Difference between revisions of "Team:HZAU-China/Improve"
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<p>We gave the surface display system from | <p>We gave the surface display system from | ||
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part is under the control of <i>lac</i> promoter. </p> | part is under the control of <i>lac</i> promoter. </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> | <p><b>Figure 1</b>. The homology modelling result of Lpp-OmpA-RGD. Red arrow shows the location of RGD motif.</p> | ||
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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 <a href="http://parts.igem.org/Part:BBa_J36850">BBa_J36850</a>. </p> | <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 <a href="http://parts.igem.org/Part:BBa_J36850">BBa_J36850</a>. </p> | ||
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<p><b>Figure 2</b>. 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 | ||
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<i>lac</i> promoter. </p> | <i>lac</i> promoter. </p> | ||
<div style="width: 90%; margin: 0px auto"> | <div style="width: 90%; margin: 0px auto"> | ||
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</div> | </div> | ||
<p><b>Figure 3</b>. 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= | + | <img src="https://static.igem.org/mediawiki/2018/f/f7/T--HZAU-China--Improve4.png" width=100% alt=""> |
</div> | </div> | ||
<p><b>Figure 4</b>. 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"> | ||
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</div> | </div> | ||
<p><b>Figure 5</b>. 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 | ||
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<label for="zhedie-toggle3">Method</label> | <label for="zhedie-toggle3">Method</label> | ||
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<b>Preparation of Cells for Infection</b><br> | <b>Preparation of Cells for Infection</b><br> | ||
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7.Add 16 μg/mL ATc for remainder of experiment.<br> | 7.Add 16 μg/mL ATc for remainder of experiment.<br> | ||
Observation is taken after 5 min, 30 min, 1.5 h.<br><br> | Observation is taken after 5 min, 30 min, 1.5 h.<br><br> | ||
| − | + | <div class="h1">Basic Part Table</div> | |
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<table class="table table-bordered table-hover"> | <table class="table table-bordered table-hover"> | ||
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| − | <tr><td><a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2632008">BBa_K2632008</a></td><td>Coding</td><td>Plac-Lpp-OmpA-RGD</td><td width="100px">Lingyu Zhong</td><td align="right">619</td> | + | <tr> |
| − | </tr> | + | <td><a class="noul_link part_link" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2632008">BBa_K2632008</a></td> |
| − | + | <<td>Coding</td><td>Plac-Lpp-OmpA-RGD</td><td width="100px">Lingyu Zhong</td><td align="right">619</td> | |
| − | + | </tr> </div> | |
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<div class="h1">Reference</div> | <div class="h1">Reference</div> | ||
<p>1 Park, S. H. et al. RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic | <p>1 Park, S. H. et al. RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic | ||
Revision as of 18:29, 17 October 2018
We gave the surface display system from 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.
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.
| Name | Type | Description | Designer | Length(bp) |
|---|---|---|---|---|
| BBa_K2632008 | <Coding | Plac-Lpp-OmpA-RGD | Lingyu Zhong | 619 |
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).
