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

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                 </p>
 
                 </p>
 
                 <img src="" width="100%" alt="">
 
                 <img src="" width="100%" alt="">
                 <p><b>Figure 1.</b> Overall Design Circuit</p>
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                 <p><b>Figure 1.</b> Overall Circuit Design.</p>
  
 
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             </div>
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                 <p>
 
                 <p>
 
                     <i>sifA</i> locates in <i>Salmonella</i> pathogenicity island,
 
                     <i>sifA</i> locates in <i>Salmonella</i> pathogenicity island,
                     taking the role of maintaining the stability of <i>Salmonella</i>-Containing Vacuole (SCV) where <i>Salmonella</i>
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                     taking the role of maintaining the stability of <i>Salmonella</i>-Containing Vacuole (SCV) when <i>Salmonella</i>
 
                     survive and replicate in host cells.
 
                     survive and replicate in host cells.
 
                     Because of the unstable SCV, growth inhibition of <i>ΔsifA</i> mutant in macrophage is remarkable<sup>1</sup>.
 
                     Because of the unstable SCV, growth inhibition of <i>ΔsifA</i> mutant in macrophage is remarkable<sup>1</sup>.
                     Thus, with the view of reducing 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.
 
                     project.
 
                 </p><br><br>
 
                 </p><br><br>
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                     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
 
                     v beta 3 (αvβ3) integrin,
 
                     v beta 3 (αvβ3) integrin,
                     which is a biomarker of cancer cells and widely overexpressed on cancer cells and blood vessels
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                     which is a biomarker of cancer cells and widely overexpressed in cancer cells and blood vessels
 
                     during cancer angiogenesis<sup>2</sup>.
 
                     during cancer angiogenesis<sup>2</sup>.
 
                     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

Revision as of 18:09, 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. 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.



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.



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 2).



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 2. 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 upstream gene3. With the presence of anhydrotetracycline (ATc), repressor TetR which under the control of tet promoter (Ptet) will integrate with ATc and Mg2+ result in expression of GSDMD-N275 (Figure 3). Finally, this system is successfully used to express GSDMD-N275 in Salmonella and induce host cell pyroptosis. (See more details in Results.)

Figure 3. 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 are existed in Salmonella4. Therefore, this event shows an approach for us to implement specific expression of GSDMD-N275. We utilized regulatory part from the upstream of sifA (PsifA) to control the expression of GSDMD-N275 (Figure 4). Ultimately, we successfully demonstrated the intracellular specificty of PsifA .(See more details in Results.)

Figure 4. 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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Results Demonstrate