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

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         <div class="zhengwen">
 
         <div class="zhengwen">
 
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             <div id="float01" class="cur">
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                 <p>We gave the surface display system from BBa_J36850 a new function through displaying a RGD motif on
+
                 <p>We gave the surface display system from <a href="http://parts.igem.org/Part:BBa_J36850">BBa_J36850</a> a new function through displaying a RGD motif on
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                     the Lpp-OmpA. RGD motif can specifically bind to αVβ3, a biomarker of cancer cells<sup>1</sup>. We
+
                     the Lpp-OmpA which contains a signal sequence, the N-terminal of the lipoprotein (Lpp) and the residual 46-159 amino acids of the OmpA. In addition, lipoprotein executes the function of targeting to the outer membrane and OmpA constructs an anchor on the outer membrane. RGD motif can specifically bind to αVβ3, a biomarker of cancer cells such as melanoma, neuroblastoma, glioma, adenocarcinoma<sup>1</sup>. We
 
                     determine
 
                     determine
−
                     the surface display site on the third loop of Lpp-OmpA through homology modelling (Figure 1). This
+
                     the surface display site on the third loop of Lpp-OmpA through homology modelling (<b>Figure 1</b>). This
 
                     part is under the control of <i>lac</i> promoter. </p>
 
                     part is under the control of <i>lac</i> promoter. </p>
 
                 <div style="width: 30%; margin: 0px auto">
 
                 <div style="width: 30%; margin: 0px auto">
 
                     <img src="https://static.igem.org/mediawiki/2018/5/5f/T--HZAU-China--Improve1.png" width=100% alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/5/5f/T--HZAU-China--Improve1.png" width=100% alt="">
 
                 </div>
 
                 </div>
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                 <p>Figure 1. Homology modelling result of Lpp-OmpA-RGD. Red arrow shows the location of RGD motif.</p>
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                 <p><b>Figure 1</b>. The homology modelling result of Lpp-OmpA-RGD. Red arrow shows the location of RGD motif.</p>
 
             </div>
 
             </div>
 
             <div id="float02">
 
             <div id="float02">
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                 <p>Microscopy shows that <i>E. coli</i> expressed Lpp-OmpA-RGD induced by 0.1mM IPTG can bind to αVβ3-positive
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                 <p>Microscopy shows that Lpp-OmpA-RGD overexpressed by <i>E. coli</i> with 0.1mM IPTG can bind to αVβ3-positive MDA-MB-231 cell. Red arrow point the location of <i>E. coli</i> (<b>Figure 2</b>). But can not bind to αVβ3-negative MCF7 cell (<b>Figure 3</b>). We also use BBa_J36850 as a control. This strain can not bind to Vβ3-positive MDA-MB-231 cell line (<b>Figure 4</b>) and αVβ3-negative MCF7 cell line (<b>Figure 5</b>). These results suggest that we successfully improve the part BBa_J36850. </p>
−
                    MDA-MB-231 cell line. Red arrow points the location of bacteria (Figure 2). But cannot bind to
+
−
                    αVβ3-negative MCF7 cell line (Figure 3). We also use BBa_J36850 as a control. This strain cannot
+
−
                    bind to αVβ3-positive MDA-MB-231 cell line (Figure 4) and αVβ3-negative MCF7 cell line (Figure 5).
+
−
                    These results suggest that we successfully improve the part BBa_J36850. (Click here to see the
+
−
                    method)</p>
+
 
                 <div style="width: 90%; margin: 0px auto">
 
                 <div style="width: 90%; margin: 0px auto">
 
                     <img src="https://static.igem.org/mediawiki/2018/b/b1/T--HZAU-China--Improve2.png" width=100% alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/b/b1/T--HZAU-China--Improve2.png" width=100% alt="">
 
                 </div>
 
                 </div>
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                 <p>Figure 2. αVβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constructively
+
                 <p><b>Figure 2</b>. αVβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constructively
 
                     expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of
 
                     expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of
 
                     <i>lac</i> promoter. </p>
 
                     <i>lac</i> promoter. </p>
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                     <img src="https://static.igem.org/mediawiki/2018/c/cf/T--HZAU-China--Improve3.png" width=100% alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/c/cf/T--HZAU-China--Improve3.png" width=100% alt="">
 
                 </div>
 
                 </div>
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                 <p>Figure 3. αVβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constructive expressed RFP
+
                 <p><b>Figure 3</b>. αVβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constructive expressed RFP
 
                     and contained BBa_J36850. This improved part expressed RGD motif under the control of <i>lac</i> promoter.</p>
 
                     and contained BBa_J36850. This improved part expressed RGD motif under the control of <i>lac</i> promoter.</p>
 
                 <div style="width: 90%; margin: 0px auto">
 
                 <div style="width: 90%; margin: 0px auto">
 
                     <img src="https://static.igem.org/mediawiki/2018/f/f7/T--HZAU-China--Improve4.png" width=100% alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/f/f7/T--HZAU-China--Improve4.png" width=100% alt="">
 
                 </div>
 
                 </div>
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                 <p>Figure 4. αVβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constructively
+
                 <p><b>Figure 4</b>. αVβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constructively
 
                     expressed RFP and contained BBa_J36850.</p>
 
                     expressed RFP and contained BBa_J36850.</p>
 
                 <div style="width: 90%; margin: 0px auto">
 
                 <div style="width: 90%; margin: 0px auto">
 
                     <img src="https://static.igem.org/mediawiki/2018/c/ce/T--HZAU-China--Improve5.png" width=100% alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/c/ce/T--HZAU-China--Improve5.png" width=100% alt="">
 
