell by making membrane pores. The lysate of cell rupture during pyroptosis destroys the tumor microenvironment and attracts immune cells into tumor bed to kill tumor cells. Our project which aims to induce pyroptosis to tumor cells provides a new approach for cancer therapy (Figure 1).

Figure 1. Overall Circuit Design.

Chassis

This year, we chose Salmonella enterica serovar Typhimurium str. SL1344 as our chassis. Why we chose Salmonella as our carrier is based on the following reasons. First, GSDMD-N275 can only induce pyroptosis from the inside of a cell, therefore Salmonella is a brilliant candidate as an intracellular parasite. Second, Salmonella is a widely used carrier to cancer therapy because its natural taxis to tumor (Figure 2). However, feedbacks from human practice suggested that we should consider more about the safety in our design and experiment. (See more details in Human Practice.) Therefore, we make efforts to improve safety of our project through knocking out sifA and displaying RGD motif on Salmonella.


Figure 2. Salmonella have natural taxis to tumor.

sifA locates in Salmonella pathogenicity island, taking the role of maintaining the stability of Salmonella-Containing Vacuole (SCV) when Salmonella survive and replicate in host cells. Because of the unstable SCV, growth inhibition of ΔsifA mutant in macrophage is remarkable1. Thus, to reduce virulence of Salmonella, sifA was knocked out in our project (Figure 3).

Figure 3. Schematic diagram of sifA mutant in macrophage.


RGD motif (Arg-Gly-Asp) is a well-studied tumor homing tripeptide that specifically binds to alpha v beta 3 (αvβ3) integrin, which is a biomarker of cancer cells and widely overexpressed in cancer cells and blood vessels during cancer angiogenesis2. In order to enhance targeting of bacteria to tumor, RGD motif is displayed on OmpA, an outer membrane protein of bacteria (Figure 4).


Finally, the safety of our project is successfully demonstrated by a set of experiments using engineered bacteria mentioned above. (See more details in Results.)

Figure 4. Realization of tumor targeting through surface displaying RGD motif.

ATc-dependent expression of GSDMD-N275

We use anhydrotetracycline transcriptional regulation system to regulate the expression of GSDMD-N275 in our project because of its low expression noise, high response speed and great linear relation between the inducer and the expression of downstream gene3. With the presence of anhydrotetracycline (ATc), the repressor TetR which is under the control of tet promoter (Ptet) will combine with ATc and Mg2+, resulting in expression of GSDMD-N275 (Figure 5). Finally, this system is successfully used to express GSDMD-N275 in Salmonella and induces host cell pyroptosis. (See more details in Results.)

Figure 5. Schematic diagram of ATc-dependent expression of GSDMD-N275.

Intracellular environment-dependent expression of GSDMD-N275

As an intracellular parasite, some intracellular environment-dependent genes such as sifA exist in Salmonella4. Therefore, this feature gives an approach for us to implement specific expression of GSDMD-N275. We utilized the regulatory part from the upstream of sifA (PsifA) to control the expression of GSDMD-N275 (Figure 6.) Ultimately, we successfully demonstrated the intracellular specificty of PsifA .(See more details in Results.)

Figure 6. Schematic diagram of intracellular environment-dependent expression of GSDMD-N275.

Reference

1. Thurston, T. L. et al. Growth inhibition of cytosolic Salmonella by caspase-1 and caspase-11 precedes host cell death. Nature communications 7, 13292, doi:10.1038/ncomms13292 (2016).

2. Danhier, F., Le Breton, A. & Preat, V. RGD-based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis. Mol Pharm 9, 2961-2973, doi:10.1021/mp3002733 (2012).

3. Nevozhay, D. Negative autoregulation linearizes the dose–response and suppresses the heterogeneity of gene expression. PNAS 106 5123-5128, doi:10.1073/pnas.0809901106 (2008).

4. Garmendia, J., Beuzon, 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, doi:10.1099/mic.0.26397-0 (2003).

Description
Design

Overview

Chassis

ATc-dependent expression of GSDMD-N275

Intracellular environment-dependent expression of GSDMD-N275

Reference

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