                 </div>
 
                 </div>
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                 <p>Figure 5. αVβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constructively expressed RFP
+
                 <p><b>Figure 5</b>. αVβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constructively expressed RFP
 
                     and contained BBa_J36850. </p>
 
                     and contained BBa_J36850. </p>
 
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                 <div class="collapseDiv">
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                         <b>Preparation of Cells for Infection</b><br>
 
                         <b>Preparation of Cells for Infection</b><br>
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                         1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO2 tissue-culture incubator.<br>
+
                         1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO<sub>2</sub> tissue-culture incubator.<br>
 
                         2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow
 
                         2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow
 
                         overnight.<br>
 
                         overnight.<br>
 
                         <b>Preparation of Bacteria</b><br>
 
                         <b>Preparation of Bacteria</b><br>
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                         1. Grow bacteria overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking
+
                         1. Grow bacterial cells overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking
 
                         incubator (200 rpm).<br>
 
                         incubator (200 rpm).<br>
−
                         2. Subculture bacteria by transferring 300 μL of the overnight culture into 5 mL of LB in a
+
                         2. Subculture bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB in a
 
                         loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log
 
                         loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log
 
                         phase.<br>
 
                         phase.<br>
−
                         3. Pellet 1 mL of the Salmonella subculture by centrifugation at 1000 g in a microfuge for 2
+
                         3. Pellet 1 mL of the bacterial cells subculture by centrifugation at 1000 g in a microfuge for 2
 
                         min at room temperature.<br>
 
                         min at room temperature.<br>
 
                         4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.<br>
 
                         4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.<br>
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                         2. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the cell-culture
 
                         2. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the cell-culture
 
                         supernatant.<br>
 
                         supernatant.<br>
−
                         3. Incubate for 2 h at 37 °C in 5% CO2.<br>
+
                         3. Incubate for 2 h at 37 °C in 5% CO<sub>2</sub>.<br>
 
                         4. Aspirate media and wash<br>
 
                         4. Aspirate media and wash<br>
−
                         5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37 °C in 5% CO2 for 2
+
                         5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37 °C in 5% CO<sub>2</sub> for 2
 
                         h.<br>
 
                         h.<br>
 
                         6. Replace GM with fresh GM containing 20 μg/mL gentamicin for 1 h.<br>
 
                         6. Replace GM with fresh GM containing 20 μg/mL gentamicin for 1 h.<br>

Revision as of 17:34, 17 October 2018

We gave the surface display system from BBa_J36850 a new function through displaying a RGD motif on the Lpp-OmpA which contains a signal sequence, the N-terminal of the lipoprotein (Lpp) and the residual 46-159 amino acids of the OmpA. In addition, lipoprotein executes the function of targeting to the outer membrane and OmpA constructs an anchor on the outer membrane. RGD motif can specifically bind to αVβ3, a biomarker of cancer cells such as melanoma, neuroblastoma, glioma, adenocarcinoma1. We determine the surface display site on the third loop of Lpp-OmpA through homology modelling (Figure 1). This part is under the control of lac promoter.

Figure 1. The homology modelling result of Lpp-OmpA-RGD. Red arrow shows the location of RGD motif.

Microscopy shows that Lpp-OmpA-RGD overexpressed by E. coli with 0.1mM IPTG can bind to αVβ3-positive MDA-MB-231 cell. Red arrow point the location of E. coli (Figure 2). But can not bind to αVβ3-negative MCF7 cell (Figure 3). We also use BBa_J36850 as a control. This strain can not bind to Vβ3-positive MDA-MB-231 cell line (Figure 4) and αVβ3-negative MCF7 cell line (Figure 5). These results suggest that we successfully improve the part BBa_J36850.

Figure 2. αVβ3-positive MDA-MB-231 cell line was incubated with E. coli which constructively expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of lac promoter.

Figure 3. αVβ3-negative MCF7 cell line was incubated with E. coli which constructive expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of lac promoter.

Figure 4. αVβ3-positive MDA-MB-231 cell line was incubated with E. coli which constructively expressed RFP and contained BBa_J36850.

Figure 5. αVβ3-negative MCF7 cell line was incubated with E. coli which constructively expressed RFP and contained BBa_J36850.

Preparation of Cells for Infection
1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow overnight.
Preparation of Bacteria
1. Grow bacterial cells overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the bacterial cells subculture by centrifugation at 1000 g in a microfuge for 2 min at room temperature.
4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.
Infection
1. Aspirate media and rinse the monolayer twice with PBS.
2. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the cell-culture supernatant.
3. Incubate for 2 h at 37 °C in 5% CO2.
4. Aspirate media and wash
5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37 °C in 5% CO2 for 2 h.
6. Replace GM with fresh GM containing 20 μg/mL gentamicin for 1 h.
7.Add 16 μg/mL ATc for remainder of experiment.
Observation is taken after 5 min, 30 min, 1.5 h.

Reference

1 Park, S. H. et al. RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic Efficacy of Attenuated Salmonella-mediated Cancer Therapy. Theranostics 6, 1672-1682, doi:10.7150/thno.16135 (2016).

Experiments
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